Preface

Microtubules are one of the main components of the eukaryotic cytoskeleton. Together with the actin-myosin system, and among other functions, they organize the intracellular transport, forming the filaments along which dyneins and kinesins travel. These motor proteins use the energy produced by ATP hydrolysis to walk along microtubule, carrying loads. The hallmark of kinesins is a relatively well-conserved motor domain, which comprises both the binding site for the nucleotide and the binding site for microtubule. Based on the position of the motor domain in the sequence, at the N-terminus, internal or at the C-terminus, kinesins can be divided into three subfamilies, N-type, internal and Ctype, which are further divided into 14 classes according to differences in the motor domain sequence. N-type kinesins, including classes 1 to 12, move towards the plus end of microtubule, i.e. broadly speaking from the center to the periphery of interphase cells; whereas C-type kinesins, which kinesin-14 belongs to, move towards microtubule minus end. Kinesin-13s, internal kinesins, are not motile but depolymerize microtubule, showing that kinesins have roles beyond transport, for instance in the regulation of microtubule dynamics.

Kinesin binding to or dissociating from microtubules is tightly coupled with its nucleotide cycle. ADP-kinesin has a low affinity for microtubules and a low ADP dissociation rate. Binding to microtubules enhances ADP dissociation in kinesin and converts kinesin to a high affinity state for microtubules. Subsequent ATP binding triggers the conformational changes leading to the mechanical step. After ATP hydrolysis and Pi release, kinesin, back to ADP bound state, detaches from microtubules to complete the cycle. Although this general framework is well established, our understanding of several steps of this cycle is still incomplete, including how ADP dissociation is accelerated by microtubules and how ATP binding triggers the movement. Structurally, the conformation of ADP-kinesin detached from microtubule is known from X-ray crystallography whereas both crystal and electron microscopy structures have been determined for kinesin in an ATP state, bound to a microtubule (or to tubulin, which is the microtubule building block). What is missing are high resolution structural data on nucleotide-free kinesin, both isolated and as a complex with microtubules. These data should highlight the mechanism of ADP release from kinesin and complete our view of the kinesin structural cycle related to its mechanochemical cycle. This is the objective of my work.

Introduction 1. Microtubules and tubulin

In eukaryotic cells, combined with microfilaments and intermediate filaments, microtubules serve as cytoskeleton, providing cells with an inner framework, maintaining the shape of cells. Microtubules contribute to the structural formation of mitotic spindle, centrosome, cilia and flagella. Also, microtubules serve as a track for intercellular transport. Motor proteins, as kinesin and dynein, move along microtubules to transport cargoes. Microtubules are observed in all eukaryotes, playing important roles in different cellular functions including cell division, cell transport, morphogenesis etc.

Microtubules

Structure of microtubules

Microtubules have a cylinder-shaped structure with an external diameter of 24 nm. They are typically formed by 13 linear and parallel protofilaments that interact laterally. In vitro, they can consist of 9 to 16 protofilaments. Each of the protofilaments is composed of α/β tubulin heterodimers arranged head-to-tail (Fig. 1.1a), so in a protofilament, one end is an exposed α tubulin and the other is an exposed β tubulin. Because of the parallel arrangement of the protofilaments, microtubules are polarized: the end with the β subunit exposed is called the plus end; the other end with α tubulin exposed is called the minus end. Microtubules can be elongated at both the plus and minus ends, but it extends more rapidly at its plus end [START_REF] Walker | Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies[END_REF].

A standard microtubule with 13 protofilaments has a pitch of 3 monomers; consequently microtubule has a "seam". Laterally, protofilaments contact each other with the same subunit (i.e. α tubulin to α tubulin, β tubulin to β tubulin), except the contacts in the "seam", where α subunits are adjacent to neighbor β subunits (Fig. 1.1a).

Dynamics of microtubules

Dynamic instability of microtubules is studied as a foundation of cell physiology [START_REF] Brouhard | Dynamic instability 30 years later: complexities in microtubule growth and catastrophe[END_REF]. Figure 1.1b presents the dynamics of microtubule including several main processes: polymerization, depolymerization, catastrophe and rescue [START_REF] Conde | Microtubule assembly, organization and dynamics in axons and dendrites[END_REF]. b) The microtubule cycle.

In a tubulin heterodimer, each subunit binds a nucleotide, one of which is neither hydrolysable nor exchangeable GTP bound to α tubulin. Concerning the nucleotide in β tubulin, if it is a GTP, then the tubulin is called GTP-tubulin; if it is a GDP instead, the tubulin is called GDP-tubulin. That GTP-tubulins keep binding to microtubule ends is responsible for the growth of microtubules. This event is also called "polymerization". The hydrolysis of the GTP molecule in GTP-tubulin, accompanying microtubule assembly, leads to the paradox that microtubules are composed mainly of GDP-tubulin, which tends to fall off from the tip of microtubules. The paradox is resolved because of the delay between microtubule assembly and GTP hydrolysis, which leads to the formation of a "GTP-cap", preventing microtubules from depolymerization at microtubule ends. When the GTP in the cap is hydrolyzed, microtubules will go through rapid shrinkage. The loss of the GTP-cap and its induced disassembly is called a "catastrophe". It is observed by cryo-electron microscopy that protofilaments peel from the ends into ring-like structures during shrinkage [START_REF] Mandelkow | Microtubule dynamics and microtubule caps: a time-resolved cryo-electron microscopy study[END_REF]. GTP tubulin can also bind to the shrinking end to form a new cap preventing the microtubule from depolymerizing. The process that a microtubule stops shrinking and starts to grow is called a 'rescue'.

Microtubule growth and shrinkage is the basis of its functions.

Tubulin

Tubulin belongs to a protein family of several members sharing a conserved GTPase domain. There are seven different kinds of tubulin in eukaryotic cells: α, β, , , ,  and  [START_REF] Oakley | An abundance of tubulins[END_REF]Among them, , ,  and tubulin are considered as rare tubulins and do not have a ubiquitous distribution in eukaryotic organisms [START_REF] Dutcher | The tubulin fraternity: alpha to eta[END_REF]. The other tubulins are found in all eukaryotes. tubulin, localized primarily in centrosomes and spindle pole bodies, is associated with microtubules nucleation and polar orientation. α and β tubulin are always associated in an heterodimer which is the building block of microtubule in all eukaryotic cells. The tubulin talked about in this thesis refers to the α/β tubulin heterodimer.

Characters of tubulin heterodimers

The sequence of α and β tubulins is highly conserved across species. For example, the comparison of tubulin sequences from human, Drosophila, Chlamydomonas, Trypanosoma and Paramecium indicates that α tubulins are 89-95% similar and that β tubulins are 88-94% similar [START_REF] Dutcher | The tubulin fraternity: alpha to eta[END_REF].

α tubulins and β tubulins, both having a molecular weight about 50 000 Dalton, share 40% amino-acid sequence identity. The isoelectric point of these subunits is between 5.2 and 5.8. Each monomer consists of about 440 amino acids upon which post-translational modifications play an important role in its dynamics and its organizations [START_REF] Song | Post-translational modifications of tubulin: pathways to functional diversity of microtubules[END_REF].

In cells, α/β tubulin colliding with the end of a protofilament extends a microtubule. The higher the concentration of tubulin is, the more frequently these collisions occur, and therefore the microtubule grows faster. For a single protofilament, α/β tubulin binds to its ends rapidly (at about 4 μM -1 s -1 ), but it falls off as rapidly when there is no lateral bond formed. Usually α/β tubulins entombed by their neighbors with a low off rate form open sheets at the ends of microtubules, which close into cylinders later. This process is usually accompanied by GTP hydrolysis, though the hydrolysis of GTP is not obliged. With GMPCPP, a GTP non-hydrolysable analog, tubulin heterodimers can still form microtubules [START_REF] Nogales | Structural intermediates in microtubule assembly and disassembly: how and why?[END_REF].

In solution, tubulin is instable and tends to form polymers. This process is GTP consuming and sensitive to the temperature. At 4°C, tubulins do not self-assemble and exist as heterodimers in the solution. When incubated at 37 °C with GTP and magnesium ions (Mg 2+ ) in the solution, tubulin heterodimers polymerize to form microtubules. If the temperature is decreased, microtubules start to depolymerize. This behavior of tubulin is used to purify it by several cycles of polymerization and depolymerization but also gives trouble for tubulin crystallization. Protein crystallization usually requires high homogeneity of samples, while tubulin heterodimers tend to polymerize and form various polymers. As a result, tubulin crystallization without any stabilizer is very difficult. Actually, so far there is no tubulin heterodimer alone without stabilizer that has been reported to be crystallized.

Structural studies on tubulin

Tubulin can exist in three major forms: straight protofilaments, as found in microtubule, heterodimers and curved oligomers. Different approaches have been used to characterize them structurally. The first near-atomic model of tubulin was determined by electron crystallography [START_REF] Nogales | Structure of the alpha beta tubulin dimer by electron crystallography[END_REF]. In the presence of zinc ion, tubulin assembles into two-dimensional sheets made of antiparallel protofilaments. These Znsheets have been studied by electron diffraction. More recently, thanks to the progress in Cryo-electron microscopy, the structure of microtubules has been determined at about 3.5 Å resolution [START_REF] Zhang | Mechanistic Origin of Microtubule Dynamic Instability and Its Modulation by EB Proteins[END_REF]. The structure of non-microtubular tubulin has also been determined but by X-ray crystallography with different tubulin stabilizers. Our lab published a crystal structure of a complex of GDP tubulin, two tubulins arranged head to tail, with the stathmin-like domain of RB3, a stathmin family protein [START_REF] Ravelli | Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain[END_REF]. Following this, more high resolution crystal structures of tubulin oligomers and heterodimers in complex with stabilizers have been determined [START_REF] Nawrotek | The determinants that govern microtubule assembly from the atomic structure of GTP-tubulin[END_REF].

As expected from their ~40% sequence identity, α tubulin and β tubulin present a similar overall structure (Fig. 1.2). They are globular proteins with a diameter of about 4 nm. Each subunit consists of two interacting β sheets surrounded by α helices. The subunit can be divided into three structural domains, including the nucleotide binding domain, an intermediate domain and the C-terminal domain. The nucleotide binding domain, also referred to as the N-terminal domain, forms a Rossmann fold where parallel β strands alternate with α helices. The Rossmann fold is typical for nucleotide-binding proteins. The nucleotide binding site on α-tubulin is called N-site (Non-exchangeable site); the one on βtubulin is called E-site (exchangeable site). The intermediate domain, containing the taxol binding site, also has a β sheet surrounded by α helices. The C-terminal domain, consisting of 2 α helices contacting the previous domains, is exposed to the outside surface of the microtubule and is usually the target of microtubule associated proteins (MAPs) and motor proteins [START_REF] Downing | Tubulin and microtubule structure[END_REF]. The last C-terminal residues of each subunit are flexible and are usually not seen in the structures. They are enriched in acidic residues. This part is also where the most differences among isotypes across species occur. Besides, these residues are the target of most of the post-translational modifications, including the removal of the last tyrosine of the α subunit and the addition of glutamate chains [START_REF] Wloga | Post-translational modifications of microtubules[END_REF]. Nevertheless, these residues are removable by a protease called subtilisin which will reduces the total number of isotypes for tubulin consequently [START_REF] Lobert | Subtilisin cleavage of tubulin heterodimers and polymers[END_REF], so it's likely that subtilisin treatment may improve the possibility of tubulin's crystallization. For β subunit, there are more than one subtilisin site which have different accessibility to the protease corresponding to the heterodimer or polymer form of the tubulin.

Ligands of tubulin

Tubulin is the target of agents which can perturb microtubule dynamics with either a stabilizing or a destabilizing effect [START_REF] Cormier | The binding of vinca domain agents to tubulin: structural and biochemical studies[END_REF]). Some of these ligands are used clinically for cancer treatment. Different ligands target various sites in the tubulin heterodimer. Here I will present only two ligands of tubulin that I have used during my work either as a tubulin stabilizer for crystallization purpose, or as a microtubule stabilizer needed for kinetic studies.

Colchicine

Colchicine is originally extracted from plants of the genus Colchicum. It prevents tubulin heterodimer from polymerizing into microtubules. Structural studies identified the colchicine binding site on β tubulin; it is mostly buried in the β tubulin's intermediate domain [START_REF] Ravelli | Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain[END_REF]). So as a tubulin stabilizer, colchicine has probably no direct impact on kinesin or kinesin-like protein binding. It has been cheeked afterwards by gel filtration chromatography that with or without colchicine a peak of kinesin-tubulin-DARPin complex shows at a similar position.

Taxane

Taxanes are originally found in plants of the genus Taxus. Taxol and Taxotere are used widely as chemotherapy agents because of their ability to interfere with cell division by stabilizing microtubules. These compounds are used to generate stable microtubules, when they are bound to tubulin heterodimers in a one to one ratio. The taxol binding site was identified by electron crystallography [START_REF] Nogales | Structure of the alpha beta tubulin dimer by electron crystallography[END_REF] on Zn-sheet tubulin, as a hydrophobic pocket located also in β tubulin.

DARPin

DARPin is the abbreviation for 'Designed Ankyrin Repeat ProteIN' (Fig. 1.3). Anti-tubulin DARPins have been selected in the lab to interfere with microtubule assembly in a unique manner. The ideal DARPins used in this thesis should be able to bind tubulin stably with a ratio of one to one forming a tubulin DARPin trimer, while not preventing kinesins from binding to tubulin. D1 is a typical selected DARPin, able to stabilize the tubulin heterodimer. It contains an N-cap (blue) and a C-cap (red), between which there are three internal ankyrin repeats. Its N-terminus also has six His (wheat) to facilitate purification.

Selection of DARPins

The DARPin library initially used for screening consists of DARPins having four or five ankyrin repeats. The N-terminal and the C-terminal ankyrin repeats stabilize the structure, between which there are two (N2C) or three (N3C) variable repeats. Tubulin was fixed on the surface of microtiter plates through its α subunit; consequently, its β subunit was exposed. The library was screened through four rounds of ribosome display. Then the cDNAs of selected DARPins were subcloned and clones producing tubulin binders were identified by ELISA on crude cell extracts [START_REF] Pecqueur | A designed ankyrin repeat protein selected to bind to tubulin caps the microtubule plus end[END_REF].

Several DARPins generating a strong signal during screening were expressed and purified. Because of the way tubulin was immobilized, the likelihood to obtain β subunit-specific DARPins was enhanced. However, the precise binding site remains unknown until the DARPin-tubulin complex structure is determined.

DARPin-tubulin complex

Several structures of DARPins in complex with tubulin heterodimer have been determined.

D1-tubulin structure at 2.2 Å resolution has been determined [START_REF] Pecqueur | A designed ankyrin repeat protein selected to bind to tubulin caps the microtubule plus end[END_REF]. D1, binding a tubulin heterodimer with a one to one ratio, has a high affinity for tubulin regardless the nucleotide state of the tubulin. Its dissociation equilibrium constant from GDP tubulin is about 120 nM; that from GTP tubulin is about 155 nM. The structure shows D1 binds to the longitudinal interface of the β subunit. Five ankyrin repeats of D1 contact the helices H6, H11 and loop T5 of β tubulin, which explains the reason why D1 prevents tubulin from polymerizing. In D1-tubulin, the kinesin binding site located on the lateral surface of a heterodimer remains uncovered. Hence, D1 is likely a good candidate to be a crystallization chaperone for tubulin-MAP complexes, in particular tubulin-kinesin complexes.

Another anti-tubulin DARPin structure published is D2-tubulin in complex with kinesin and an ATP-analog at 3.2 Å (Gigant et al. 2013). It's the first successful case where a DARPin is used to help crystallizing a tubulin-kinesin complex, in order to observe the conformational changes of kinesin when bound to tubulin. D2 binds to the β subunit's longitudinal interface, with a different binding mode compared with D1 (Fig. 1.4), stabilizing tubulin. It's proved that D2 doesn't modify the interaction between kinesin and tubulin, because kinesin's ATPase activities stimulated by tubulin or by D2-tubulin are similar with nearly identical catalytic constants and similar Km. In the structure, kinesin with an ATP analog binding tubulin fits the previous 8 Å Cryo-EM map where a microtubule was decorated with kinesin-AMPPNP. Details of this structure, especially of kinesin, will be presented in the kinesin-1 part of this introduction. This complex structure points to a promising way to generate tubulin-kinesin crystal structures using DARPins as tubulin stabilizers.

Kinesin in general

Cytoskeletal motors play important roles in eukaryotic cells. They convert chemical energy into mechanical work and take nanometer steps along their tracks to transport a variety of cargos in the cytoplasm. Failures of motors can cause severe diseases or be lethal in some cases. Cytoskeletal motors are divided into three families: myosins, using actin filaments as tracks, dyneins and kinesins, both moving on microtubules to travel inside the cell [START_REF] Schliwa | Molecular motors[END_REF].

Kinesin superfamily proteins (KIFs) are reported to be able to transport organelles, protein complexes and mRNAs to specific destinations, using the energy produced by ATP hydrolysis. Thanks to them, the intracellular transport is spatially and temporally well controlled in cells. They also contribute to chromosomal and spindle movements during mitosis and meiosis [START_REF] Hirokawa | Intracellular transport and kinesin superfamily proteins, KIFs: structure, function, and dynamics[END_REF].

KIFs share a conserved catalytic motor domain, also called head, containing an ATP binding site and a microtubule binding site. This part, where the chemical energy is converted into work, is responsible for ATP hydrolysis and kinesin's directional motility. Beside from the motor domain, kinesin also have a quiet variable non-motor domain, usually containing a coiled-coil segment, known as "stalk" region and a globular "tail" region. The tail region has a wide variety of functions in different kinesins, including interacting with cargo, regulating the ATPase activity of the motor domain; containing ATP independent MT binding sites and targeting kinesins to different locations [START_REF] Ovechkina | Unconventional motoring: an overview of the Kin C and Kin I kinesins[END_REF]. In some kinesins, the "tail" doesn't contact cargoes directly, but through light chains or associated protein instead [START_REF] Hirokawa | Intracellular transport and kinesin superfamily proteins, KIFs: structure, function, and dynamics[END_REF].

Classification of the kinesin superfamily

Different KIFs have various structures and functions. Whereas the motor domain sequence of kinesins is relatively conserved and is the hallmark of this protein superfamily, sequence differences have led to the classification of kinesins in 14 sub-families (Fig. 2.1). The organization of their polypeptide chains has also led to the definition of 3 types of kinesins [START_REF] Hirokawa | Kinesin superfamily motor proteins and intracellular transport[END_REF]. The motor domains are colored in light green. Kinesin-1-12s, whose motor domain is located at kinesin's N-terminus, move toward the plus end of microtubule; Kinesin-14s, whose motor domain is located at its C-terminus, undergo minus end-directed motility; Kinesin-13s, whose motor domain is in the middle of the polypeptide sequence, don't move along microtubule actively; they diffuse instead. Kinesin-6's motor domain has a unique loop with approximately 65 amino acids insertion, colored in light green.

Many kinesins are oligomerized by their coiled-coil segments, colored in dark green. Most kinesins exist as dimers; some exist as either monomers or dimers (Kinesin-3 for example); some kinesins exist as tetramers (Kinesin-5 for example).

Non-motor domains, colored in dark blue, vary in kinesins. They are responsible for their isoform-specific regulation or functional properties or both. In addition to its-motor containing heavy chain, Kinesin-1 has a light chain contacting the C-terminal non-motor domain of the dimerized heavy chain. The light chain allows Kinesin-1 to bind its cargoes indirectly. Some Kinesin-2s are heterotrimers, containing two motor subunits (KIF3A and KIF3B) and a Kinesin Associated Protein (KAP3). KAP3, either interacting with cargoes or regulating motor activity, contacts heterodimerized motor subunits through their non-motor tails [START_REF] Marszalek | Understanding the functions of kinesin-II[END_REF].

N-type kinesins, constituted of kinesins in classes 1 to 12, whose motor domains are located at the N-terminus of the sequence, have been found either to move towards the microtubule plus end or not to move. Most N-type kinesins, which move towards microtubule plus end, are processive. It means they are capable of taking hundreds of steps before dissociation. During the cycle of translocation, it's required that kinesin keeps attaching to microtubule. In this case, there is at least one motor domain that always binds to the microtubule. This type of kinesin is believed to move along microtubule in a handover-hand manner. The details of its motility will be introduced later.

Kin-Is, for internal kinesins, all belonging to the 13 th sub-family, are characterized by a motor domain that is internal to the polypeptide sequence. They hydrolyze ATP and use the energy to disassemble microtubules from both ends. This kind of kinesin doesn't generate movement; instead, they reach microtubule ends, either by direct binding or by diffusing along microtubules. There, they either induce or stabilize a depolymerizing-favoring conformational change in GTP tubulin dimer. Kinetic analysis also suggests that some Kin-Is can depolymerize microtubules processively, removing approximately 20 tubulin dimers from the end of a protofilament one by one before dissociation [START_REF] Hunter | The kinesin-related protein MCAK is a microtubule depolymerase that forms an ATP-hydrolyzing complex at microtubule ends[END_REF].

The third type, called C-type, which coincides with the kinesin-14 sub-family, has a Cterminal motor; these kinesins move towards the microtubule minus end. Some of them contain a non-ATP dependent microtubule binding site in their non-motor region so that they can cross link microtubules. When their motor domain hydrolyzing ATP generates a movement, they are able to slide one microtubule with respect to another. Moreover, some members of kinesin-14 are also reported to have microtubule destabilizing ability (Wordeman et al., 2003).

Conserved motor domain

Although kinesin superfamily has a large number of members with different functions, they all share a conserved motor domain which is responsible for nucleotide consumption and microtubule binding. Among various KIFs, the motor domains have amino acid sequence similarity of about 30-60%. The sequence alignment of some most studied members is presented in Fig. 2.2 [START_REF] Sablin | Crystal structure of the motor domain of the kinesin-related motor ncd[END_REF].

Four conserved nucleotide binding motifs are marked, named N-1 to N-4. N-1, also called the P-loop (Phosphate binding loop) motif, with the conserved sequence GQTxxGKS/T, interacts with the α and β phosphates of the nucleotide; N-2, known as the Switch1 motif, whose pattern is NxxSSR, can bind the  phosphate of ATP; N-3, Switch 2 motif (DxxGxE), is supposed to contact the  phosphate as well; N-4, with the conserved sequence RxRP located near the N terminus of the kinesin, interacts with the adenine ring of the nucleotide through hydrophobic interactions. These nucleotide associated motifs are also observed in myosin and some GTPase proteins [START_REF] Sack | Motor proteins of the kinesin family. Structures, variations, and nucleotide binding sites[END_REF]. In kinesin, central strands (blue) are surrounded by α helices (pink). α helices associated with microtubule binding are colored in green; the nucleotide binding region (purple) and the ADP are shown.

As the motor domains of various members in the kinesin superfamily are conserved, they share a similar structure. A typical kinesin motor domain structure (pdb id 2kin, [START_REF] Sack | X-ray structure of motor and neck domains from rat brain kinesin[END_REF]) is presented in the Figure 2.3. Generally, the kinesin motor domain consists of a series β strands locating in the middle of the structure with three α helices on each side. The nucleotide binding pocket with loops where conserved nucleotide binding motifs embed is clearly identified because in most cases the kinesin is co-crystallized with ADP.

Other cytoskeletal motors

In addition to kinesin, there are two other cytoskeletal motor families: myosins which move along actin filaments and dyneins which move along microtubules but usually towards the minus end. All of three use ATP as the source of energy. Among them, kinesins and myosins share a structural homology and a similarity in their mechanochemistry.

Despite their low sequence identity, the topologies and tertiary structure of kinesin and myosin indicate they may derive from a common ancestor [START_REF] Rayment | Kinesin and myosin: molecular motors with similar engines[END_REF]. By contrast, dyneins deviate from kinesins and myosins in both energy generation mechanism and in overall structure [START_REF] Bhabha | How Dynein Moves Along Microtubules[END_REF]. In this section, a brief introduction on both myosin and dynein will be given.

Myosin

Myosin, responsible for actin-based motility, plays important roles in muscle contraction and motility processes in eukaryotes. It was found in both striated muscle tissue and smooth muscle tissue by [START_REF] Pollard | Acanthamoeba myosin. I. Isolation from Acanthamoeba castellanii of an enzyme similar to muscle myosin[END_REF]. Myosins constitute a large and divergent protein family. Usually, myosins are heteromers consisting of one or two heavy chains and a variable number of light chains. A general structure of myosin V is presented in figure 2.4. Myosin heavy chain includes motor (head), neck and tail domain (Maravillas-Montero and Santos-Argumedo 2012). The motor domain is responsible for both actin filament binding and ATP hydrolysis. The neck domain serves as a linker between the catalytic motor domain and the tail; also, it is able to act as a lever arm for force transduction. The neck domain usually contains the myosin light chain binding site.

The tail domain plays various roles in different myosin subfamilies. Most myosins move from the actin pointed (minus) end to its barbed (plus) end, except myosin VI which moves in the reverse direction [START_REF] Sweeney | Myosin VI rewrites the rules for myosin motors[END_REF].

The motor domain structures of various myosins have been determined in different nucleotide state. In general, the myosin motor domain can be divided into four subdomains which are able to generate nucleotide-state associated movement [START_REF] Coureux | A structural state of the myosin V motor without bound nucleotide[END_REF].

The motor domain in myosin superfamily shares a similar catalytic pocket with that of the kinesin superfamily, including three conserved nucleotide associated motifs: P-loop, Switch 1 and Switch 2. Moreover, the secondary structure elements of kinesin are also found in myosin with a central β sheet surrounded by several α helices (Fig. 2.5). The head domain of myosin contains the nucleotide binding site and actin binding site. The neck domain is composed of α helices able to bind a calmodulin molecule. The tail domain consists of a coiled-coil stalk dimerizing the heavy chains, followed by two globular tail domains (GTDs). GTDs are responsible for cargo binding. In myosin, movement corresponds to the nucleotide cycle of motor domains [START_REF] Vale | The way things move: looking under the hood of molecular motor proteins[END_REF]. The cycle alternates through high and low affinity for actin. Usually, the myosin motor domain in nucleotide-free state or ADP state is able to bind actin tightly; while in ATP or ADP and inorganic phosphate (Pi) state it dissociates from actin easily.

Here, the processive movement of a myosin Va, corresponding to its nucleotide cycle, is presented as an example (Fig. 2.6). Myosin Va dwells in a state with both motor domain binding ADP attaching to the actin filament. The two motors exert intramolecular strain on each other. ADP releases from the trailing motor so that this motor is able to bind ATP.

After ATP binding, the dissociation of this motor occurs and so does ATP hydrolysis; meanwhile, the actin binding motor undergoes a power stroke, leading the dissociated motor, turning to its ADP-Pi state, to its forward binding site. Rebinding to actin filament, the ADP-Pi motor releases its Pi quickly and attaches to actin tightly. The cycle resets to the beginning and restarts all over again with myosin Va moving forward about 36 nm in each cycle.

Dynein

Cytoplasmic dynein belongs to the AAA (ATPases Associated with diverse cellular Activites) family. Although it is able to move along microtubules using the energy produced by hydrolysis of ATP, it is quite different from kinesin and myosin for the following aspects. First, structurally, its catalytic core (AAA ring) is separated from its microtubule binding domain (MTBD) by a long coiled coil. In both myosin and kinesin, the same motor domain is responsible for both ATP hydrolysis and microtubule binding. Second, unlike in kinesin and myosin in which two motor domains cooperate with each other during movement, the two dynein heads step independently of each other. Third, usually the movement of kinesin and myosin is processive; while dynein exihibits a weak directional bias and variable step sizes.

Kinesin-1

Kinesin-1, also called conventional kinesin, has been found in squid giant axon in 1985 [START_REF] Vale | Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility[END_REF]. It was regarded as the only kinesin for a long time. It is the most studied kinesin, both through in-depth characterization of kinetic and motile aspects and through structural study.

As a typical N-type kinesin, once attached, kinesin-1 moves toward the (+)-end along the microtubule surface in 8 nm increments as it hydrolyzes one ATP per step [START_REF] Howard | The movement of kinesin along microtubules[END_REF]).

As mentioned above, kinesin-1 can be described as a homodimer of heterodimers. It comprises two identical polypeptides which dimerize to form a rod-shaped coiled-coil stalk, with a tail at the C-terminal end and twin globular heads at the other end (Fig. 3.1). Each heavy chain contacts cargoes with the help of a partner "light chain", which binds its Cterminal tail. Each head comprises the binding sites for both nucleotide and microtubule, which allows it to attach to microtubules with nucleotide-dependent affinity [START_REF] Asbury | Kinesin: world's tiniest biped[END_REF].

Consequently, studies of kinesin-1's motor domain of its heavy chain (KHC), bring insights into Kinesin's conserved mechanisms, including those for ADP release and ATP binding and also into the mechanism of N-type kinesin's motility. Kinesin-1 light chain (KLC), connecting to the C-terminal tail of kinesin-1 heavy chain (KHC), contributes to cargo binding. Motor domains (magenta), located on the other side of the coiledcoil stalk, are responsible for walking along the microtubule. A neck linker embedded in the neck domain (yellow) of each motor plays an important role in kinesin-1's motility. Kinesin-1's motor domain plus its neck linker (orange cycle) is studied in this thesis.

The kinetic mechanism of kinesin

Kinesin is an ATPase whose motility is driven by its ATP turnover. Study of kinesin's kinetic mechanism is essential for understanding chemical energy consumption and motile force generation. The kinetic mechanism and relevant constants on kinesin-1 are summarized below (Cross 2004).

In the absence of microtubule

In the absence of microtubule, kinesin-1 binds ATP and hydrolyzes it on its own. When it is detached from the microtubule, kinesin-1 is trapped in an ADP-bound state, characterized by a low ADP dissociation rate (koff about 0.01 s -1 for wild type dimers). ADP dissociation is the rate limiting step for an ATP turnover by isolated kinesin. The ADP dissociation rate is usually determined using the fluorescence variation signal of Mant-ADP which is a fluorescent analog of ADP with the same dissociation rate as ADP (Fig. 3.2).

After ADP dissociation, ATP is able to bind kinesin. For apo-kinesin-1, the rate of ATP binding is about 4 M -1 s -1 . For this measurement, apo-kinesin is required. To generate apokinesin in the absence of microtubules, EDTA and apyrase are used to accelerate the release of ADP and to hydrolyze it. But it is reported that apo-kinesin is prone to denature in the absence of a stabilizer [START_REF] Sadhu | A kinetic study of the kinesin ATPase[END_REF][START_REF] Huang | Drosophila kinesin minimal motor domain expressed in Escherichia coli. Purification and kinetic characterization[END_REF]. The KD for ATP dissociation is about 75 M.

The rate of ATP hydrolysis by a microtubule-unbound single motor is about 7 s -1 (Ma and Taylor 1997). After ATP hydrolysis, phosphate releases before ADP dissociation, as ADP is trapped tightly by kinesin.

In the presence of microtubules

The relation of the nucleotide cycle with the interaction between kinesins and microtubules is summarized in Figure 3.3 (Ma and Taylor 1997;[START_REF] Vale | The way things move: looking under the hood of molecular motor proteins[END_REF][START_REF] Wang | Kinesin, 30 years later: Recent insights from structural studies[END_REF].

ADP kinesin is not only characterized by its low ADP dissociation rate but by its relatively low affinity for microtubules as well (KD is about 10-20 M). However, once it binds to microtubules, its ADP release is accelerated thousands of times (koff increases from about 0.01 s -1 to about 50 s -1 for a monomer). A key discovery was made by Hackney that ADP release is sequential in a kinesin-1 dimer (Hackney 1994). He found that upon microtubule binding only one of the motors releases ADP directly, while the other head releases its ADP only after ATP binding to the first head (Cross 2004).

After ADP release, the apo-kinesin motor binds microtubules tightly with an unbinding rate constant of about 0.002 s -1 . The apo-kinesin in complex with microtubule, often called "rigor" following the equivalent state of myosin, is able to rebind ATP at a rate of 2-4 M -1 s - 1 . ATP unbinding rate is about 150 s -1 . Actually ADP is also able to bind microtubuleattached kinesin with a rate of about 1.5 M -1 s -1 . Hence ATP and ADP actually bind to and dissociate from rigor complex with similar rates. So ADP is a competitive inhibitor of ATP binding to kinesin with an inhibition constant Ki of about 150 M. Indeed it has been shown that ADP tends to detach kinesin-1 from microtubules and is able to reduce both the velocity and run length of walking kinesin-1 [START_REF] Yajima | Direct long-term observation of kinesin processivity at low load[END_REF]. However, in physiological conditions, the concentration of ATP is at least five times higher than that of ADP [START_REF] Tantama | Imaging energy status in live cells with a fluorescent biosensor of the intracellular ATP-to-ADP ratio[END_REF], so microtubule-bound apo-kinesin binds ATP preferentially and in this way completes its cycle.

Microtubule binding doesn't only stimulate kinesins' ADP dissociation but also accelerates ATP hydrolysis in an ATP turnover. The hydrolysis rate increase from 7 s -1 to about 250 s -1 (Ma and Taylor 1997). Then phosphate releases quickly with a rate higher than 100 s -1 . The low affinity between microtubule and the ADP-kinesin leads to its detachment from the microtubule. Although the frame of kinesin's nucleotide cycle is clear (Fig. 3.3), detail information on the effects caused by microtubule binding, including for instance how ADP dissociation is accelerated and how ATP hydrolysis is favored, is still missing.

Mutagenesis studies gives insights into kinesin's ADP release mechanism

Despite the unclearness of how ADP dissociation is accelerated by microtubule binding, some kinesin mutants are reported to have a higher ADP dissociation rate. These mutants may provide insights into kinesin's ADP release mechanism.

Most of these mutations, leading to a high spontaneous ADP release rate, are associated with its P-loop motif that interacts with the phosphates of the nucleotide. P-loop motif, a conserved nucleotide associated motif present in NTPases, including kinesin, myosin and G protein, has the sequence consensus motif GxxxxGKT/S. For kinesins there are two extra conserved residues in their P-loop motif so that their P-loop motif pattern is GQTxxGKS/T [START_REF] Sack | Motor proteins of the kinesin family. Structures, variations, and nucleotide binding sites[END_REF].

Higuchi et al. mutated the first threonine of the P-loop motif to serine (GQTxxGKS/T) in Drosophila kinesin-1, because in myosin the equivalent residue is a serine (Higuchi et al. 2004). They found the T>S mutant releases ADP (koff ≈ 0.0128 s -1 ) faster than wild type (koff ≈ 0.0035s -1 ) by 3.6 times without microtubules being involved.

A conserved residue of the so-called Switch 2 motif, E236 (in human kinesin-1 numbering) interacts with this P-loop threonine residue T87 (in human kinesin-1 numbering). If this glutamic acid is mutated to an alanine, its connection with the P-loop will be interfered. In the absence of microtubules, the E236A mutation increases the ADP dissociation rate from 0.01 s -1 to 1.1 s -1 (Rice et al. 1999). Interestingly, this mutation doesn't influence the microtubule-stimulated ADP release rate.

A related mutation is G234A, also located in the Switch 2 motif. This glycine is conserved in myosin and G protein; it forms a hydrogen bond with the γ phosphate of a tri-phosphate nucleotide. The G234A kinesin-1 mutant releases ADP very quickly with a koff of around 10 s -1 . In addition, and in contrast to the case of the E236A mutant, in the case of G234A the microtubule-stimulated ADP release is disturbed. Moreover, this mutant is reported to be unable to undergo a conformational change corresponding to ATP binding so that its ADP dissociation cannot be accelerated by microtubules (Rice et al. 1999).

Another interesting mutant is associated with T92, another conserved threonine of the Ploop (GxxxxGKT/S). Nakata et al. found that if this threonine is mutated to an isoleucine or an asparagine, the mutant will bind microtubule strongly without capacity of detaching even in the presence of a high concentration of ATP (Nakata and Hirokawa 1995). Because of this character, T92N and T92I are used as a roadblock on microtubules is some studies (Crevel et al. 2004). It's also reported that T92N hydrolyzes ATP much more slowly (about 100 times) than wild type kinesin-1 [START_REF] Krylyshkina | Modulation of substrate adhesion dynamics via microtubule targeting requires kinesin-1[END_REF]. Interestingly, if the threonine is mutated to serine, as is the case in some kinesin subfamilies, i.e. kinesin-3, the mutant is still capable of moving along microtubules as wild type (Nakata and Hirokawa 1995).

These results highlighted the importance of nucleotide binding motifs on nucleotide trapping in kinesin. Nevertheless, in order to translate these results into microtubuleaccelerated ADP release mechanism, more studies, especially structural studies, are required.

Motility model of kinesin-1

Kinesin-1 moves in a hand-over-hand way

Two main models of kinesins motility have been proposed [START_REF] Yildiz | Kinesin: walking, crawling or sliding along?[END_REF].

One is called the inchworm model. Kinesin dimer walks with a leading motor always leading and always followed by the rear motor. The first motor steps about 8.3 nm once, which is about the length of a tubulin heterodimer; then the second motor steps the same distance while never bypassing the leading one. It suggests only one motor is catalytically active. It also means that both motors revert to the same conformation without requiring the rotation of the stalk.

The other model is called hand-over-hand, in which kinesin moves like people walk. The rear motor bypasses the leading motor to take a step, binding to the next binding site while becoming the leading motor. In this case, if the centroid position of the dimer moves 8.3 nm, a single motor moves twice this distance, 16.6 nm, as a step. In the next step, the former leading motor which is the rear motor now moves 16.6 nm forward, becoming the leading motor again. During this process, the other motor always sticks to its original binding site without moving until it's its own turn to step.

Evidences presented so far mostly support the hand-over-hand model. For example, Kaseda et al. found the movement of a heterodimeric construct, in which one motor contains a mutation decreasing its ATPase activity (18 times slower than WT) while the other one has the normal catalytic rate, is not uniform. The heterodimer makes one step with long dwell time following one step with short dwell time, because one of the motors takes more time to hydrolyze ATP. The overall moving speed of the heterodimer is 9 times slower than that of a wild type, which matches its average speed of ATP consumption [START_REF] Kaseda | Alternate fast and slow stepping of a heterodimeric kinesin molecule[END_REF]. If kinesin moves in an inchworm way, for each step the dwell time would be constant and the velocity of its movement would either be normal (with a normal motor as a leading head), or 18 times slower (with a mutated motor as a leading head). In another set of experiments, Yildiz et al. labeled one motor of a kinesin dimer with a Cy3 fluorophore, so the movement of the marked individual motor in the dimer can be visualized (Fig. 3.4). They found that instead of moving about 8 nm for each step, as predicted by the inchworm model, one motor moves 16 nm in a step. This result matches the hand-over-hand model [START_REF] Yildiz | Kinesin walks hand-over-hand[END_REF]). In

Kinesin's motility coupled with its nucleotide cycle

Kinesin-1 walks towards microtubules plus end for hundreds of steps before dissociation. The processivity and unidirectional movement relies on the communication between the two motors. The neck linker model has been proposed and is generally accepted to account for this communication (Fig. 3.5a). The neck linker is a peptide of about 15 residues located at the C-terminus of a kinesin-1 motor domain and connecting each motor to the coiled-coil stalk. Previous studies indicate it can be either rigid and docked on the motor domain or flexible. Furthermore, the conformational change of the neck linker is associated with kinesin's nucleotide state and also with its attachment to microtubule.

EPR spectroscopy and FRET studies on isolated ADP-kinesin suggest the neck linker is not attached to the catalytic core in a uniform rigid way. The neck linker may have a docked [START_REF] Sindelar | Two conformations in the human kinesin power stroke defined by X-ray crystallography and EPR spectroscopy[END_REF] or an undocked (Kull et al. 1996) conformation, both of which were observed in ADP-kinesin crystal structures. Upon kinesin binding to a microtubule coupled to ADP release, the neck linker is mainly in a detached and disordered conformation. However, when ATP binds to kinesin, the neck linker docks on the catalytic core. Neck linker docking requires both ATP (or an ATP analog, as AMP-PNP or ADP-AlF -4,) and microtubule binding. After phosphate release, the neck linker turns back to a mobile state again and the ADP-bound motor dissociates from the microtubule to complete the cycle (Rice et al. 1999).

Taking both motors in a dimer into consideration, the processive and unidirectional movement of kinesin can be summarized as the followings (Fig. 3.5b). In the absence of microtubule, each motor of a kinesin dimer traps an ADP. Once one of the motors (motor A) binds a microtubule, its ADP dissociation is accelerated. Apo-motor A binds to the microtubule tightly, while being able to accept a nucleotide. During this process, the other motor (motor B) is always in its ADP state and keeps unbound from the microtubule, until ATP, whose concentration in vivo is higher than that of ADP, binds motor A. As discussed above, in a microtubule-and ATP-bound motor, the neck linker interacts with (docks on) the catalytic core, pointing towards the microtubule plus end. Neck linker docking on motor A narrows the distance between ADP-bound motor B and its next microtubule binding site, which is closer to microtubules plus end, favoring the attachment of motor B to the next binding site. Then ADP dissociates from motor B and ATP rebinding is allowed on apo-motor B; meanwhile, in motor A, ATP is hydrolyzed and ADP-motor A is able to detach from the microtubule. Afterwards, ATP binding induces neck linker docking on motor B, while ADP-motor A is driven to its next binding site. ADP-motor A rebinds to microtubule and its motile cycle continues, so does the cycle on motor B. Therefore, a kinesin dimer, with two motors going through alternative stages of the same cycle, walks step by step processively in one direction.

Advancement of kinesin-1 structural studies ADP kinesin-1

Since the first kinesin-1 structure was published, several ADP-bound kinesin-1 structures from different species have become available in the PDB. They are either monomers or dimers, from various species, with either a flexible or a rigid docked neck linker.

One of them is shown in figure 3.6 (pdb id 1BG2) (Kull et al. 1996). It consists of a central β sheet with a group of 3 α helices on each side. Most of the helices of kinesin are somehow unusual. One of them is interrupted by an inserted loop. At least two of them are 'distorted'. The nucleotide-binding pocket is formed by four regions of the polypeptide chain: P-loop, Switch 1, Switch 2 and N-4. The P-loop motif, as presented above, also called N-1 in kinesin, with the conserved sequence GQTxxGKS/T, interacts with α and β phosphates of the nucleotide. Switch1 motif forms a short α helice H3a in this ADP binding kinesin. Switch 2 motif is followed by a long flexible loop and the H4 (also called α4) helix which has 4 turns. N-4, with the conserved sequence RxRP near the N terminus of the kinesin interacts with the adenine ring of the nucleotide through hydrophobic interactions [START_REF] Sack | Motor proteins of the kinesin family. Structures, variations, and nucleotide binding sites[END_REF].

Sindelar et al., using site-specific EPR measurement to detect the conformation of kinesin's neck linker in the absence of microtubules, found that both docked and undocked neck linker exists in the ADP-kinesin population. Following this, they determined the structure of ADP-kinesin (pdb id 1MKJ) with a docked neck linker [START_REF] Sindelar | Two conformations in the human kinesin power stroke defined by X-ray crystallography and EPR spectroscopy[END_REF]. In general, this structure has a similar conformation as 1BG2: P-loop motif binds the ADP phosphates; Switch 1 forms the short H3a; Switch 2 is embedded in L11, most of which is disordered. By contrast, H4 tilts slightly and creates a space for the first residue of the neck linker to dock (Fig. 3.7). ADP-Kinesin-1 structures from different species also show a similar conformation (e.g. pdb id 2KIN).

Kinesin-1 dimers were also successfully crystallized with ADP bound (pdb id 3KIN, 2Y5W) [START_REF] Kaan | The structure of the kinesin-1 motor-tail complex reveals the mechanism of autoinhibition[END_REF]. In both cases, the neck linker is rigid, which makes their motors' structure resemble to that of a monomer with a docked neck linker. For example, the r.m.s.d. of Cα between 1MKJ and 2Y5W is only about 0.4 Å, despite the two kinesins are from different species (human and drosophila). a) The neck linker in this structure is rigid and interacts with the catalytic core in the same way as observed in microtubule-bound ATP-kinesin (Parke et al. 2010b). The nucleotide binding motifs (Switch1, Switch2, P-loop and N-4), which are labeled in different colors to indicate their locations, have similar conformations as those in the neck linker undocked ADP kinesin-1.

b) View of 1MKJ from another side. The C-terminal neck linker in this structure is ordered and attached to the catalytic core. The nucleotide is marked.

ATP analog kinesin-1

The structure of the kinesin motor domain in an ATP-like state in complex with tubulin (Fig. 3.8) has been determined in the laboratory (Gigant et al. 2013). It explains the mechanism of microtubule-stimulated ATP hydrolysis and also uncovers the connection between ATP, microtubule binding and the conformation of the neck linker. The main findings are summarized here:

-Upon tubulin binding, the Switch2 loop interacts with α tubulin and becomes ordered. This leads to the elongation of the H4 helix at its N-terminus, which in turns reinforces the binding to the microtubule. The analog of the third phosphate and the nucleotide-bound Mg 2+ form several hydrogen bonds with conserved residues in Switch1 and Switch2, leading to significant conformational changes, mostly of Switch1.

-The conserved Glu236 residue, which is the C-terminal residue of Switch2, relocates and makes a salt-bridge with a conserved residue, Arg 203, of Switch1. The relocation of Glu236 puts it at the right place to activate the water molecule abstracting a proton from the water that has been proposed to be the nucleophile attacking the - phosphodiester bond. As a consequence, the chemical step of ATP hydrolysis is accelerated. This two water ATP hydrolysis mechanism had been initially proposed based on the isolated Kinesin-5-ATP structure (Parke et al. 2010b).

-The structure also explained how the conformational change of ATP-kinesin allows the interaction of the neck linker with the core of the motor domain, which causes the docking of the neck linker and leads to the power stroke.

-It also defines the tubulin-kinesin interface (Fig. 3.9). Residues of L11, H4 and H6 in the kinesin contact H'3, H'11 and H'12 in α tubulin. On β tubulin, most of the interaction zone is located on H12, which interacts with L8, L12 and S5a on the kinesin. The contacting areas are highlighted on tubulin (left) and on kinesin (right). In the upper figure, the contacting area on α tubulin is colored in blue, on β tubulin it is colored in purple and on kinesin in green. In the lower figure, color code on tubulin remains the same, while in kinesin the interface is colored corresponding to the tubulin subunit contact. Relevant secondary structure elements are labeled on both tubulin and kinesin.

The missing structures of apo kinesin-1

Cryo-EM data of nucleotide-free kinesin-1 bound to microtubule structure have been obtained at 9 Å resolution [START_REF] Sindelar | The beginning of kinesin's force-generating cycle visualized at 9-A resolution[END_REF]. At this resolution, only a rough orientation of the motor domain as well as the extension of H4 could be inferred. The resolution is too low to determine an atomic model. More detailed information can be offered by an atomic structure of nucleotide-free kinesin bound to a microtubule or to tubulin, especially to reveal the mechanism of ADP release.

Some kinesin subfamily has a relatively high spontaneous ADP release rate. An internal kinesin, pKinI (spontaneous ADP release koff ≈ 8 s -1 ), whose motor domain is in the middle of the sequence instead of at its N-terminus like most kinesins, was crystallized and reported to have a similar conformation as ADP kinesin (pdb id 1RY6 [START_REF] Shipley | Structure of a kinesin microtubule depolymerization machine[END_REF]).

Its insight on kinesin's ADP release mechanism in a general case is limited, because this kind of kinesin has a different power stroke mechanism from N-terminus kinesins, including kinesin-1.

There are only two microtubule-unbound apo N-type kinesin crystal structures in the PDB, probably due to the difficulty of generating stable apo-kinesin [START_REF] Sadhu | A kinetic study of the kinesin ATPase[END_REF][START_REF] Huang | Drosophila kinesin minimal motor domain expressed in Escherichia coli. Purification and kinetic characterization[END_REF].

One is a structure o Eg5 bound to an inhibitor (pdb id 3WPN). In this case, an allosteric inhibitor able to suppress ATP binding is co-crystallized with Eg5 [START_REF] Yokoyama | Structural basis of new allosteric inhibition in Kinesin spindle protein Eg5[END_REF]. This inhibitor binds to the H4/H6 pocket which is 15 Å from the nucleotide binding pocket, leading also to a conformational change of the neck linker. As a consequence, conformational changes also occur around the nucleotide binding pocket and ATP binding is interfered. Hence, this structure doesn't represent a natural apo-like conformation.

The other apo-kinesin crystal structure (pdb id 3WRD) is generated by soaking ADP-Kif5C crystals with kinesin's C-terminal peptide [START_REF] Morikawa | X-ray and Cryo-EM structures reveal mutual conformational changes of Kinesin and GTPstate microtubules upon binding[END_REF]. The structure is claimed to have an ATP-like conformation, in which a C-terminal neck linker docks into kinesin itself. In microtubule binding apo-kinesin EM structure, neck linker docking is not observed. The apo-Kif5C structure and ADP-Kif5C structure are very similar with an RMSD equaling 0.36 Å.

Determining the crystal structure of the complex of apo-kinesin-1 with tubulin is one of the objectives of this thesis. It is an intermediate state between ADP binding and ATP binding, and the main missing part in kinesin's nucleotide cycle coupled with microtubule binding (Fig. 3.10). Compared with ADP kinesin, it can give an insight to the ADP dissociation stimulated by microtubule binding; compared with ATP-analog-kinesin in complex with tubulin, it can help to explain the reason for neck linker docking. The atomic structure of microtubule-unbound ADP kinesin has been determined long time ago. Recently, ATP analog-kinesin in complex with a tubulin heterodimer has been crystallized as well. As the main missing part of kinesin's cycle, the nucleotide-free kinesin in complex with tubulin can provide more information on i.e. microtubule stimulated ADP release mechanism and ATP associated neck linker docking.

Kinesin-14 4.1 Kinesin-14 in general

Kinesin-14, as presented before, is a subfamily of kinesins whose motor domain locating at the C-terminus of its sequence. They are dimerized through an N-terminal coiled-coil. Unlike most kinesins using an asymmetric hand-over-hand mechanism for a microtubule plus-end-directed processive movement, kinesin-14 subfamily members travel in a reverse direction, going through a different mechanism of motility. The force, allowing kinesin-14 to slide one MT relative to another, is generated by a rotation or bending of the coiled-coil stalk; while the rotation is likely coupled with the kinesin's nucleotide cycle.

Kinesin-14s play important roles during mitosis and meiosis. They are involved in spindle assembly and maintenance, are known to cross-link two parallel microtubules at the spindle poles, while cross-linking antiparallel interpolar microtubules in the spindle overlap zone.

The two most studied kinesin-14 motors are a Drosophila motor, Ncd (non-claret disjunctional), and a Saccharomyces cerevisiae motor, Kar3, serving as kinesin-14 models.

Here, Ncd is taken as example to introduce kinesin-14's structure and motility cycle.

Ncd

Ncd (non-claret disjunctional) is a kinesin-14 in Drosophila melanogaster, playing a role in spindle organization and integrity during the early cleavages of the embryo and during female meiosis. It cross-links two microtubules, either parallel or antiparallel by its Cterminal ATP-dependent motor domains and its N-terminal ATP-independent tail. It is able to slide one microtubule relatively to another.

Ncd's motor domain has approximately 350 amino acid residues with 40% identity to kinesin-1, and previous studies indicate it has a kinetic mechanism similar to kinesin-1 as well [START_REF] Pechatnikova | Kinetic mechanism of monomeric non-claret disjunctional protein (Ncd) ATPase[END_REF]. The rate-limiting step for both kinesins is the ADP dissociation in the absence of microtubules, while upon microtubule binding these kinesins can release ADP significantly faster. Although in drosophila kinesin-1, ADP dissociation is much faster after binding to microtubules and is no longer the rate limiting step [START_REF] Milic | Kinesin processivity is gated by phosphate release[END_REF], in microtubule activated Ncd the hydrolysis of ATP is always rate-limited by ADP release [START_REF] Mackey | Moving a microtubule may require two heads: a kinetic investigation of monomeric Ncd[END_REF]. In general, Ncd's motor performs like a slow kinesin, its ATP hydrolysis speed and ADP dissociation rate are 15-20 times slower than those of a kinesin. In addition, the velocity of Ncd movement is about 100 nm s -1 , which is much slower than that of Kinesin-1 (about 1800 nm s -1 ).

Structural studies of Ncd

First, the ADP bound Ncd monomer crystal structure was published in 1996 [START_REF] Sablin | Crystal structure of the motor domain of the kinesin-related motor ncd[END_REF], showing a remarkable similarity with kinesin-1, with a r.m.s.d of 1.21 Å. The position of Mg-ADP in both Ncd and kinesin-1 is identical. So are the central β sheet and six α helices, constituting the core in both motors. Moreover, the similarity of both structures suggests Ncd and kinesin-1 share a similar tubulin binding site. The main differences between kinesin-1 and Ncd happen in their surface loops. A group of loops (L1, L5 and L9) surrounding the entrance to the nucleotide-binding site are directed closer to the nucleotide in Ncd than in kinesin-1. Another different area in Ncd is found in L2, containing 10 more residues compared with kinesin-1. This loop extends from the main body of Ncd's motor. Sequence alignments indicate the length of the loop varies among members of the kinesin superfamily. L11, which is disordered in ADP-Kinesin-1 and is most other ADP-bound kinesins, is ordered in this structure, which turned out in a later study to be caused by crystal packing [START_REF] Sablin | Direction determination in the minus-end-directed kinesin motor ncd[END_REF].

A crystal structure of an Ncd homodimer binding Mg-ADP (Fig. 4.1a) was determined two years later [START_REF] Sablin | Direction determination in the minus-end-directed kinesin motor ncd[END_REF]. In that structure, the two motors are connected through their N-terminal "neck", 13 residues preceding the first β strand of its catalytic core. The neck is helical and symmetrical so that it forms a parallel coiled-coil connected to the coiled-coil stalk domain without intervening loops, despite of being disordered in its ADPmonomer structure [START_REF] Sablin | Crystal structure of the motor domain of the kinesin-related motor ncd[END_REF]. Together with part of the coiled-coil stalk, the neck contacts H1 and L6 in Ncd's catalytic core. Based on the structure, mutations were introduced in the "neck" and in the part contacting the "neck" in Ncd's catalytic core. Among these mutants, one whose 12 residues of its "neck" are randomly replaced by 12 hydrophilic residues moves towards microtubules plus end slowly, despite the mutant has a normal ATPase activity. These results indicate that the "neck" is essential for a minusend-directed movement in Ncd.

Low angle X-ray scattering and Cryo-electron microscopy were used to study Ncd's structure as well. These studies suggest that the ADP-Ncd dimer is symmetric in both crystal and solution; but once Ncd is bound to microtubules, the symmetry is broken [START_REF] Endres | A lever-arm rotation drives motility of the minus-end-directed kinesin Ncd[END_REF]). These studies indicate microtubule binding and ADP release from the microtubule-bound motor is associated with a symmetry breaking conformational change.

At least three Ncd mutants also show an asymmetric conformation even without a microtubule bound, although all these mutants bind ADP in the structure (Fig. 4.1b). In dimeric Ncd N600K mutant (pdb id 1N6M), where the mutation is located in the tubulin binding site, one of the motors and its coiled-coil stalk rotate about 75° while the other motor was superposed with one head of the symmetric Ncd dimer. The mutant dissociates ADP faster than wild type Ncd dimer [START_REF] Yun | Rotation of the stalk/neck and one head in a new crystal structure of the kinesin motor protein, Ncd[END_REF]. Another mutant, T436S (pdb id 1CZ7), shows a similar rotation of one of the motors together with its coiled-coil stalk as well. The mutation is located at the nucleotide binding site, leading to a high ADP dissociation rate (Heuston et al. 2010). A similar coiled-coil stalk rotation was also observed in the Ncd G347D mutant (pdb id 3L1C). In this case, a conserved glycine, the pivot point of stalk rotation was mutated to alter the stalk's normal rotation [START_REF] Liu | Neck-motor interactions trigger rotation of the kinesin stalk[END_REF]. The asymmetric conformation was generated by interfering with different aspects, microtubule binding, nucleotide binding and neck rotation, in these ADPbinding structures. A tubulin or microtubule bound Ncd structure would be most helpful to answer the remaining questions about how the asymmetric conformation is generated in wild type Ncd. b) In some mutants, the ADP-bound Ncd homodimer has an asymmetric conformation. The asymmetric conformation is also observed by EM when Ncd binds to a microtubule.

Studies on Ncd's motility

Further studies on microtubule bound Ncd dimer's structure by Cryo-electron microscopy reveal the general mechanism of Ncd's motility [START_REF] Endres | A lever-arm rotation drives motility of the minus-end-directed kinesin Ncd[END_REF]. Three dimensional density maps of microtubule-bound dimeric Ncd in nucleotide-free state or in ATP analog state were calculated. A significant conformational change of Ncd's structure with or without ATP analog happens to its neck. Compared with those of apo-Ncd, the neck and unbound head of Ncd in ATP analog state rotate about 70° towards microtubules minus end (Fig. 4.2a). However, this clear ATP-caused rotation of the neck is not observed on an Ncd mutant, N340K, which can move along microtubule bidirectionally. Cryo-EM maps suggest that after ATP binding, the neck of the mutant rotates towards microtubules plus end or minus end with roughly equal probability.

These results lead to a theory that Ncd uses its neck as a lever arm to generate its power stroke [START_REF] Endres | A lever-arm rotation drives motility of the minus-end-directed kinesin Ncd[END_REF]). To prove it, the velocity of Ncd with different length of neck was measured and the results were compared, because for a lever-arm mechanism the velocity should be proportional to the length of the lever arm. Indeed, Ncd with a longer neck turns out to move faster than that with a shorter neck, having the same ATPase turnover. If its neck is too short to form a dimer, Ncd almost loses its capability of movement, even consuming ATP normally. If a coiled-coil neck is kept while one of the motors is truncated, Ncd can still generate a "normal" movement with the same speed as a dimeric Ncd having the same length of neck. It is suggested that a stable coiled-coil stalk is required as a lever-arm to generate Ncd's movement. But one motor seems to be sufficient for Ncd's movement.

In general, Ncd moves in the following way (Fig. 4.2b). Ncd crosslinks two microtubules with one motor binding one microtubule while its N-terminal ATP-independent tail anchors to another microtubule. Once ATP binds to its microtubule-bound apo-motor, the neck rotates toward the motor-bound microtubule minus end. The neck plays a role as a lever-arm so that the motor-bound microtubule is drawn to slide over the tail-bound microtubule. Then ATP is hydrolyzed and the neck rotates back; as a consequence, the motor detaches from the microtubule and is able to bind to the next binding site, which is closer to the microtubule minus end. In this case Ncd slides one microtubule with respect to another, with its motor moving towards the motor-bound microtubule minus end. [START_REF] Endres | A lever-arm rotation drives motility of the minus-end-directed kinesin Ncd[END_REF]. When ADP-Ncd motor (blue) binds a microtubule, its neck (red) points toward the microtubule plus end. Then the motor releases its ADP and is able to bind ATP. ATP binding leads a rotation of the neck (yellow) toward the microtubule minus end. After ATP is hydrolyzed, the neck rotates back to its original position (red) along with motor dissociation from the microtubule. Tubulin heterodimers are colored in green and white.

b) The general model of Ncd's motility. Ncd's N-terminal ATP-independent microtubule binding tail (pink) attaches to one microtubule (blue), while its C-terminal motor (orange) binds to another microtubule. Once ATP binds to the microtubule-bound motor, a rotation happens to Ncd's neck (from red to yellow). As a result, the motor-bound microtubule is drawn to slide with respect to the other microtubule. After ATP hydrolysis, the neck rotates back (from yellow to red), allowing ADP-Ncd to detach from the microtubule and to rebind to its next binding site.

Objectives

I am interested in the motility mechanism of kinesin, including its movement generation and its nucleotide consumption. Studying the structure of motor domain should provide information on the conformational changes of kinesin as a function of the nucleotide state and on their relation to the interaction with microtubules. Despite the advancement on kinesin-1's structure study, the main missing piece in the structural description on kinesin's cycle is an atomic structure of nucleotide-free kinesin in complex with tubulin. As an intermediate state between isolated ADP-kinesin and microtubule-bound ATP-kinesin, this structure will bring insights into microtubule stimulated ADP release mechanism, by comparison with ADP-kinesin structure. By comparison with tubulin-bound kinesin in the ATP state, such a structure should also identify the structural changes upon ATP binding which lead to neck linker docking.

The main objective of this thesis is to determine the crystal structure of a (+)-end directed motile kinesin in a nucleotide-free state, both isolated and in complex with tubulin heterodimer. In the latter case, a DARPin will be used as a tubulin stabilizer.

These structural data will be complemented by the study of structure-based kinesin mutants. Moreover, mutagenesis study will be performed on kinesin-1 to test these analysis established on apo-kinesin-tubulin structure. Mutants and wild type kinesin will be studied structurally and biochemically. By analyzing the data on spontaneous ADP dissociation, ATPase activity of both kinesin mutants and wild type, I will know which residues are essential for kinesin's kinetic mechanism. For this objective, I have worked mainly with human kinesin-1 constructs. The results I have obtained are presented in the first (structure of apo-kinesin-1 bound to tubulin) and second (structures of isolated apokinesin-1) chapters of the results section.

The second objective is associated with kinesin-14, which differs from (+)-end directed kinesin as it moves towards the microtubule (-)-end. So far, there are only isolated kinesin-14s (not bound to tubulin) that have been crystallized. I will try to co-crystallize kinesin-14 in different nucleotide state (nucleotide-free or ATP-analog) in complex with tubulin. These data should allow one to compare the way that ATP binding triggers movement in these two kinds of kinesins (plus and minus-end directed kinesins). I will use both Ncd and Kar3 as representatives of kinesin-14 motors. Different DARPins will be used to enhance the chances of crystallization. Also crystallization of Vik1 (Kar3's motor-homology partner) in complex with tubulin will be tried. The advancement I have achieved so far will be presented in the third chapter of the results section.

Results and Discussion

Structure of apo-kinesin-1 bound to tubulin 1.1 Context

Kinesin-1 (kinesin or KHC) walks processively along microtubules in a hand-over-hand manner (cf. Introduction). The processive movement of kinesin requires each motor domain of the homodimer following the same mechanochemical cycle while in alternative stages.

The following short description summarizes the kinetic cycle relevant to the analysis made in this part of work (Cross 2004). The kinetic rate may vary because of different kinesin constructs studied.

In solution, kinesin in the ADP state has a slow release of ADP (off rate, koff is about 0.01 s -1 ) and weak microtubule binding (dissociation constant, Kd ≈ 10-20M). Once the ADPkinesin binds a microtubule, ADP release is stimulated. The off rate (koff > 100 s -1 ) of microtubule-activated ADP release is several thousand folds of the ADP dissociation rate in the absence of microtubules (Hackney 1988;[START_REF] Hackney | Modulation of kinesin halfsite ADP release and kinetic processivity by a spacer between the head groups[END_REF]. Then, the affinity for microtubules of the kinesin, now in nucleotide-free state, is enhanced. After ADP release, Mg-ATP is allowed to bind the kinesin-microtubule complex (on rate, kon ≈ 2 M -1 s -1 ).

When ATP-kinesin is bound to microtubule, ATP hydrolysis is favored: the ATP hydrolysis in an ATPase turnover is accelerated from 7 s -1 (in the absence of microtubules) to 200-300 s -1 . Then, phosphate release happens (with a speed > 100 s -1 ). The low affinity for microtubule of the ADP-kinesin leads to its detachment from the microtubule. Although the framework of kinesin's nucleotide cycle is known, how microtubule stimulates both ADP release and the chemical step of ATP hydrolysis remains unclear. Structural study will help to figure out the conformational variations in the cycle.

Another point that remains to be clarified is how ATP binding to microtubule-bound nucleotide-free kinesin induces "neck linker docking". Neck linker, located at the Cterminus of kinesin motor domain, is a 15-amino-acid-long "linker" between kinesin motor domain and the coiled-coil stalk needed for kinesin dimerization. It's responsible for the communication between two motors.

In ADP-kinesin detached from microtubule, the conformation of neck linker varies. It can be either rigid and docked into the catalytic core or disordered. Both conformations have been observed in ADP-kinesin crystal structure [START_REF] Sindelar | Two conformations in the human kinesin power stroke defined by X-ray crystallography and EPR spectroscopy[END_REF]. Using EPR spectroscopy and FRET approaches, Rice et al. have reported that, once kinesin binds microtubule, the neck linker is disordered after ADP release (apo-kinesin) and becomes ordered upon ATP binding (Rice et al. 1999).

The ATP-induced neck linker docking in microtubule-bound kinesin is crucial for kinesin's processivity and unidirectional movement to microtubule plus end. Because when in the leading motor, ATP binds to the microtubule-attached catalytic core; the other motor in ADP state either binds microtubule untightly or is detached from microtubule. The neck linker docking ensures: 1) there is always at least one motor domain attaches tightly to the microtubule, because ATP doesn't hydrolyze efficiently without neck linker docking and ATP-or apo-kinesin, unlike ADP-kinesin, always has high affinity with microtubule. 2) the rear ADP-motor moves unidirectional. As the direction of neck linker docking points to the plus end of microtubule, the rear motor is led toward the plus end. 3) kinesin homodimer moves processively. The rebinding of the "rear" head to microtubule makes sure the mechanochemical cycle on that motor continue. In other words, when the "rear" head moves forward and rebinds microtubule, it become the new "leading" head releasing ADP and accepting ATP. The neck linker docking will happen in the new "leading" head, accompanied with the movement of the new "rear" head. However, details on the mechanism of neck linker docking remains ambiguous. In particular it remains unclear how the conformation of neck linker changes upon microtubule binding and nucleotide state.

Structural study on kinesin's motor domain in different nucleotide state should provide detail information.

Although recently several structures of microtubule-bound kinesin in different nucleotide states have been obtained by Cryo-electron microscopy (Atherton et al. 2014;Shang et al. 2014); when I started my work, there was only one EM structure of nucleotide-free kinesin-1 binding microtubule [START_REF] Sindelar | The beginning of kinesin's force-generating cycle visualized at 9-A resolution[END_REF]. Limited by resolution, it was hard to generate an atomic model. One can only infer a rough orientation of the motor domain and the extension of H4 helix.

Until then, most kinesin high-resolution structures obtained by X-ray crystallography in different nucleotide states are in the absence of microtubule or of tubulin (e.g. see (Kull et al. 1996;Parke et al. 2010b)). But some of the properties of the kinesin change so much upon interaction with microtubules that the structure of microtubule-unbound kinesin is likely not to be relevant to several steps of its nucleotide cycle. The first crystal structure of ATP analog-kinesin in complex with tubulin, as a previous work in our lab (Gigant et al. 2013), provided insights to the mechanism of ATP hydrolysis and neck linker docking. The main missing piece on the structural study of kinesin's microtubule associated nucleotide cycle is a microtubule-bound apo-kinesin structure, which is the intermediate state between ADP-kinesin and microtubule-bound ATP-kinesin.

Here, I further the structure study by determining a 2.2 Å crystal structure of a nucleotidefree kinesin in complex with tubulin. Compared with ADP kinesin structure, this structure shall provide information explaining how binding microtubule accelerates the ADP dissociation in kinesin motor. In addition, comparing apo-kinesin and ATP-analog kinesin, both in complexes with tubulin, we can distinguish the conformational changes caused by ATP binding from those led by microtubule binding. Furthermore, we may answer the questions including how neck linker docking is induced by ATP binding, how ATP hydrolysis is favored in microtubule-bound kinesin etc.

Paper: "

The structure of apo-kinesin bound to tubulin links the nucleotide cycle to movement."

Discussion

Kinesin's processivity

When kinesin-1 homodimer moves along microtubules, each head goes through the same mechanochemical cycle (Fig. 5.1A) continuously and alternately. This is the key point for kinesin's processive movement which evinces that a single kinesin homodimer is capable of taking hundreds of steps before dissociation from microtubule. The processivity of kinesin relies on gating mechanisms which include a series of tightly coordinated biochemical and mechanical events [START_REF] Block | Kinesin motor mechanics: binding, stepping, tracking, gating, and limping[END_REF]). binding to the forward binding site. When the rear head moves forward, the binding gate favors its binding, while preventing the unbinding of the bound head. When both heads bind microtubule, the unbinding gate promotes the unbinding of the rear head and prevents the unbinding of the leading head.

To simplify the gating mechanism (Fig. 5.1B), there are three principles ensuring the processivity of kinesin movement (Andreasson et al. 2015a):

1) If there is only one head bound to microtubule, its dissociation should be prohibited.

2) The on-pathway transitions, including attaching towards the forward binding and detaching from the rear binding site, are promoted.

3) The off-pathway transitions, including rebinding of the rear head to its rear binding position and dissociation of the head from its leading position, are prohibited.

Tubulin-bound apo-kinesin structure presented here, compared with ADP-kinesin structure and tubulin-bound ATP-like kinesin structure, may provide some structural interpretations to the gating mechanism, including in the one-motor-bound state how the dissociation is prohibited and how the off-pathway transition is avoided.

One-motor-bound state

In the mechanochemical cycle of kinesin dimer, the one-motor-bound state includes "ATPwaiting-state" following the dissociation of ADP bound rear head and "ATP-state" leading the rebinding of the former rear head towards the forward binding site. During this procedure, the microtubule bound head is either in nucleotide-free state or binding ATP.

Previous biochemical study has already shown that both apo-kinesin motor (KD ≈ 0.62 M) and ATP-kinesin motor (KD ≈ 0.27 M) are able to bind microtubule tightly [START_REF] Crevel | Weak and strong states of kinesin and ncd[END_REF]. Analyzing both tubulin-bound apo-kinesin and ATP-analogkinesin structures, we confirmed an elongated α helix (H4) is responsible for their high affinity with microtubule. H4, located in the tubulin-binding subdomain, in microtubuleunbound ADP kinesin is short and followed by a flexible loop. When kinesin binds tubulin part of the disordered loop becomes rigid leading to the extension of H4. The extended part of H4 interacts with tubulin. The interaction between H4 extended part and tubulin is observed in both ATP analog-kinesin and apo-kinesin, despite of slight differences. In ATPanalog kinesin, the N-terminus of H4 helice is curved, instead of straight, so that N-terminal L11 can connect Switch 1 which is ordered and contacting the γ phosphate of ATP (or ATP analog). The elongation of H4 is also observed in electron microscopy studies (Atherton et al. 2014;Shang et al. 2014).

In physiological conditions, microtubule-bound apo-kinesin binds ATP (and not ADP) because 1) of a lower concentration of ADP compared with that of ATP [START_REF] Tantama | Imaging energy status in live cells with a fluorescent biosensor of the intracellular ATP-to-ADP ratio[END_REF]) and 2) of a slightly higher affinity of microtubule-bound apo-kinesin for ATP (KD ≈ 75 M) than for ADP (KD ≈ 120 M) (Cross 2004).

Off-pathway transitions are prohibited

There are two main off-pathway transitions that should be avoided in kinesin cycle.

1) When there is one head attached to microtubule, the rebinding of the rear head upon the rear-binding site should be prohibited. 2) When both heads bind microtubule, the ATP hydrolysis in the leading head should be prohibited so that the leading head will not detach from the microtubule.

For the first coordination mechanism, it has been shown that the rebinding of the rear head is controlled by the interhead tension caused by the neck linker docking. In kinesin mutants whose neck linker is elongated artificially, the rear head has more chance to rebind to its former binding site (Isojima et al. 2016).

The second coordination mechanism is much more complicated. There are two controversial gating mechanisms. One is the "front-head gating" model in which ATP binding or hydrolysis in the front head is prohibited. The other is the "rear-head gating" model which suggests ATP hydrolysis or MT release in the rear head are accelerated. Furthermore, there are two theories about the gating mechanism that is either tension dependent or orientation dependent. When both heads bind microtubule, the heads are separated by 8.2 nm. The neck linker of the front head points backwards, while that of the rear head points forwards and docks onto the catalytic core. Consequently, intramolecular tension is developed between the heads. Some people proposed the tension "pulls" the rear head, accelerating the dissociation of the rear head, responsible for the "rear-head gating" mechanism. The other interpretation is that the backward orientation of the neck linker in the front head inhibits the ATP binding or ATP hydrolysis in the front head, causing the "front-head gating".

Recently, more and more results on single-molecule study support the front-head gating mechanism caused by the neck linker backward orientation. Isojima et al. found that the dwell time of the two-heads-bound state is independent of the concentration of ATP. It indicates that the two-heads-bound state is not gated by the fast ATP hydrolysis in the rear head. They also found it's rare that the leading motor dissociates from microtubule before the dissociation of the rear head in both wild type kinesin dimer and a mutant where the neck linker is elongated. This result rules out the possibility that the gating mechanism is led by the interhead tension (Isojima et al. 2016). Dogan et al. found that a small backward tension, as low as 2pN, is necessary to inhibit the nucleotide binding to a single head, although the tension between two microtubule-bound motor domains is up to 15 pN.

Based on that, they proposed that the gating mechanism is caused by the back ward orientation of the front head's neck linker [START_REF] Dogan | Kinesin's front head is gated by the backward orientation of its neck linker[END_REF].

My results, indicating that neck-linker docking in the forward head is the necessary condition for ATP hydrolysis, are able to provide a structural interpretation to this theory. For a single microtubule-bound motor domain, there is no space for neck linker to dock in apo-state; while the subdomain rotations induced by ATP binding create a potential hydrophobic site allowing the first residue of neck linker to dock into. The docked, even if partially docked, neck linker, as a pin, stabilizes the structure at a subdomain-rotated state in which ATP hydrolysis is favored. This theory is supported by the facts that if neck linker is truncated or the first residue is mutated, the ATPase hydrolysis efficiency is significantly interfered (see table 1 of the article).

Taking both motor domains into consideration, we find that if the rear head still binds its rear binding site or doesn't move forward enough, the neck linker is always backward oriented, which means the neck linker cannot dock into the front head. The front head will act as the KHC 324 mutant, in which there is no neck linker docking, with very low ATPase activity. As a result, the binding or hydrolysis of ATP in the leading motor domain is prohibited.

Furthermore, the partial docking of the neck linker, promoting the ATP hydrolysis, probably leads neck linker to fully dock into the catalytic core. In this case rebinding of the rear head to its original binding site is prohibited and binding of the rear head to its forward binding site is favored. But more evidence should be provided to support this hypothesis.

Kinesin and myosin

The first determined kinesin structure reveals the topological similarity between kinesin and myosin, despite that these two proteins have a low sequence identity (Kull et al. 1996).

Myosin motor domain in different nucleotide state have been analyzed structurally [START_REF] Coureux | A structural state of the myosin V motor without bound nucleotide[END_REF]). These results have revealed that myosin can be divided into several subdomains. The rotation of these domains, including N-terminal subdomain, upper and lower 50 kDa subdomains, the converter subdomain, is associated with the nucleotide state, with actin attachment and with the closure of a major cleft between upper and lower 50 kDa subdomains (Fig. 5.2). The structures of nucleotide-free myosin V, myosin II in near-rigor and transition state are presented here. The structures have been superimposed on their N-terminal subdomains. The rotation necessary to move from nucleotide-free myosin V to the near-rigor myosin II is marked on the each subdomain of myosin V; the rotation necessary to move from the nearrigor to the transition station in myosin II is indicated on the subdomains of myosin II in transition state.

However, due to lack of the intermediation state structure, the subdomains in kinesin similar to myosin had not been identified until this work. Those subdomains close to nucleotide binding site of kinesin and myosin are compared and presented in figure 5.3.

Besides, myosin and kinesin share a similar distortion of the central β sheets along with their nucleotide cycles (Fig. 5.4). These common features confirm the reliability of threesubdomain theory in kinesin, also strengthens that kinesin and myosin share a same ancestor.

a. b. The similarity of subdomains in kinesin (pdb id 4HNA) and myosin (pdb id 1W7J) is presented here, with an ADP-BeFx bound myosin (a) [START_REF] Cecchini | Allosteric communication in myosin V: from small conformational changes to large directed movements[END_REF] and an ADP-AlFx bound kinesin (b) (Gigant et al. 2013). The lower 50 subdomain of myosin and tubulin binding subdomain of kinesin are colored in green. Myosin's N-terminal subdomain and kinesin's P-loop subdomain are colored in wheat, with P-loop motifs (orange) highlighted in both structures. The upper 50 subdomain of myosin and Switch 1/2 subdomain of kinesin are shown in cyan with both Switch 1 motifs (blue) highlighted. In both structures, the loop containing Switch 2 motif is shown in grey. [START_REF] Cecchini | Allosteric communication in myosin V: from small conformational changes to large directed movements[END_REF] or in blue (nucleotide-free structures, pdb id 1OE9 [START_REF] Coureux | A structural state of the myosin V motor without bound nucleotide[END_REF]). For kinesin, strands (β1, β2 and β8, in orange) of P-loop subdomains are superimposed, and strands of Switch 1/2 subdomains are shown in cyan (ATP-like structure, pdb id 4HNA (Gigant et al. 2013), or in blue (nucleotide-free structure, pdb id 4LNU, (Cao et al. 2014)).

Nevertheless, along with these structural and mechanochemical similarities, there are a lot of differences between these two motor proteins. The major one associated with subdomain definition is the binding mode of myosin or kinesin to their track. Kinesin motor binds one tubulin with its tubulin binding subdomain; while myosin attaches to one actin molecule through its upper and lower 50 subdomains, and to another actin of the same filament through its lower 50 subdomain. As a result, myosin and kinesin are able to track actin filaments and microtubules respectively.

Perspectives

Kinesin's processivity

As discussed, kinesin's processivity is a complicated but subtle process involving kinesin's mechanochemical cycle. Each step is controlled spetially and temporally. Our work provides possible structural explanation to kinesin's processivity which matches the theory proposed based on biophysical results. But questions still remain to be answered including how the rear head detachment is favored, how the microtubule binding stimulated ADP dissociation is prevented from the rear head etc. A possible way to generate the answers is to determine ADP-kinesin structure in complex with tubulin heterodimer. Both cocrystallization of ADP-kinesin with tubulin and soaking apo-kinesin-tubulin complex crystals with ADP can be tried.

ADP release mechanism

Although we present the evidence that Switch 1/2 subdomain rotation led by elongation of H4 is involved in high ADP dissociation rate, more study is necessary to reveal the proper microtubule stimulated ADP release mechanism.

The spontaneous ADP dissociation rate of the double mutant R190A D231A (koff = 0.6 s -1 ) is still far lower than the microtubule stimulated ADP release rate of its parent constructs (koff = 50 s -1 ), despite the fact that it's already about 20 times more than the spontaneous ADP dissociation rate of the WT kinesin. Moreover, beside the Switch 1/2 subdomain rotation, there are other parts of kinesin that go through significant structural changes i.e. P-loop subdomain rotates as well. These evidences indicate that there must be other factors involved in the microtubule stimulated ADP dissociation.

Comparing the structure of apo-kinesin in complex with tubulin to that of ADP-kinesin, we can know the structural changes involved in the microtubule and nucleotide binding. Nevertheless, having a kinesin structure with nucleotide without microtubule and another kinesin structure without nucleotide with microtubule, we cannot tell which conformational change is associated with nucleotide release or which is involved in microtubule binding. As a result, mutagenesis studies are necessary to identify more residues involved in this process. As well, more structural studies on apo-kinesin will be helpful to clarify the structural influence caused by the microtubule attachment. This is the objective of the next chapter.

Structures of isolated apo-kinesin-1 2.1 Context

Previous biochemical study indicated that ADP dissociation from kinesin is significantly accelerated upon microtubule binding (Cross 2004;Hackney 1988). However the molecular mechanism remains unclear. The structure of apo-kinesin-1 in complex with tubulin which I determined, making up kinesin's mechanochemical cycle, provided important information on the linkage between ATP binding and hydrolysis and kinesin movement, and as well on the ADP release mechanism. Structural analysis indicates that, upon microtubule binding, the binding environment of magnesium in kinesin is disrupted so that ADP release is favored. This hypothesis is further supported by mutagenesis study on kinesin. When R190 and D231, residues stabilizing magnesium indirectly via its water ligand, are mutated to alanine, the mutants have higher spontaneous ADP release compared with WT. In WT kinesin these two residues' interaction with magnesium is interfered by microtubule binding, which contributes to the enhanced ADP dissociation.

However, whereas the comparison between apo-kinesin-1-tubulin complex and ADPkinesin in the absence of tubulin is very informative, questions on the ADP dissociation mechanism remain. One reason is that the presence or absence of ADP is not the only variable in these two structures. Indeed it is difficult to discriminate which conformational changes are caused by tubulin binding and which are caused by ADP release. Consequently, more study is necessary to characterize further the mechanism of microtubule-stimulated ADP dissociation from kinesin.

As presented in the introduction part, several kinesin mutants are able to release ADP much faster than WT in the absence of microtubule but without obvious structural explanations [START_REF] Rayment | Kinesin and myosin: molecular motors with similar engines[END_REF]Higuchi et al. 2004;Heuston et al. 2010). Most of these residues belong to the conserved nucleotide binding motifs, including P-loop motif and Switch 2 motif, of kinesin. Among them, there are two mutants drawing my attention. One is the T92 equivalent mutant. The P-loop residue T92 interacts directly with the Mg 2+ ion. It also interacts with D231 (from Switch 2). Further analysis on T92-associated mutants may provide supporting evidence to our previous study. The second one is the E236A mutant which has a spontaneous ADP release rate significantly higher than that of WT; meanwhile this switch 2 glutamic acid residue doesn't contact magnesium nor the nucleotide directly (Rice et al. 1999). Instead it interacts with the P-loop, in particular with T87.

In order to gain new information on ADP release from kinesin and on the underlying structural consequences, I have studied the contribution of T92 and the influence of different substitutions of this residue, and also the contribution of T87. These two groups of mutations represent two different ways to generate fast ADP-releasing kinesin.

Moreover, high resolution crystal structures on these mutants in nucleotide-free state were obtained, as well as that of nucleotide-depleted WT kinesin. Interestingly, these apokinesins adopt two different conformations. By analyzing these structures, I found that microtubule accelerates ADP dissociation from kinesin by both interfering with magnesium binding and destabilizing the P-loop.

Paper: "

The structural switch of nucleotide-free kinesin."

Kinesins are a family of microtubule-based motors that play important roles in intracellular transport and cell division. Kinesin-1 transports cargo within cells, a process tightly coupled with ATP hydrolysis (Svoboda et al. 1993;[START_REF] Coy | Kinesin takes one 8-nm step for each ATP that it hydrolyzes[END_REF].

Single-molecule studies have shown that dimeric kinesin-1 moves in a hand-over-hand manner by alternately translocating its two motor domains (Hackney 1994;[START_REF] Kaseda | Alternate fast and slow stepping of a heterodimeric kinesin molecule[END_REF]. Whereas kinesin-1 in solution is mostly loaded with ADP, ADP release is accelerated several thousand-fold upon microtubule binding (Hackney 1988;Cross 2004). ATP binding then triggers a conformational change in the microtubule-bound leading motor domain, following which the rear head is drawn forward in the direction of the (+)-end of the microtubule. The moving head then binds to the microtubule 16 nm ahead from its previous position, whereas the (now) rear head hydrolyzes ATP and eventually detaches from microtubule, achieving a step (Andreasson et al. 2015b;Hancock 2016;Isojima et al. 2016).

X-ray crystallographic studies have defined the structures of an ADP-loaded kinesin-1 motor domain (Kull et al. 1996;Kozielski et al. 1997). Structural changes in the nucleotidebinding site upon binding of a non-hydrolysable ATP analog were then identified in the kinesin-5 Eg5 (Parke et al. 2010a). Most recent X-ray structural studies have shown that a kinesin-1 motor domain comprises 3 subdomains that reorient as a function of the nucleotide content and upon binding to tubulin (Cao et al. 2014;Gigant et al. 2013).

Because the three nucleotide-binding motifs (the P-loop, Switch 1 and Switch 2) do not belong to the same subdomain, the nucleotide environment gets remodeled along with the kinesin mechanochemical cycle. The P-loop is embedded in the so-called "P-loop subdomain" that comprises elements of the N-terminal and of the C-terminal parts of the motor domain. The C-terminal part of Switch 1, together with the first residue of the Switch 2 motif, has been ascribed to the "Switch 1/2 subdomain", internal in the sequence of the motor domain, whereas most of Switch 2 is N-terminal to the α4 helix, one of the main elements of the "tubulin-binding subdomain" (Cao et al. 2014). These X-ray structural studies have been conducted in parallel with electron microscopy characterizations of what occurs in a motor domain as a function of its nucleotide, culminating in about 6 Å studies of kinesin bound to microtubules that were broadly consistent with the X-ray results, either when conducted on kinesin-1 (Shang et al. 2014;Sindelar and Downing 2010;Atherton et al. 2014) or on a kinesin-3 (Atherton et al. 2014).

One of the points that remained uncertain from these studies is that microtubule binding and nucleotide release were characterized in the same structure and, therefore, it was difficult to ascertain which structural changes were due to each of the two steps of the mechanism. To answer this question, apo-kinesin in the absence of microtubule has to be studied. Mutations have been identified that accelerate nucleotide release by a kinesin from several times (Cao et al. 2014;Higuchi et al. 2004;Heuston et al. 2010;Rice et al. 1999) to several thousand-fold (Rice et al. 1999) but the structural consequences of these mutations on the kinesin apo-form are not known. Here we characterized P-loop mutations in kinesin-1 that interfere with ADP binding and determined the structure of the corresponding mutated nucleotide-free kinesins. Notably, these structures are mostly similar to those of ADP-kinesin or of tubulin-bound nucleotide-free kinesin. Most remarkably these conformations are also adopted by the parental, nucleotide-depleted, wild-type protein.

The consequences of these findings will be discussed.

Results

Mutational approach to enhance nucleotide release from kinesin-1.

Mutations in two general areas of kinesin have been found to facilitate nucleotide release.

The first ones are in the environment of the Mg 2+ ion that interacts with the ADP ligand in most kinesins (see, for instance, (Kull et al. 1996)). Indeed, initial studies demonstrated that modulating the Mg 2+ free concentration changes the ADP release rate in kinesin-1 [START_REF] Cheng | Interaction of mant-adenosine nucleotides and magnesium with kinesin[END_REF] and in kinesin-3 [START_REF] Nitta | Structural model for strain-dependent microtubule activation of Mg-ADP release from kinesin[END_REF]. In kinesin-1, the only residue that interacts directly with the Mg 2+ ion is T92 (Supplementary Fig. 1), the last residue of the P-loop motif. Either a threonine or a serine, this residue is conserved in all nucleotide-binding proteins' P-loop motif (GxxxxGK(S/T)), which is known as Walker A motif and required for coordinating α and β phosphates (Walker et al. 1982).

In kinesins, when a serine is found at the last P-loop position, a higher ADP release rate is observed. Such is the case of the kinesin-3 Kif14, where a high ADP release rate was deduced from the spontaneous ATPase rate of the isolated motor domain (0.88 sec -1 ) (Arora et al. 2014). As expected, when the T92S mutation was introduced in kinesin-1, ADP release was substantially accelerated, about 10-fold from 0.034 s -1 in wild-type kinesin to 0.33 s -1 in T92S (Table 1). Other mutations that interfere with the kinesin mechanism have been identified at this position in kinesin-1. It has been shown that when this residue is mutated to isoleucine or to asparagine, the corresponding kinesin binds to microtubules without dissociating, even in the presence of a large concentration of ATP (Nakata and Hirokawa 1995;Crevel et al. 2004). We also found that when T92 is mutated to a valine, the fluorescence of the kinesin does not increase upon incubation with mant-ADP, suggesting that this kinesin mutant had lost its ability to bind ADP. To confirm this, we quantified the nucleotide content of the same amount of mutant and wild-type kinesin on an ion exchange column. We found indeed that the T92V kinesin has essentially lost its ability to bind ADP (Fig. 1A).

An initial indication that residues on the other side of ADP as compared to Mg 2+ are important for ADP binding came from the study of the kinesin E236A mutant, which releases ADP about 100-fold faster than wild-type kinesin (Rice et al. 1999), a result we were able to confirm (Fig. 1B, Table 1). In ADP-kinesin-1, E236 forms two hydrogen bonds with T87 in the P-loop motif, one with the main chain and the other one with the side-chain (Supplementary Fig. 1), whereas these interactions do not exist in tubulin-bound apokinesin (Cao et al. 2014). Kinetic studies show that the kinesin conserved T87 residue is important for ADP binding, though it does not interact directly with the nucleotide: the T87S mutant releases ADP 10 times faster than wild-type (Fig. 1B, Table 1), similar to the equivalent mutation in drosophila kinesin-1 (Higuchi et al. 2004) and in kinesin-14 (Heuston et al. 2010). More importantly, when more mutations of residue 87 were tested, the T87A and T87G mutants were found to release ADP much faster than wild-type (koff= 2.77 s -1 and 4.80 s -1 , respectively) (Fig. 1B, Table 1).

To gain further understanding of the effect of nucleotide release on the kinesin, we have determined the nucleotide-free structures of the two single amino acid mutants of kinesin-1, T87A and T92V. Remarkably, we were also able to remove the nucleotide from wild-type kinesin and to crystallize it in the same buffer conditions as the two previous mutants. The corresponding structures will now be presented.

The main difference between T92V and wild-type ADP-kinesin is the missing nucleotide and magnesium ion in the T92V nucleotide binding cleft. Instead of the nucleotide, an electron density, well accounted for by a sulfate ion that likely comes from the crystallizing solution, is located close to the missing ADP β phosphate (Fig. 2C). Therefore, we conclude from these observations that the T92V mutation is sufficient to interfere with Mg 2+ binding to the kinesin and, consequently, with ADP binding. This interference does not require an overall rearrangement of the structure of the kinesin motor domain. These results agree with an ADP release rate limited by that of Mg 2+ [START_REF] Cheng | Interaction of mant-adenosine nucleotides and magnesium with kinesin[END_REF].

We were also able to crystallize in the same buffer conditions the nucleotide-free parental kinesin-1 (without the T92V mutation), following nucleotide digestion with apyrase, and to determine its structure (Supplementary Table 1, Supplementary Fig. 3). Remarkably this structure is isomorphous to that of ADP-kinesin (Kull et al. 1996). Therefore an ADPkinesin like conformation can be adopted both by nucleotide-free wild-type kinesin and by the T92V mutant.

Isolated apo-kinesins in a tubulin-bound apo-kinesin-1 conformation.

Apo-T87A kinesin-1 was crystallized in a new crystal form following nucleotide digestion with apyrase and gel filtration to remove any trace of nucleotide. The crystals diffracted to 2.6 Å (Supplementary Table 1) and there were six identical molecules in the asymmetric unit (r.m.s.d. of Cαs in pairs of molecules varies between 0.3 Å and 0.55 Å). Using these T87A crystals as seeds (see methods), we were also able to crystallize wild-type apokinesin-1, prepared as above by digestion of the nucleotide with apyrase followed by gel filtration, and the T92V mutant and to determine their structures (Supplementary Table 1, Switch 2 motif, and its environment. As already mentioned, in ADP-kinesin-1 (and in apokinesin-1 in an ADP-kinesin like conformation, see Fig. 2B), this residue establishes two hydrogen bonds with T87. In apo-kinesin-1 bound to tubulin, the structure of L11, where this residue is embedded, changes and E236 interacts with residue R203, in the Switch 1 motif. But in apo-T87A, the structure of L11 has changed again (see above). As a consequence, the R203-E236 interaction is lost and the E236 side-chain is disordered and not visible beyond the Cβ (Fig. 3C). Since it is disordered in isolated T87A apo-kinesin-1, E236 does not make any visible interaction with the P-loop and with residue 87 in particular. It is therefore not surprising that the P-loop temperature factors become higher than those of the kinesin central β-sheet, taken as a reference, in apo-T87A (average temperature factor of the P-loop Cs is 47 Å 2 , over the six molecules in the asymmetric unit, to be compared to an average temperature factor of 35 Å 2 for the central β-sheet Cs), whereas these temperature factors of the P-loop are much closer to that of the central βsheet both in ADP-kinesin (Kull et al. 1996) and in apo-kinesin in an ADP-like conformation (this work) (Supplementary Table 2). Because in this last case kinesins are nucleotide-free, our temperature factors comparison rules out the possibility that the P-loop is mainly stabilized by ADP binding. The second difference between the structures of apo-T87A kinesin and that of apo-kinesin bound to tubulin concerns the N-terminal end of α4. In a complex with tubulin, this part of the kinesin is close to the tubulin α subunit, with which residues at the C-terminal end of L11 (such as E244 and G242, Fig. 3B), i.e. close to α4, interact. But in the structure of isolated apo-T87A kinesin-1, such an interaction is not made and, probably as a consequence, the temperature factors of residues in the first two turns of α4 (residues 246 to 253) are significantly higher than those of residues in the rest of the helix: the temperature factors of their Cs are larger than 70 Å 2 on average, whereas they are about 32 Å 2 in the rest of the helix. Such a difference of temperature factors is not observed among α4 residues in tubulin-bound apo-kinesin-1 (average Cα temperature factor for the C-terminal part of the helix is 44 Å 2 whereas that of the first two turns is 53

Å 2 ).
The structure of isolated apo-kinesins similar to that of apo-kinesin-1 in its complex with tubulin provides an additional opportunity to probe the changes that occur when ADPkinesin binds to tubulin (or microtubules). In particular, residue Q86 that interacts with residue N308 in the ADP-kinesin conformation, points towards the carbonyl of S235 when the tubulin-bound apo-kinesin like conformation is adopted (Fig. 3D). Mutating the Q86 residue to an alanine would clearly destabilize the interaction of this residue with N308 in ADP-kinesin, favoring ADP release, as witnessed by an increase of this reaction rate (from 0.034 s -1 to 0.14 s -1 ). This effect is further confirmed by the observation that the rate of ADP release by the double mutant Q86A-T87A is higher than that of the T87A point mutant (3.5

s -1 vs. 2.77 s -1 , Table 1).

Apart from the T92V substitution, which leads to a kinesin unable to bind nucleotides, the rates of ADP release by all the mutants tested so far remain much lower than the rate of microtubule-stimulated ATP hydrolysis by kinesin-1 (kcat=50 s -1 , see (Gigant et al. 2013)).

These mutants involve in particular mutations at two positions in the P-loop, 87 and 92, as described above. Since most of ADP is embedded in the P-loop subdomain and interacts with the P-loop, it is not surprising that interactions of that loop with the rest of the motor domain that are lost in the tubulin-bound apo-kinesin-1 conformation are important for ADP binding. As a consequence, ADP release is accelerated when they are prevented. This is in particular the case of the interactions of T87 and T92 (in the P-loop) with E236 and D231, respectively (Fig. 2B, Supplementary Fig. 1). Interestingly, a substantially higher rate of ADP release was reached by the T87A-T92S kinesin double mutant (koff=19 s -1 ) compared to the single mutants (Fig. 1, Table 1).

Discussion

ADP release from kinesin is a slow process that is accelerated upon microtubule binding.

Here we have studied the effect of several mutations on spontaneous ADP release and determined the structures of two point mutants and of wild-type kinesin-1 in the apo form.

Our results point to two features of ADP binding. The first one is the importance of Mg 2+ stabilization. In particular, mutating the Mg 2+ interacting residue T92 to a valine leads to an intrinsically nucleotide-free kinesin (Fig. 1A) without the need of an overall structural change towards the tubulin-bound apo-kinesin conformation (Fig. 2A). An intermediate effect is observed with the T92S mutation. When this mutation is introduced in kinesin-1, ADP release is accelerated 10 times (Table 1) to reach a rate similar to those of kinesin-3s, as deduced from the spontaneous ATP hydrolysis rate of these kinesins (Arora et al. 2014;[START_REF] Nitta | Structural model for strain-dependent microtubule activation of Mg-ADP release from kinesin[END_REF]. Additionally, the mutation to an alanine of residue 231, which interacts with T92, also accelerates ADP release substantially (Cao et al. 2014).

These results are consistent with a spontaneous dissociation of ADP from wild-type kinesin that is limited by that of Mg 2+ [START_REF] Cheng | Interaction of mant-adenosine nucleotides and magnesium with kinesin[END_REF]. The second feature is the importance of P-loop stabilization for ADP binding. Although the most obvious direct interactions of ADP with the P-loop are with the side chain of K91 and with the peptidic nitrogens of residues 88 and 90 to 93 (Supplementary Fig. 1) (Kull et al. 1996), mutations of T87 to alanine or glycine have among the strongest effects measured so far on ADP release; the effect of the destabilization of the interactions of Q86 (through a mutation of this residue to an alanine) is intermediate (Table 1). P-loop destabilization, together with the removal of the nucleotide, leads to a tubulin-bound apo-kinesin like conformation, as seen in the structure of the T87A kinesin (Fig. 3A).

It is remarkable that both crystal forms of apo-kinesin characterized here were also obtained from wild-type kinesin, following removal of the nucleotide, using crystallization conditions similar to those for the T92V and T87A mutants. In a natural context, the release of ADP from kinesin upon microtubule binding proceeds in two steps, a transition from weak to strong microtubule binding followed by ADP release (Cross 2004;Ma and Taylor 1997). The corresponding structural changes lead both to the destabilization of the P-loop and of Mg 2+ binding, hence ADP release is favored (Cao et al. 2014;Shang et al. 2014).

Interestingly, a double mutant (T87A-T92S) of kinesin, in which Mg 2+ binding and the Ploop are destabilized, releases ADP spontaneously at a rate similar to that of tubulinstimulated ATP hydrolysis by the kinesin (Gigant et al. 2013).

Whereas our results demonstrate that, in the absence of microtubules, apo-kinesin-1 may adopt two conformations (ADP-kinesin like and tubulin-bound apo-kinesin like), the effect of tubulin binding is to shift the balance between them and to stabilize the tubulin-bound apo-kinesin conformation, which is primed to bind ATP. The wild-type sequence also ensures that kinesin-1's interaction with microtubules is modulated by the bound nucleotide. During processive movement, multiple cycles are performed, where this nucleotide gets hydrolyzed then replaced by ATP, as required.

Methods

Constructs and protein purification

The constructs and mutants used in this study were obtained by standard molecular biology from a monomeric cys-light version of human kinesin-1 (Rice et al. 1999). These constructs comprise the motor domain and the neck linker (construct 1-149, used for kinetic experiments) or the motor domain catalytic core only (construct 1-325, used for crystallization). Proteins were produced in BL21 Escherichia coli cells grown in 2YT medium. The expression was induced with 0.3 mM isopropyl -D-1-thiogalactopyranoside at 18 °C overnight, after the absorbance at 600 nm of the culture reached 0.7 to 0.8. Cells were harvested and suspended in buffer A (25 mM Pipes pH 6.8, 1 mM MgCl2, 0.5 mM EGTA, 25 µM ATP, 1 mM DTT) plus 1 mM phenylmethylsulfonyl fluoride and protease inhibitors mix (complete EDTA free, Roche Applied Science). Two ion exchange columns were used for purification, a Hitrap SP FF column (GE Healthcare), using 20% buffer B (buffer A + 1 M NaCl) for the elution, then a Mono Q column (GE Healthcare) with a 10 to 40% buffer B gradient for elution.

To generate nucleotide-free kinesin-1 (wild-type and T87A mutant), the proteins were treated with apyrase (Sigma, 0.5 U/ml) at 4 °C overnight. For crystallization, an additional gel filtration column (Superdex 200 10/300 GL, GE Healthcare) in a buffer consisting of 16 mM Pipes pH 6.8, 0.5 mM MgCl2, 0.2 mM EGTA, 100 mM NaCl was performed. The protein was then concentrated, quantified using the absorbance value at 280 nm and stored in liquid nitrogen until use.

Crystallization and structure determination

Crystallizations were performed at 293 K by vapor diffusion using the hanging drop method. T92V and nucleotide-depleted wild-type kinesin were crystallized in the ADPkinesin like form in a buffer consisting of 0.1 M Na-acetate pH 4.5 to 5.5, 20 to 25% (W/V) polyethylene glycol 4000, 0.2 M (NH4)2SO4 and 0.1 mM SrCl2. Apo-T87A was crystallized in a tubulin-bound apo-kinesin like conformation in a buffer containing 50 mM Mes pH 6.5, 0.16 to 0.2 M (NH4)2SO4 and 30% (W/V) polyethylene glycol 5000 monomethyl ether. T92V

and wild-type apo-kinesin-1 were also crystallized in these same conditions following streak seeding using T87A crystals as seeds. Crystals were harvested in the crystallization buffer supplemented with 20% glycerol, then flash-cooled in liquid nitrogen until data collection.

Data sets were collected at 100 K at the Proxima1 and Proxima 2 beam lines (Soleil Synchrotron, Saint Aubin, France). The data were processed with XDS (Kabsch 2010) and Aimless (Winn et al. 2011). The structures were solved by molecular replacement with PHASER (McCoy et al. 2007) using either ADP-kinesin (pdb id 1BG2 (Kull et al. 1996)) or tubulin-bound apo-kinesin (pdb id 4LNU (Cao et al. 2014)) as starting models. The structures were iteratively refined with Buster [START_REF] Bricogne | BUSTER version 2.10[END_REF] with model building in Coot [START_REF] Emsley | Features and development of Coot[END_REF]). Some refinement cycles were also performed using Phenix [START_REF] Adams | PHENIX: a comprehensive Python-based system for macromolecular structure solution[END_REF] corresponding to the dissociation of mant-ADP from kinesin and yields the rate constant for ADP release (Table 1). The fit in the case of the wild-type protein, which releases ADP much more slowly, is presented in Supplementary Fig. 4. a.u., arbitrary units. kinesin-1 mutant (green) superimposed on apo-kinesin-1 (wheat, the side chain of residues T87 and T92 is drawn) in complex with tubulin (blue) (pdb id 4LNU). In both structures, L9 is disordered whereas most of L11 is ordered, leading to full elongation of the α4 helix. (B) The structure of the L11 loop. Whereas in the absence of tubulin, a stretch of the kinesin L11 loop

Discussion

To further study the mechanism of ADP dissociation from kinesin-1, I did mutagenesis study on kinesin-1 motor domain to generate mutants mimicking the conditions when it binds microtubule. Two series of mutants with high ADP dissociation rates represent two pathways to interfere with ADP binding. In addition, after ADP digestion by apyrase, I was able to prepare nucleotide-free kinesin (either WT or mutants) and to determine several structures in the absence of tubulin. Because isolated apo-kinesin was reported to be unstable (Cross 2004;[START_REF] Hackney | The kinetic cycles of myosin, kinesin, and dynein[END_REF], I further studied the stability of the apo-kinesins I prepared.

Stability of kinesin and its mutants in the absence of nucleotide

Kinesins in nucleotide-free state were reported to be unstable [START_REF] Sadhu | A kinetic study of the kinesin ATPase[END_REF]Hackney 1994). When the nucleotide in kinesin-1 is removed, some motor protein denatures and precipitates. The same instability of apo-kinesin was also observed in kinesin-14 [START_REF] Lockhart | Origins of reversed directionality in the ncd molecular motor[END_REF].

The observation that the intrinsically nucleotide-free T92V mutant could be purified suggests that apo-T92V is more stable and soluble than anticipated. So I tried to crystallize this mutant. The final success in the crystallization of apo-T92V convinced me that at least with T92 mutated, apo-kinesin is stable and well-folded. Furthermore, I obtained the crystal structure of another apo-kinesin mutant, T87A, after treatment with apyrase to remove the remaining nucleotide. It suggested that with T87 mutated kinesin is stable and well-folded as well, despite of its distinct crystal structure compared to T92V. These results make me wonder whether these mutations play a role in kinesin stability.

Therefore I have studied the stability of apo-kinesin mutants and apo-kinesin wild type by differential scanning calorimetry (DSC) with ADP-kinesin wild type as a control (Fig. 6.1).

The results show that apo-kinesin is less stable and easier to denature than ADP-kinesin, as expected from results from the literature (Fig. 6.2). However the melting temperature is only 3 to 4°C lower in the case of apo-kinesin wild type compared to ADP-kinesin. In addition it is more stable than the T92V and T97A mutants. Overall these results indicated that apo-kinesin wild type, as well as its mutants, remains soluble and well-folded at 20°C, i.e. at the temperature where most of the biochemical experiments were performed, also where the protein was crystallized.

Figure 6.1 An example of DSC analysis of kinesin

The DSC thermographs (black) of kinesin are asymmetric; they were fitted (red) by two symmetric peaks with two melting temperature values (Tm) and two values of enthalpy of transition (H) (red dash lines). The asymmetry of thermographs may be due to an intermediate state during the process of kinesin denaturation.

The solubility and stability of apo-kinesin, which is found higher than expected, could be due to the concentration of salt (150-300 mM NaCl) used. Indeed during purification kinesin elutes from the ion exchange columns at about 30% Buffer B, i.e. about 300 mM NaCl. I tried to desalt the samples when preparing them for crystallization, but I noticed some precipitation when the concentration of salt went down to 100 mM. Therefore for crystallization and DSC analysis, there was always 150 mM NaCl in the system to prevent kinesin from precipitating. In the ADP-release experiments, the concentration of kinesin is low (0.5 -1 M) and there is always extra nucleotide in the system, so no salt is added during the measurement. The structural switch of nucleotide-free kinesin.

The DSC analysis indicates that the stability of apo-kinesin wild type is higher than that of apo-kinesin T92V and T87A mutants. Because useful crystals were obtained with these mutants, I wondered whether wild type apo-kinesin could be crystallized as well. I tried to crystallize it in the conditions where the apo-kinesin mutants crystallized spontaneously. Indeed, wild type apo-kinesin was crystallized and the structure indicates that it can adopt the two different conformations, ADP-like and tubulin-bound apo-like.

ADP-like apo-kinesin, with solvent replacing ADP in its nucleotide binding pocket, is able to crystallize spontaneously. It suggests that ADP-like is likely the dominant conformation in kinesin. It explains the stability and solubility of kinesin with the presence of salts as well.

Wild type apo-kinesin (so is T92V mutant) is able to be crystallized with an apo-like conformation only under the circumstance of seeded with apo-T87 mutants' crystals in the same condition where apo-T87 mutant is crystallized with apo-like conformation. This conformation of apo-kinesin with a relatively open nucleotide binding pocket, is probably a short-lived state which allowing apo-kinesin to rebind nucleotide. Binding to microtubule stabilizes this apo-like conformation by keeping the conformational changes of tubulinbinding subdomain, including the elongation of H4 helix.

Nevertheless, these results suggest the existence of various apo-kinesin conformations. More efforts should be made to investigate the states of apo-kinesin in the solution.

Structural study on kinesin motility

Kinesin-1 walks along microtubule as a homodimer in a hand-over-hand manner. That two motor domains go through the same cycle but in different states is crucial for kinesin's processive and unidirectional movement. Investigating the communication between two motor domains during kinesin movement will be interesting and meaningful.

So far, we obtained the crystal structure of kinesin monomer in different nucleotide states. The fact that T92V doesn't bind any nucleotide provides a possibility to study the communication between two motor domains of kinesin directly by having a structure of kinesin dimer with two motor domains in different nucleotide state, i.e. with one motor domain in nucleotide free state and the other in ATP-analog or ADP state.

Recent advancement on recombination and expression of tubulin suggests the possibility of obtaining a tubulin linear tetramer through introducing mutations into tubulin. In other words, one can obtain a tubulin polymer having two kinesin binding sites.

In general, a structure presented in figure 6.3 is accessible in theory and will bring new insights into kinesin's processive motility. T92V and wild type kinesin heterodimer may provide an opportunity to observe the communication between two motors domains in different mechanochemical states directly. With mutations (red points) introduced into recombined and expressed tubulin heterodimer, it's possible to generate heterodimeric tubulin heterodimer, a linear tubulin tetramer having two kinesin binding sites. Therefore, a complex, in which both kinesin motor domains bind tubulin but one in nucleotide-free state while the other in ATP-analog state (for example), can be generated. This work will be very challenging but possible theoretically. Structural studies on Kar3 will provide more insight into Kar3's partner-modulated motility. The first crystal structure published revealed that the Kar3 motor domain has a structure similar to those of other kinesin family members [START_REF] Gulick | X-ray crystal structure of the yeast Kar3 motor domain complexed with Mg.ADP to 2.3 A resolution[END_REF]). Vik1's crystal structure also shows that it has a fold similar to that of the kinesin catalytic core, while it lacks a functional ATP binding site [START_REF] Allingham | Vik1 modulates microtubule-Kar3 interactions through a motor domain that lacks an active site[END_REF]). The structure also points the conserved Gly373 Vik1 residue as a key residue for neck rotation (Fig. 7.2). It has suggested that the Kar3/Vik1 heterodimer may go through the same neck rotation as the Ncd homodimer (Cf. Introduction). The similarity between the Kar3/Vik1 heterodimer and the Ncd homodimer is also confirmed by the crystal structure of the Kar3/Vik1 heterodimer (Fig. 7.3).

To generate this structure, some artificial modifications were introduced in the heterodimer [START_REF] Rank | Kar3Vik1, a member of the kinesin-14 superfamily, shows a novel kinesin microtubule binding pattern[END_REF]. First, different lengths of stalk were tried to generate a soluble and stable heterodimer. Second, a Synthetic HeteroDimer (SHD), with an acidic helix and a basic one, was added to the stalk in order to increase the yield and stability of Kar3/Vik1 heterodimer. These two peptides of SHD are able to form a dimer with hydrophobic interaction, while those charged residues on SHD prevent them from forming homodimers. Third, an intramolecular cross-link was introduced in Vik1, preventing Vik1's globular domain from moving relative to its helix.

The ADP-bound Kar3/Vik1 heterodimer is in a conformation similar to that of the asymmetric Ncd (cf. Introduction), which likely represents the pre-powerstroke position instead of the post-powerstroke one. The pre-powerstroke conformation was further supported by docking the heterodimer structure into the cryo-EM map of microtubulebound Kar3/Vik1. ADP-Kar3/Vik1 heterodimer crystal structure, treated as a rigid body, was docked into both nucleotide-free and AMPPNP-Kar3/Vik1 decorated microtubule cryo-EM maps. The structure fitted well into the map of nucleotide-free Kar3/Vik1 bound to a microtubule. Docking of the Kar3/Vik1 heterodimer into the map of AMPPNP-Kar3/Vik1 decorated microtubule indicates there is a large structural rearrangement of Vik1 and the coiled-coil stalk. In ATP-analog-Kar3/Vik1, the stalk together with Vik1 rotates about 90° towards the minus end of microtubule [START_REF] Rank | Kar3Vik1, a member of the kinesin-14 superfamily, shows a novel kinesin microtubule binding pattern[END_REF]. A similar rotation in ATP-analog-Ncd with the presence of microtubule has also been observed by cryo-EM [START_REF] Endres | A lever-arm rotation drives motility of the minus-end-directed kinesin Ncd[END_REF]). Left: The structure of an asymmetric Ncd homodimer [START_REF] Liu | Neck-motor interactions trigger rotation of the kinesin stalk[END_REF] is shown here, with two ADP molecules (yellow) binding to two motor domains. The similarity between Kar3/Vik1 and asymmetric Ncd homodimer is obvious.

Distinct from Kar3 and other motor proteins, Vik1 doesn't have a nucleotide binding site. In addition a non-conventional binding of Vik1 to microtubules, different from that of kinesins, has been proposed (Fig. 7.1, bottom). Moreover, the rotation of Vik1's neck hasn't been observed so far at an atomic level. A Vik1 motor-homology domain-tubulin complex structure may address these pending issues.

As in the case of Ncd, the structural information of Kar3 in different nucleotide states complexed with tubulin will be helpful to understand the details of Kar3 heterodimer's motility and more generally to gain insights into the motility of (-)-end directed kinesin-14s.

So, my main goal in this part was to crystallize Kar3 or Vik1 or Ncd in complex with a tubulin heterodimer.

Constructs

Motor or motor-homology domain

In the case of Vik1, the motor-homology domain (Vik1 MHD, Fig. 7.2) whose crystal structure has been published [START_REF] Allingham | Vik1 modulates microtubule-Kar3 interactions through a motor domain that lacks an active site[END_REF]) was purified and a complex with tubulin was prepared.

In the case of Kar3 and Ncd, my main objective is to study the dimers. As mentioned in the introduction, kinesin-14s require the dimeric stalk for movement. But Kinesin-14 homodimers (in vitro Kar3 can form a homodimer as well [START_REF] Chu | Kar3 interaction with Cik1 alters motor structure and function[END_REF])) obviously have two microtubule binding sites, which may lead to the difficulty of co-crystallizing their complexes with tubulin. Therefore I have worked with Kar3 and Ncd heterodimers. Indeed previous experiments suggest that, as long as a dimeric stalk is formed, Ncd can still move at a normal speed even with only one functional motor domain (Fig. 7.4). Hence a onemotor kinesin-14 heterodimer construct is a suitable surrogate to study the kinesin-14 mechanochemical cycle. The two SHD chains are in black and white. Hydrophobic residues are shown in yellow; acidic and basic residues are in red and blue, respectively.

heterodimer which was crystallized finally. It suggests that the heterodimer is stable in the purification-crystallization process.

Afterwards, as the bigger chain of the kinesin-14 heterodimer is detected by SDS-PAGE, the small peptide is considered existing in the construct by default. However, the possibility that the small peptide may be lost later on cannot be ruled out.

Affinity to microtubules

To verify that the kinesin-14 heterodimers are functional, I first checked whether they bind microtubules and in which conditions the binding is favored. A centrifugation assay was used to analyze the interaction between microtubules and the Ncd heterodimers loaded with the ATP-analog, AMPPNP.

First, the binding ability of Ncd to microtubules was checked with various concentrations of microtubule (Fig. 7.6). The results suggested that in the presence of AMPPNP, Ncd is able to bind to microtubules tightly. In the presence of 2 mM AMPPNP, 4 M Ncd heterodimer alone or with 4 M microtubules were incubated at 25°C for 30 min at the indicated salt concentrations. The supernatant and pellet content were analyzed by SDS Page respectively after ultracentrifugation.

Then, I checked the influence of salt concentration because the stability of Ncd is slightly improved in the presence of salt. The results are presented in Fig. 7.7 and indicate that Ncd binds slightly less tightly to microtubules along with the increase of the salt concentration.

I have also tried to study the interaction without added nucleotide. To generate nucleotidefree Ncd heterodimer, the kinesin was incubated with 0.5 U/ml apyrase. After centrifugation all the motor protein went to the pellet even without microtubules having been added, indicating that spin down assay is not a proper method to study the interaction between nucleotide-free Ncd and microtubules. It also suggested that it may not be a good idea to generate an apo-kinesin-14 tubulin DARPin complex by a treatment with apyrase. As a result, despite the fact that the ADP dissociation from kinesin-14 is slower than that from kinesin-1, I prepared apo-kinesin-14-tubulin-DARPin complexes by simply mixing the components together without treating with apyrase, the same method which I used to generate an apo-kinesin-1-tubulin-DARPin complex which I was able to crystallize (Cao et al. 2014). The nucleotide state of the complex generated was analyzed to confirm in the complex the kinesin-14 doesn't bind an ADP. The results will be presented later.

Complex

Kinesin-14 in different nucleotide states was mixed with tubulin-DARPin complexes and the mixture was purified by a gel filtration chromatography. The gel filtration chromatography allowed me to 1) determine which DARPins are suitable candidates to make ternary complexes with kinesin-14 and tubulin; 2) have an idea on the motor protein:tubulin stoichiometry (a 1:1 complex is expected); 3) estimate the stability of the complexes.

Representative results are shown in Figures 7.8 and 7.9. They suggest that kinesin-14s, mentionned in the summary above, in different nucleotide states are able to form a complex with DARPin-stabilized tubulin with a ratio of 1 to 1 to 1 (kinesin-14 to tubulin to DARPin). However, kinesin-14 motor protein induces also some aggregation, which varies from batch to batch. So far, I haven't found a method to avoid the aggregation. For crystallization experiments, the main peak containing the expected complex was collected and concentrated. Top: gel filtration chromatography of Ncd (Hetero-Ncd 700) in different nucleotide states in complex with a tubulin-DARPin complex (tubulin-H10IV). The tubulin-DARPin complex was also injected as a control. Both apo-and AMPPNP-Ncd in complex with this tubulin-DARPin eluted at a similar position, earlier than tubulin-DARPin complex.

Bottom: SDS Page analysis of the AMPPNP-Ncd-tubulin-DARPin complex. The tubulin heterodimer (Het Tub), the Ncd motor domain and the DARPin are shown on the gel. The first two lanes correspond to the peak of aggregates (void volume of the column, about 8 ml).

Fractions corresponding to the second and main peak were analyzed as well. The results confirmed that the main peak is composed of Ncd, tubulin and the DARPin proteins with a ratio around 1 to 1 to 1. Bottom: SDS Page analysis confirmed the presence of Kar3 and tubulin (or Vik1 and tubulin) in the main peak (DARPin is not shown here) with a ratio about 1 to 1. For Kar3, there is still a part of the complex that came out as aggregation in the void volume of the column.

Nucleotide state analysis of the apo-ncd-tubulin-DARPin complex

I prepared the nucleotide-free kinesin-14 complex by mixing directly the kinesin construct with tubulin-DARPin, instead of treating kinesin-14 with apyrase to digest the nucleotide

Data 1

Apo Het Kar3 AMPPNP Het Kar3 Vik1

Volume (mL)

trapped in the motor. There are several reasons for this procedure. First, in the absence of microtubules or of tubulin the nucleotide-free kinesin-14 motor is very unstable and aggregates easily (see above). Second, although apyrase digests ATP and ADP in priority, it also hydrolyzes GDP and GTP. Therefore apyrase treatment cannot be performed on the complex because hydrolysis of GDP or GTP may destabilize the tubulin heterodimer. Finally, this procedure has worked in the case of kinesin-1.

However, although the same method was used to produce the apo-kinesin-1 tubulin DARPin complex, I wanted to check its efficiency in the case of a kinesin-14. The nucleotide content of a kinesin-14-tubulin-DARPin complex was checked by ion exchange chromatography (Fig. 7.10), after protein denaturation to release the bound nucleotide. The results indicated that the complex contains only GDP and GTP that come from tubulin. Therefore mixing kinesin-14 with tubulin-DARPin leads to a complex with nucleotide-free kinesin-14.

Crystallization

Kinesin-14 constructs in different nucleotide states were mixed with tubulin stabilized by different DARPins, then the complexes were purified by gel filtration chromatography as described above. Commercially available crystallization kits were used to screen for crystallization conditions of concentrated kinesin-14-tubulin-DARPin complexes. Some home-made kits were tried as well. However, whereas crystals have been obtained, they were of tubulin-DARPin complexes only and not of the ternary complexes.

As a means to increase the likelihood of obtaining crystals of kinesin-14-tubulin-DARPin complexes, I also digested tubulin with subtilisin. The C-terminal part of both tubulin subunits is flexible. Moreover, it is heterogeneous because of differences between tubulin isotypes and because of post-translational modifications. These features are a priori unfavorable for crystallization. These C-terminal tails can be removed by subtilisin limited proteolysis. Unfortunately it turned out to be unsuccessful as well. After several different DARPins and different commercial kits have been tried, there is no crystal of kinesin-14-DARPin in complex with subtilisin-treated tubulin has been obtained.

In addition I tried to crystallize Hetero-Kar3 in the AMPPNP state alone (without tubulin).

The initial idea was to observe the effect of ATP binding on the orientation of the stalk. Crystals were obtained in several conditions and a high resolution dataset was collected. However, molecular replacement with the Kar3 motor domain failed, suggesting that these were probably not crystals of Kar3. I tried to analyze the component of crystals by SDS Page, but nothing was observed on the gel. What crystalizes is probably either a protein contaminant or the motor-less peptide of the heterodimer. But this should be confirmed by further analysis.

Discussion

Despite a lot of attempts, kinesin-14s still have not been successfully crystallized in complex with tubulin (and a DARPin). This could be due to several reasons, including the quality of the kinesin-14 constructs and the quality of the kinesin-14 tubulin DARPin complexes.

SHD heterodimer stability

A successful case where Kar3/Vik1 heterodimer was crystallized with the help of SHD has been reported. The Kar3/Vik1 heterodimer was purified and crystallized at a pH varying from 7.5 to 8 [START_REF] Rank | Kar3Vik1, a member of the kinesin-14 superfamily, shows a novel kinesin microtubule binding pattern[END_REF].

Another previous study indicated that the SHD is sensitive to pH, with a lower stability at pH 5 compared to pH 7 (Fig. 7.11). In my case, crystals were usually obtained at pH below 7, which may have led to the destabilization of the kinesin-14 heterodimer. In addition, the K3 peptide is linked to a small part of Vik1 stalk, without a motor-homology domain. What crystallized is probably the self-polymerized K3 Vik1 peptide. More sensitive methods should be used to check the components in the crystals. In neutral solution, the two peptides of SHD form a stable heterodimer. But at lower pH, the heterodimer is less stable and E3 homotrimer, K3 monomers and dimers (not shown here) as well as E3/K3 heterodimers coexist in the same solution.

Quality of Kinesin-14 motor domain

Kinesin-14 motor domains are much less stable than the conventional kinesin-1 motor domain. First, they are unstable in the nucleotide-free state, as shown above. Moreover, without excess nucleotide kinesin-14 motors precipitate easily. Precipitates are also observed even with excess nucleotide. Second, the solubility of kinesin-14 motors is salt dependent. Although a spin down assay with 4 µM Ncd did not indicate a substantial difference in solubility according to the salt conditions (Fig. 7.6), at the protein concentration needed for crystallization experiments (usually more than 100 M), aggregates are observed when the concentration of salt is lower than 100 mM. Different salt or other additives may be tried to generate stable kinesin-14 motors.

Quality of Kinesin-14 in complex with tubulin-DARPin

Even in complex with tubulin-DARPin, kinesin-14 motors precipitate with its partner proteins. Both Kar3 and Ncd are able to form ternary complexes with tubulin-DARPin no matter the nucleotide state of the kinesin-14. But gel filtration chromatography indicates that some aggregates are formed. Even though the aggregates are separated from the 1:1:1 kinesin-14:tubulin:DARPin complex by gel filtration chromatography, aggregation may still happen later on, especially during concentration. The quality of kinesin-14-tubulin-DARPin complex should be further checked in order to know the stability of complex during the process of crystallizing screening, i.e. the complex should be analyzed by gel filtration after concentration, after 1 day incubation at room temperature and after a few days incubation.

The case of Vik1 seems more favorable. The gel filtration chromatography indicates that a ternary complex with tubulin-DARPin is formed; no obvious aggregation was detected: a single peak was observed (Fig. 7.9). This result suggested that the vik1-tubulin-DARPin complex is stable and homogeneous. Nevertheless, so far, no useful crystal of Vik1-tubulin-DARPin has been obtained.

I think more efforts can be tried to crystallize a kinesin-14 complex. For example, more covalent DARPin-kinesin constructs can be generated. As ATP-analog kinesin-1 in complex with tubulin-D2 has been crystallized, how D2 binds tubulin is known. D2-kinesin constructs can be designed based on this information in order to generate a stable kinesin-14-tubulin-D2 complex. Moreover, I may also try screening with the crystallization kits at 4 °C, considering the complex is unstable. Lowering the crystallizing temperature could increase the chances of crystals growth.

Step 1: One motor domain (A) in nucleotide-free state binds to microtubule, with another motor domain (B) in ADP state detached.

Step 2: ATP binds to apo-motor A, causing the subdomain movement identified in this thesis. The movement of subdomains creates a space, which is remote from the nucleotide binding site, in motor A, allowing the first residue of neck linker (I325) to dock into.

In the meantime, ADP-motor B binds to microtubule. Microtubule brings conformational changes to motor B. In the tubulin binding subdomain, binding to microtubule makes the H4 helix extend and the whole L11 loop become ordered. Conformational changes in the tubulin binding subdomain induce the change of other parts. Elongation of H4 helix clashing with Switch 1 leads to the melting of L9 loop. The residue E236 located on L11 can no longer anchor the P-loop by forming hydrogen bonds with P-loop residue T87. P-loop destabilization together with P-loop subdomain rotation interferes with ADP binding. On the other hand, rotations of P-loop subdomain and Switch 1/2 subdomain, separating T92 and D231, disturb the magnesium binding environment. ADP release is further accelerated.

Step 3: In motor A, I325, as a pin, preventing subdomains from rotating back to the apo-like conformation, stabilizes the conformation in an ATP-like state where ATP can be hydrolyzed efficiently.

Step 4: Pi is released, ADP-motor-A detaches from microtubule with a flexible neck linker. The kinesin homodimer is reset back to the step 1 but with one step further.

The structure of apo-kinesin-1 in complex with tubulin

As the intermediate state between isolated ADP-kinesin and ATP-kinesin bound to microtubule in the kinesin cycle, the atomic structure of apo-kinesin bound to a microtubule (or tubulin) is expected to give insights into the mechanism of microtubulestimulated ADP release, of the conformational changes accompanying ATP binding and leading to neck linker docking, and of ATP hydrolysis.

When I started this work, the structures of isolated ADP-kinesin (Kull et al. 1996;[START_REF] Sindelar | Two conformations in the human kinesin power stroke defined by X-ray crystallography and EPR spectroscopy[END_REF][START_REF] Sack | X-ray structure of motor and neck domains from rat brain kinesin[END_REF]) and of tubulin-bound ATP-like kinesin (Gigant et al. 2013) were known. These structures were very useful to precise the kinesin conformational changes upon microtubule binding, whose general features had been previously inferred from EM studies [START_REF] Sindelar | The beginning of kinesin's force-generating cycle visualized at 9-A resolution[END_REF][START_REF] Goulet | The structural basis of force generation by the mitotic motor kinesin-5[END_REF]. These data led somehow to the division of the kinesin motor domain into two subdomains: a tubulin-binding one and a second and main block comprising the Switch1/2 and the P-loop subdomains. Because these two subdomains rotate roughly along the same axis when going from microtubule-bound ATP-kinesin to isolated ADP-kinesin (see Fig. 3 of Article 1), they were not discriminated at that time. In addition, the elements that interact with microtubule undergo a substantial conformational change upon binding (this includes in particular the ordering of L11 and the lengthening of the H4 helix), that might have precluded the identification of more subtle changes. By contrast, in ATP-like-kinesin-tubulin and apokinesin-tubulin complexes, kinesin is always attached to tubulin, so the tubulin-binding subdomain does not vary much and the main variable is whether ATP is bound or not.

By comparing these two high-resolution tubulin-bound kinesin structures, the kinesin's three subdomains have been identified. ATP, located between the Switch 1/2 subdomain and the P-loop subdomain, is able to induce distinct rotations of these subdomains. Furthermore, the three-subdomain movement provides a structural explanation for the docking of the neck linker and for the efficiency of ATP hydrolysis in microtubule-bound ATP kinesin.

When ADP-kinesin and apo-kinesin-tubulin are compared, the nucleotide state is not the only variable. Having a structure of kinesin with nucleotide in the absence of tubulin and another one of tubulin-bound kinesin but without nucleotide, I found it's hard to isolate the conformational changes caused by tubulin binding or by nucleotide dissociation. Consequently, further studies were necessary to understand the kinesin's ADP dissociation mechanism and to dissociate the structural consequences linked to ADP release from those linked to microtubule binding. That's the reason I further did the mutagenesis study and structural study of isolated kinesin-1.

Moreover, it is worth trying to obtain the crystal structure of ADP-kinesin in complex with tubulin. As presented in the introduction part of this thesis, in kinesin-1's mechanochemical cycle, ADP-kinesin-1 binds to a microtubule, following which ADP is released. After ATP hydrolysis and the release of Pi, there is also a (transient) ADP-kinesinmicrotubule complex. It is therefore of interest to obtain such a structure. By comparing isolated and tubulin-bound ADP-kinesin, we can have direct information on how microtubule binding and ADP binding influences each other. Whereas the affinity between ADP-kinesin and microtubules is relatively low, the KD being about 10-20  (Cross 2004), and the ADP dissociation rate is high (about 50 s -1 ), using highly concentrated protein and high concentration of ADP, it might be possible to crystallize an ADP-kinesin-tubulin-DARPin complex. There are several EM structures of different ADP-kinesins bound to microtubule reported, indicating that it's possible to generate such stable ADP-kinesin microtubule complexes so that it might be also possible to have a stable ADP-kinesin tubulin complex (Atherton et al. 2014;[START_REF] Goulet | The structural basis of force generation by the mitotic motor kinesin-5[END_REF].

Another step of the kinesin cycle which could be studied structurally with apo-kinesintubulin crystals is the hydrolysis of ATP in crystallo. Because in this complex the kinesin is nucleotide-free, it might be possible to soak crystals with ATP. By freezing crystals in liquid nitrogen using different lengths of soaking time, theoretically, different stages of ATP hydrolysis can be observed. By analyzing these series of structures, we may obtain structural information from nucleotide-free kinesin accepting ATP up to ADP-kinesin tending to detach from tubulin. There are several prerequisites for this experiment to work. First, the hydrolysis rate there should be slow enough to trap an ATP state or some intermediate states. Then, the ATP molecule should be able to diffuse up to the kinesin nucleotide-binding site. Moreover the kinesin structural changes upon ATP binding should be compatible with the crystal packing.

Mutagenesis study on kinesin-1

A mutagenesis study on kinesin-1 was performed to precise further the ADP release mechanism in kinesin motor domain. Residues, predicted to be involved in the mechanism of microtubule-stimulated ADP dissociation based on structural studies, were mutated in order to mimic the effect of microtubule binding. Furthermore, structures of some mutants in nucleotide-free state were determined. These results indicate that microtubule binding interferes with ADP binding in kinesin mainly through 1) disturbing its magnesium binding environment and 2) breaking the anchor which stabilizes the P-loop.

Interestingly, during this study I found that isolated wild type kinesin-1 in nucleotide-free state can be generated by ADP digestion with apyrase. Furthermore, with the presence of extra salt in the solution, apo-kinesin-1 can be almost as stable as ADP-bound kinesin-1, although it was reported that apo-kinesin can denature easily [START_REF] Sadhu | A kinetic study of the kinesin ATPase[END_REF]Hackney 1994). Remarkably I crystallized apo-WT kinesin-1 in the conditions where apokinesin mutants have been crystallized and I obtained two apo-WT kinesin-1 structures, one of which has an ADP-like conformation and the other one is similar to that of tubulinbound apo-kinesin. These results suggest that, in solution, apo-kinesin probably alternates between these two conformations. One possibility is that kinesin-1 with the apo-like conformation is less stable, probably a short-lived state with a relatively open nucleotide binding pocket which facilitates nucleotide rebinding; while kinesin-1 with the ADP-like conformation is as stable as ADP-kinesin with solvent replacing nucleotide occupying the binding pocket, guaranteeing the stability of apo-kinesin-1. In my opinion, it's accessible and worthy to further investigate the states of apo-kinesin-1 in solution.

These results also raise the following question: could ADP-kinesin adopt an apo-like conformation? When kinesin switches to the tubulin-bound apo-like conformation, upon subdomain rotation, the E236 interaction with the P-loop is broken so that the P-loop becomes unstable. In addition the magnesium binding environment is also disrupted. As a result, ADP dissociates from kinesin. Nevertheless, without microtubule binding, there is no stimulus favoring the conformational change. Interestingly, in the case of the kinesin-3 Kif14, an "apo-like" conformation of isolated Kif14 was obtained with bound ADP (but without magnesium) (Arora et al. 2014). In this case, although the E236 equivalent residue still interacts with the ADP-bound P-loop, the loop where the glutamic acid is embedded is drawn with the movement of P-loop subdomain instead that P-loop is anchored by the glutamic acid. By contrast such a conformation has not been seen in the many other ADPkinesin structures that have been determined. Is this a specific feature of Kif14? To try answering this question, I soaked the "apo-like" apo-kinesin crystals with nucleotides. But after incubation with 1mM ADP or AMPPNP for about 12 hours, no signal could be attributed to a bound nucleotide in the electron density maps, suggesting that kinesin-1 may not share this Kif14 feature. Alternatively the crystal packing might have precluded the entrance of the nucleotide. Therefore, to confirm this result, if different apo-like apokinesin crystals are obtained, it will be interesting to try to soak them with nucleotide.

In my opinion, conformations of apo-kinesin and ADP-kinesin in solution should be checked by experiments. If possible, apo or ADP kinesin in solution may be simulated by dynamic simulation. It may provide supporting evidence to our theory on kinesin's ADP dissociation. It will bring an idea on how kinesin exchanges its nucleotide in a dynamic system as well.

Crystallization of kinesin-14 in complex with tubulin-DARPin

Kinesin-14s are peculiar because they move towards the microtubule minus end, a property shared by the dyneins whereas the other motile kinesins move towards the plus end. In addition they are the only members in the kinesin superfamily that have the motor domain positioned at the C-terminal end of the protein. Therefore a structure of tubulinbound kinesin-14 would be very useful to gain insights not only into how the movement in kinesin-14 is generated but also into the direction control in kinesin superfamily. Although substantial efforts have been made, kinesin-14s or its non-motor partner vik1 haven't been co-crystallized with tubulin-DARPin successfully yet. Several reasons could be invoked, including the instability of kinesin-14s per se and the instability of the complex.

Nonetheless I have shown that kinesin-14s are able to form complexes with tubulin-DARPin and that these complexes are stable enough to be purified by gel filtration. Based on these results, I think it's worth trying to obtain a kinesin-14-tubulin crystal structure. Among different possibilities that have not been tested so far, kinesin-14s different from Ncd or Kar3 could be tried. In addition, other DARPins could be used to stabilize the kinesin-14-tubulin complex for crystallization. Similarly, tubulin-stabilizing proteins targeting other tubulin surfaces compared to the current DARPins that bind the tubulin longitudinal interface could be tried as well. All these approaches should provide us with different kinesin-14-tubulin complexes, therefore with new chances to obtain useful crystals.

However, I think before more crystallizing conditions are screened and more DARPins are tried, the character of kinesin-14s and kinesin-14-tubulin-DARPin complexes should be further studied. Efforts should be made to find a condition where more stable homogeneous kinesin-14-tubulin-DARPin complexes can be formed and be maintained.

One option would be to generate additional kinesin-14-DARPin chimerical constructs, in which the two proteins are linked together. Beside from D1, D2 is another DARPin whose binding site on tubulin has been determined (Gigant et al. 2013). D2 forms a stable complex with tubulin heterodimer. It should be possible to design a D2-kinesin-14 covalent construct in order to generate stable and homogenous 1:1:1 tubulin:kinesin-14:D2 complexes.

Finally another possibility to study kinesin-14 interacting with microtubule would be through cryo-electron microscopy. Recent development on cryo-EM pushes the resolution for protein structure determined by this method to new levels. Especially, impressive progress has been achieved on EM studies of microtubule and microtubule associated proteins. Rui ZHANG et al. determined 3.5 Å resolution microtubule structures in different nucleotide states [START_REF] Zhang | Mechanistic Origin of Microtubule Dynamic Instability and Its Modulation by EB Proteins[END_REF]. Microtubule-bound kinesins in different nucleotide state have also been studied. The resolution achieved is in the 6 to 7 Å range (Atherton et al. 2014;Shang et al. 2014) and even an ADP-kinesin bound to microtubule has been studied at a 7 Å resolution (Atherton et al. 2014). A key feature in cryo-EM studies is the possibility to prepare homogenous samples for averaging. In particular, in the case of microtubuledecorated kinesins, microtubule should be saturated as much as possible and the kinesin molecules should be in very similar conformations. If homogenous samples can be prepared, the recent developments on cryo-EM provide an exciting and promising future for the structural study of kinesin-14 in complex with microtubule.

Materials and Methods

Differential Scanning Calorimetry assay on kinesin-1 and its mutants

Samples were prepared for Differential Scanning Calorimetry assay (DSC) by dialysis for the following goals. 1) to generate wild type and mutated kinesin (KHC 349) in nucleotidefree state, in addition to an ADP-bound wild type kinesin control; 2) to move the samples into a buffer whose pKa doesn't vary significantly with temperature, since during the experiment the system temperature increases; 3) to adjust the samples in the same condition.

1 l*200 U/ml Apyrase and 4 l * 0.25 M EDTA were added respectively into the T87A KHC 349, T87G KHC 349, T92V KHC 349 and WT KHC 349 samples with a volume about 200 l and a concentration around 200 M. Then, the samples were dialyzed against 400 ml buffer containing 30 mM NaH2PO4 pH 7.0, 150 mM NaCl, 0.5 mM EGTA, 1 mM MgCl2 with a Dialysis Cassette 10,000 MWCO (Thermo TM ) at 4 °C overnight. In the same condition, about the same amount of ADP-kinesin was dialyzed against the same buffer without Apyrase and EDTA added.

Samples were diluted until ~0.5 mg/ml before loaded to the Differential Scanning Calorimetry. The dialyzing buffer served as blank.

Purification of Kinesin-14 heterodimer

Constructs

Kar3 and Ncd

Two types of kinesin-14 are tried in co-crystalization assays with tubulin-DARPin complexes. One is Ncd, a Drosophila kinesin-14; the other is Kar3, the only kinesin-14 in Saccharomyces cerevisiae. Both of them are regarded as kinesin-14 models and well-studied biochemically. Ncd is a homodimer, having two catalytic cores locating at its C-terminus. Kar3 in cells works as a heterodimer with one catalytic core dimerized with a non-motor domain, Vik1 or Cik1.

For kinesin-14, the conformational change leading to movement is associated with its coiled-coil stalk rotation. So I designed kinesin-14 heterodimers containing one catalytic core plus a C-terminal part of dimeric coiled-coil. In other words, for ncd homodimer, one catalytic motor is truncated; for Kar3 heterodimer, the motor-homology domain of Vik1 is deleted (Fig. 9.1).

Because both Ncd and Kar3 are able to form homodimers in vitro [START_REF] Sablin | Direction determination in the minus-end-directed kinesin motor ncd[END_REF][START_REF] Chu | Kar3 interaction with Cik1 alters motor structure and function[END_REF], to generate the heterodimer, a synthetic heterodimer (SHD) is introduced at the N-terminus of the constructs, one half of the SHD in each chain [START_REF] Rank | Kar3Vik1, a member of the kinesin-14 superfamily, shows a novel kinesin microtubule binding pattern[END_REF]. The SHD containing an acidic helix and a basic helix is supposed to form a stable heterodimer with hydrophobic interactions between their paired leucine and isoleucine residues. For the short helix without motor domain, a His-tag is added to its N-terminal, followed by a cleavable TEV site, in order to purify the heterodimeric protein. The sequences of inserting fragments were checked with T7 (TAATACGACTCACTATAGGG), DuetUP2 (TTGTACACGGCCGCATAATC) and pET-RP (CTAGTTATTGCTCAGCGG) as sequencing primers. The positions where these primers bind to are shown in figure 9.2.

Ncd 679 stop

The codon of residue 680 in Ncd was mutated to a stop codon to generate a short Ncd construct by QuikChange II Site-directed Mutagenesis Method (Fig. 9.3). The same method was used to generate single mutations in Kinesin-1 as well.

A 45 bp DNA fragment, containing the target mutation in the middle of the sequence, and its reverse complement fragment are synthesized as primers for PCR amplification. A plasmid containing the mother construct is used as template. Here, both Hetero-Ncd 700 plasmid and Ncd 700 motor plasmid were used as templates. A pair of mutagenic primers containing the desired mutation is designed to produce linear fragment of the whole sequence of the plasmid. After treated by DpnI, the original plasmids are digested, while only the plasmid fragment produced by PCR is left. The linear fragment is transformed into competent cells directly. Normal plasmids with the desired mutation will be formed by nick repair in vivo.

PCR procedure:

Step 1: 95°C, 2 min

Step 2: 95°C, 30 s 55°C, 1 min 68°C, 6 min 30 s

For 20 cycles

Step 3: 68°C, 15 min

Step 4: 4°C, forever

The PCR products are purified with a GeneJET Gel Extraction and DNA Cleanup Micro Kit (Thermo Scientific TM ). Then, a fast digest enzyme DpnI is used to digest the initial template which contains methylated and hemi-methylated DNA. Fragments are purified again with GeneJET Gel Extraction and DNA Cleanup Micro Kit (Thermo Scientific TM ) and transformed into XL1-Blue competent cells by a heat shock reaction (45 seconds at 42 o C).

Sequence of the constructs containing target mutations are checked by sequencing the plasmid with T7 (TAATACGACTCACTATAGGG) as a forward primer and pET-RP (CTAGTTATTGCTCAGCGG) as a reverse primer.

Expression and purification

Transformation and Pre-culture

The competent bacteria (homemade) are transformed with the target plasmid by heat shock. E. coli Rosetta are used as competent cells because they contain plasmids (with chloramphenicol as selective antibiotics) for tRNA of rare codons as found in kinesin-14's sequence. 1 L plasmid is mixed with 100 L Rosetta and incubated on ice for 30 minutes. Then, a heat shock (45 seconds at 42 o C) is applied and afterwards the cells are incubated on ice immediately for about 2 minutes. Add 850 L preheated 2YT and incubate in a shaker (250 RPM, 37 o C) for about one hour. 2YT is an enriched medium which consists of Bacto Trypton 16 mg/mL, Yeast extract 5 mg/mL, NaCl 5 mg/mL. Streak at least 50 L on a LB agar plate containing antibiotics. 100 g/mL ampicillin and 25 g/mL chloramphenicol serve as antibiotics to select positive colonies for heterodimers (carried by pET-Duet1). Or 130 50 g/mL kanamycin and 25 g/mL chloramphenicol serve as antibiotics to select positive colonies for monomers (carried by pET-M11). The plates were incubated overnight at 37 o C.

A single colony was picked and pre-cultured in 20 mL LB liquid medium (10 mg/mL tryptone, 5 mg/mL yeast extract, 10 mg/mL NaCl) with relevant antibiotics. Pre-culture system is kept on a shaker with a speed of 200 rpm overnight at 37°C.

Culture and Inducement

The second day, 10 mL pre-culture cell is added into 1 L 2YT medium containing the same concentration of the same antibiotics as the pre-culture system. Cells are cultured on a shaker at 37°C (usually for about 3h) until its OD600 reaches 0.8-1.

0.5 mM IPTG is added to induce the expression of the target protein. The bacterial cultures are moved to 18°C overnight (about 16-18 hours).

Afterwards, cells are harvested by centrifugation 20 min at 5000g at 4°C.

Extraction

Cells are resuspended with 20 mL Lysis buffer containing Hepes pH 7.2 25 mM, NaCl 150 mM, DTT 1 mM, PMSF (Phenylmethylsulfonyl fluoride) 1mM and a tablet of EDTA free protease inhibitor cocktail (Roche TM ). Then, the cells are frozen and lysed using a French pressure cell press. DNase (1g/mL) is added after the sample has been thawed. Finally, the mixture is centrifuged at 30000g at 4 o C for one hour and the pellet is discarded. The supernatant is filtered with a 0.2 m filter right before injected into a column.

Purification

Kinesin-14s are initially purified by an affinity chromatography. The N-terminal His-tag allows kinesin-14s to bind to a Ni-column (HisTrap HP 5 ml, GE Healthcare TM ). The loading buffer contains Hepes pH 7.2 25 mM, MgCl2 2 mM, EGTA 1 mM, 150 mM NaCl, 20 mM Imidazole, 1 mM DTT and ATP 25 M. The elution buffer is prepared by adding 0.5 M Imidazole to the loading buffer. For the first purification step, the sample is eluted by a gradient elution (0% to 40% in 10 min). Target protein elutes at about 30% elution buffer. TEV (homemade) is added to the peak collected with an estimated ratio about 1:20 (w/w), in order to remove the His-tag. Then, the collected fractions mixed with TEV are dialyzed against the loading buffer overnight at 4 °C to dilute the imidazole in the solution.

For the second purification step, protein is injected to the same column. But this time, the flow through is collected. Small fragments with the cut His-tag and TEV and fragments with an uncut His-tag as well will be trapped on the column.

Protein purified is finally concentrated to 300-500 M and kept at -80°C.

50 mM, NaCl 1 M and Imidazole 15 mM; and the elution buffer contains Tris pH 8 50 mM, NaCl 100 mM and Imidazole 0.5 M. After loading the sample in loading buffer conditions, the column is washed with 100% high salt buffer. Then the DARPin is eluted with 50% elution buffer and the peak is collected. Finally, the sample collected is concentrated to a volume of 2 mL to improve the efficiency of gel filtration.

The gel filtration buffer contains K-phosphate pH 7.2 20 mM, EGTA 0.5 mM, MgCl2 1 mM and KCl 100 mM.

Purified DARPin is concentrated until the concentration reaches at least 500 M, then stored at -80°C.

Tubulin DARPin complex

Tubulin recycling

An aliquot of 500 L purified sheep tubulin in Mes pH 6.8 50 mM, GTP 0.1 mM and 1/3 Glycerol (v/v) at about 30 mg/mL is recycled by one cycle of "polymerizationdepolymerization" [START_REF] Castoldi | Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer[END_REF][START_REF] Dorleans | Studying drug-tubulin interactions by X-ray crystallography[END_REF]. A PD-10 column is then used to exchange the buffer to Pipes-K pH 6.8 50 mM, EGTA 0.4 mM, MgCl2 0.5 mM and GDP 10 M. This procedure is to recover fully functional tubulin.

Reaction

DARPin and tubulin are mixed with a ratio of 1.2 to 1, and the complex is stored in liquid nitrogen until being used.

Subtilisin-cleaved tubulin

Tubulin is treated with subtilisin protease VIII (Sigma TM ) so that the flexible C-termini of the two tubulin chains are removed. Subtilisin-cleaved tubulin is reported to have a higher homogeneity. Therefore, subtilisin treatment should be tried to increase the likelihood of complexes crystallization.

First, recycled tubulin at whatever concentration (usually 100 M) is diluted to guarantee a low salt condition (e.g. 12.5 mM Mes pH6.8, 0.25 mM MgCl2, 0.25 mM EGTA). GDP is added to reach a final concentration of 0.5 mM.

The tubulin solution is equilibrated at 25 °C about 5 min before subtilisin is added. The ratio between subtilisin:tubulin is 1:100 (W/W). Then the mixture of tubulin and subtilisin is incubated at 25 °C about 20 min. Usually, after the incubation, the solution becomes turbid.

PMSF is added to reach a final concentration of 0.1 % (W/V) in order to stop the reaction. The solution is incubated on ice for 5 min.

DARPin is added to subtilisin-treated tubulin, with a ratio about 1.2:1 (DARPin:tubulin).

The solution is still cloudy, due to some precipitated PMSF. Centrifugation 15000 rpm * 15 min is performed to get rid of the precipitant. Then the DARPin-bound cleaved tubulin is mixed with kinesin-14. The complex is further purified by a gel filtration chromatography.

Nucleotide state analysis

In theory, the nucleotide-free Kinesin-tubulin-DARPin complex shouldn't contain any ADP or ATP, while the two subunits of tubulin bind a GTP ( tubulin) and a GDP or a GTP ( tubulin), depending on the way tubulin has been prepared. The nucleotide content of the complex is analyzed by anion exchange chromatography on Mono Q (GE Healthcare TM ).

The nucleotides are detected by the absorbance at 253 nm.

Tris-HCl pH 8 20 mM is used as loading buffer, while elution buffer is the same as loading buffer supplemented with 1 M KCl. As a control, 500 L mixtures of ADP, ATP, GDP and GTP is injected and eluted by a 0 to 30% gradient of elution buffer in 15 minutes (with a flow 1 ml/min).

10 L concentrated Kinesin-tubulin-DARPin (Abs280 between 20 and 30) is diluted to 30 L so that the desalting microBiospin 6 column (Bio-Rad TM ) can be used to remove unbound nucleotide. The protein is then denatured by adding 0.5 L Trifluoroacetic acid at room temperature, which denatures the protein and releases nucleotides. The sample is centrifuged (15000g * 15 min) and the supernatant (free of denatured protein) is neutralized with KOH and injected. The sample is eluted by a 0 to 30 % gradient of elution buffer in 15 minutes (with a flow 1 ml/min).

Spin down assay

50 L Ncd (2 M or 4 M) is incubated with various concentrations of microtubules (from 0 to 4M) in the buffer containing 50 mM Pipes pH 6.8, 2 mM MgCl2, 1 mM EGTA, 2 mM AMPPNP at 25 °C for at least 30 min. 1 M Taxol is added to prevent microtubules from depolymerizing.

Then, supernatant and pellet are separated by ultracentrifugation at 85 000 rpm for 15 min at 35 °C. The pellet is resuspended in 50 L buffer.

The separated supernatant and the pellet are analyzed by SDS-PAGE respectively.

Figure 1 . 1

 11 Figure 1.1 General information on microtubules. (Conde and Caceres 2009) a) A microtubule is usually composed of 13 protofilaments, templated by the γ-tubulin ring complex. The straight arrow indicates the microtubule seam (where tubulins of adjacent protofilaments interact with a subunit of the other type).

Figure 1 . 2

 12 Figure 1.2 Structure of a tubulin heterodimer (Amos and Schlieper 2005) A tubulin heterodimer is colored based on different domains. The GTP binding domains (red) are responsible for nucleotide binding. The activation domain colored in blue provides the catalytic α-tubulin Glu 254 residue which participates in the hydrolysis of the GTP in the next βtubulin along a protofilament. The core helix (yellow) connects the two globular domains in each monomer. The C-terminal domain on the external surface is colored in green.

Figure 1 . 3

 13 Figure 1.3 Structure of a DARPin named D1.

Figure 1 . 4

 14 Figure 1.4 D1-and D2-tubulin structures. Tubulin heterodimers have been superimposed. α tubulins (magenta) and β tubulins (blue) in both structures are almost identical with a low r.m.s.d of about 0.4 Å. Although D1 (yellow) and D2 (green) have a similar structure and both contact β tubulin, they don't bind identically.

Figure 2 . 1

 21 Figure 2.1 Members of the kinesin superfamily (Verhey and Hammond 2009).

Figure 2 . 2

 22 Figure 2.2 Sequence alignment of the motor domain of some kinesin family members (Sablin et al. 1996).Residues with absolute conservation are marked in red; those with relatively lower (but still significant) conservative substitutions are marked in yellow. The positions of secondary structure elements are pointed, as well as the four nucleotide binding motifs (N-1 to N-4). Kinesins from different subfamilies and from different species are listed here as examples.

Figure 2 . 3

 23 Figure 2.3 Structure of a rat-brain kinesin (Sack, Kull, and Mandelkow 1999).

Figure 2 . 4

 24 Figure 2.4 Domain structure of a myosin V (Hammer and Sellers 2012).

Figure 2 . 5

 25 Figure 2.5 Comparison of the motor domains in kinesin and myosin (Rayment 1996). a) The kinesin motor domain. Central β strands are colored in green, with α helices (purple) surrounding. ADP is shown in balls and sticks. b) A subfragment of a chicken skeletal myosin in nucleotide-free state. A solvent (spacefilling model in red and yellow) replacing ADP locates at the nucleotide binding site. In the motor domain, secondary structural elements similar to those found in kinesin are colored in the same pattern.

Figure 2 . 6

 26 Figure 2.6 Processive movement of myosin Va motor (Hammer and Sellers 2012).

Figure 3 . 1

 31 Figure 3.1 An artistic view of Kinesin-1 transporting cargoes along a microtubule (Johnson, G., et al. 2000).

Figure 3

 3 Figure 3.2 MANT-ADP (2'-(or-3')-O-(N-Methylanthraniloyl) Adenosine 5'-Diphosphate, Na Salt).

Figure 3 . 3

 33 Figure 3.3 Kinesin's association with microtubules is coupled with its nucleotide cycle.

Figure 3 . 4

 34 Figure 3.4 Different models of kinesin-1's motility (Yildiz et al. 2004).

Figure 3 . 5

 35 Figure 3.5 Kinesin-1 monomers and processive dimers' motility models (Rice et al. 1999).The tubulins' two subunits are colored in light and dark blue. The motor domain is colored either in yellow, when it is in microtubule-bound and nucleotide triphosphate states; or in red, when it is in other states. The D marked on the motor domain refers to ADP binding; the T refers to ATP binding; and the DP refers to ADP-Pi state. A cysteine (Cysteine 333, in the neck linker, shown as a green spot) has been introduced for biophysical probes labeling.

Figure 3 . 6

 36 Figure 3.6 ADP bound human kinesin-1 (KHC) motor domain (pdb id 1BG2, rainbow colored from N-ter to C-ter) (Kull et al. 1996). a) The neck linker which is located at the C-terminal of KHC motor domain is disordered and is not shown. The nucleotide binding motifs (Switch1, Switch2, P-loop and N-4) are labeled to indicate their locations. They define the environment of the nucleotide. Part of loop 11 (L11) in which Switch2 is embedded is disordered (shown as a dashed line).

Figure 3 . 7

 37 Figure 3.7 ADP bound human kinesin-1 (KHC) motor domain (pdb id 1MKJ, rainbow colored from N-ter to C-ter)(Sindelar et al. 2002).

Figure 3 . 8

 38 Figure 3.8 ATP analog bound human kinesin-1 (KHC) motor domain complex with tubulin heterodimer (pdb id 4HNA, rainbow colored from kinesin's N-ter to C-ter) (Gigant et al. 2013). a) Loop L11 in which Switch 2 is embedded becomes ordered, extending in particular the H4 helix at its N-terminal end. The conformation of Switch 1 changes as well to contact the γ phosphate. As expected, the neck linker is docked on the catalytic core.

Figure 3 . 9

 39 Figure 3.9 Tubulin-kinesin binding interface (pdb id 4HNA) (Gigant et al. 2013).

Figure 3 .

 3 Figure 3.10 Kinesin's cycle.

Figure 4 . 1

 41 Figure 4.1 NCD homodimer in ADP state. a) Wild type Ncd homodimer structure[START_REF] Sablin | Direction determination in the minus-end-directed kinesin motor ncd[END_REF].The motor domain of Ncd is similar to that of kinesin-1. Without being bound to a microtubule, the wild type Ncd homodimer is symmetric. Two ADP (yellow)-bound motor domains connect to each other with their N-terminal necks.

Figure 4 . 2

 42 Figure 4.2 Motility model of NCD homodimer a) A cryo-EM study on the microtubule-bound Ncd homodimer indicates that a neckrotation is caused by ATP binding[START_REF] Endres | A lever-arm rotation drives motility of the minus-end-directed kinesin Ncd[END_REF]. When ADP-Ncd motor (blue) binds a microtubule, its neck (red) points toward the microtubule plus end. Then the motor releases its ADP and is able to bind ATP. ATP binding leads a rotation of the neck (yellow) toward the microtubule minus end. After ATP is hydrolyzed, the neck rotates back to its original position (red) along with motor dissociation from the microtubule. Tubulin heterodimers are colored in green and white.

Figure 5 . 1 A

 51 Figure 5.1 A general gating framework of kinesin dimer's mechanical state (Andreasson et al. 2015a). A) A summary on kinesin mechanochemical cycle. (α) The front head (red) binds to microtubule and has released its ADP. (β1) The nucleotide-free (Ø) front head attached to microtubule is able to accept ATP. ATP binding leads to the partial docking of neck linker. (β2) Afterwards ATP hydrolysis completes the neck linker docking and enables the rear head (blue) to bind the forward binding site. (γ)Microtubule binding stimulates the ADP dissociation in the new leading head; meanwhile, in the former leading head, phosphate release and motor domain dissociation happens. The dimer goes back to ATP-waiting state (α), with the former rear head moving about 16 nm forward and taking the leading position. B) A general gating framework based on kinesin's mechanochemical cycle. Stepping, binding and unbinding gates are shown. When the front head first binds to microtubule, the stepping gate prevents it from unbinding and promotes the rear head

Figure 5 . 2

 52 Figure 5.2 Positions of subdomains and connectors in different myosin states (Coureux et al. 2003).

Figure 5 . 3

 53 Figure 5.3 Comparison between subdomains in kinesin and in myosin (Wang et al. 2015)

Figure 5 . 4

 54 Figure 5.4 Distortion of the central β sheet upon ATP binding in myosin and kinesin. (Wang et al. 2015) Distortion of the central β-sheets upon ATP-binding in myosin and in kinesin is presented here. For myosin, β strands (sheet 1-4, in orange) of N-terminal subdomains are superimposed, and strands of the upper 50 subdomains are colored in cyan (ATP-like structure, pdb id 1W7J

  (a) Kinetic parameters are given as value ±s.e. (calculated from the fit).

  Figure legends.

Figure 1 .

 1 Figure 1. The interference of kinesin-1 mutations with ADP binding and release. (A) The T92V

Figure 2 .

 2 Figure 2. ADP-kinesin like structure of nucleotide-free kinesin-1. (A) Superposition of ADP-

Figure 3 .

 3 Figure 3. Isolated apo-kinesin-1 in a tubulin-bound apo-kinesin-1 conformation. (A) Apo-T87A

Figure . 1 Figure. 2 91

 12 Figure .1

Figure 6 . 2

 62 Figure 6.2 DSC analysis of kinesin-1 without or with the presence of ADPKinesin were treated with apyrase to generate apo-kinesin. The stability of apo-kinesin and ADP-kinesin are analyzed by differential scanning calorimetry parallelly. Compared to apokinesin, ADP-kinesin has higher melting temperature (Tm) and higher enthalpy of transition (H). It means ADP-kinesin can survive higher temperature and consume more energy to denature. These results indicate that ADP-kinesin is more stable than apo-kinesin.

Figure 6 . 3 A

 63 Figure 6.3 A model of kinesin-1 heterodimer in complex with tubulin tetramer.

Figure 7 . 1

 71 Figure 7.1 Motility model of Kar3/Vik1 heterodimer (Rank et al. 2012) α/β tubulin heterodimers are colored in light and dark blue. Step 1, Vik1 binds to a microtubule, leading ADP-Kar3 to attach to the same microtubule too. Step 2, microtubulebound Kar3 releases its ADP; meanwhile a strain with the coiled-coil is produced so that Vik1's tight contact with microtubule is ruined. Step 3, ATP binds nucleotide-free Kar3, causing a rotation of the stalk. Step 4, ATP hydrolysis occurs and ADP-Kar3 dissociates from microtubule. Previous studies also suggest that Kar3 motor domain and Vik1 bind to adjacent protofilaments with 'opposite orientations' on the lattice as shown in the bottom figure.

Figure 7 . 2

 72 Figure 7.2 Comparison of the neck rotation in Ncd and Vik1 (Allingham et al. 2007). The position of the neck (red) in Vik1 and the two different possible neck (red and blue) positions in Ncd are shown. For Ncd, the two motor domains' Cαs of a Ncd mutant, N600K, with an asymmetric conformation (Cf, Introduction (Yun et al. 2003)) were superimposed. For Vik1, the Cαs of Vik1's Motor-Homology Domain (MHD) without neck were superimposed with the motor domain of Ncd, but Ncd is not shown. The Gly373 in Vik1 and its equivalent residue in Ncd (Gly347) are highlighted.

Figure 7 . 3

 73 Figure 7.3 Kar3/Vik1 heterodimer (Rank et al. 2012) Right: Kar3 motor domain is colored in dark blue with a bound ADP molecule (yellow). Vik1 kinesin-like domain without a nucleotide binding site is colored in magenta. Kar3 and Vik1 are dimerized with a coiled-coil stalk. Because Kar3 can form homodimer in vitro[START_REF] Chu | Kar3 interaction with Cik1 alters motor structure and function[END_REF], an artificial heterodimerized leucine zipper (colored in cyan and wheat) is introduced to yield a Kar3/Vik1 heterodimer. In Vik1, an iodoacetamide based reagent (EBI) was used as a symmetric covalent cross-link between E355C and K423C (Red) in order to favor crystallization and improved crystal diffraction.

Figure 7 . 4

 74 Figure 7.4 Different Ncd constructs and their characterization (Endres et al. 2006). Top: Domain organization of full length Ncd. Ncd comprises an N-terminal tail, a coiled-coil stalk, a C-terminal catalytic core and a short neck linking the stalk and the catalytic core. Bottom: ATPase activity and microtubule gliding velocity of an Ncd monomer, a one-motor heterodimer and of a homodimer. N281, comprising Ncd residues 281 to 700, forms a homodimer with two motor domains; N280_Het refers to an Ncd Heterodimer construct, with one chain comprising one motor domain (Ncd residues 281 to 700), the other chain being restricted to the C-terminal part of the stalk and to the neck region (residues 281 to 347); N325 (Ncd residues 325 to 700) is monomeric. The constructs' general structures are indicated by blue triangles (catalytic core) and orange lines (neck plus the C-terminal part of the stalk). The results indicate that a full length motor domain is responsible for ATP hydrolysis whereas a dimeric stalk with one motor domain only is necessary and sufficient for kinesin-14's movement.

Figure 7 . 5

 75 Figure 7.5 Synthesized Heterodimer (SHD)(Litowski and Hodges 2002).

Figure 7 . 6

 76 Figure 7.6 Ncd binds to microtubules (MT).

Figure 7 . 7

 77 Figure 7.7 Ncd binding to microtubules (MT) under various salt concentrations.

Figure 7 . 8

 78 Figure 7.8 Ncd complex with tubulin stabilized by a DARPin (H10IV).

Figure 7 . 9

 79 Figure 7.9 Hetero-Kar3 in different nucleotide states or Vik1 in complex with tubulin-DARPin.Top: the gel filtration chromatography profile of Hetero-Kar3 in different nucleotide states in complex with tubulin and a DARPin (named H5II) is presented. The elution volumes of apoand AMPPNP-Hetero Kar3 in complex with tubulin-DARPin are similar. By comparison, the peak of Vik1-Tubulin-DARPin complex is slightly shifted, as expected because Vik1 is smaller than Kar3 and does not comprise the hetero coiled-coil.

Figure 7 . 10

 710 Figure 7.10 Nucleotide analysis of an "apo-"ncd tubulin-DARPin complex. Left: control with a mix of ADP, ATP, GDP and GTP injected to a Mono Q column and eluted with a salt gradient. Right: nucleotide content analysis of apo-ncd-tubulin-DARPin complex. By comparing the positions of peaks in the upper and lower figures, I could figure out the nucleotide component in an apo-ncd tubulin DARPin complex.

Figure 7 .

 7 Figure 7.11 SHD conformation changes according to the pH. (Apostolovic and Klok 2008)

  Moreover, for Ncd, two different lengths of motor domain are tried, Ncd 700 stop and Ncd 679 stop. One is the full length Ncd motor domain; in the other construct, the C-terminal part of Ncd (from 680 to 700), which is not observed in the crystal structures of either wild type ADP-Ncd or Ncd mutants which mimic Ncds in different nucleotide state, is truncated to facilitate crystallization.

Figure 9 . 1 A

 91 Figure 9.1 A model of Kar3 heterodimerKar3's catalytic motor domain and a part of its N-terminus stalk, both colored in orange, are dimerized with Kar3's non-motor partner Vik1 (grey) by forming a coiled-coil. The motorhomology domain of Vik1 is deleted; otherwise, this domain is able to bind another tubulin heterodimer. To improve the yield of heterodimer, as Kar3 can form a homodimer in vitro, a synthetic heterodimer (SHD) is introduced into the construct. An acidic helix (green) and a basic helix (yellow) in a SHD tend to bind each other and reinforce the helix-helix interaction.

Figure 9 . 3

 93 Figure 9.3 Overview of the QuikChange II Site-directed Mutagenesis Method (Agilent Technology TM ).

this figure, the motors of kinesin are colored in different colors to indicate the movement of each one. Although in both models, kinesin moves about 8.3 nm for each step; in hand-over- hand model, a single motor moves about 16.6 nm as a step, while in inchworm model, a step of a single motor is about 8.3 nm. Yildiz, et al. labeled one motor with a dye to observe the movement

of a single motor.

Table 1 .

 1 . Data collection and refinement statistics are reported in 31. Bricogne G, Blanc E, Brandl M, Flensburg C, Keller P, Paciorek W, Roversi P, Sharff A, Smart OS, Vonrhein C, et al. (2011) BUSTER version 2.10.0 Cambridge, United Kingdom: Global Phasing Ltd., 32. Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Mant-ADP release rate from kinesins, estimated by fluorescence spectroscopy(a) .

	Acta Crystallogr D Biol Crystallogr 66: 486-501
	33. Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW,
	Kapral GJ, Grosse-Kunstleve RW, et al. (2010) PHENIX: a comprehensive Python-based
	system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66: 213-
	221
	34. DeLano WL (2010) The PyMOL Molecular Graphics System.
	Construct	Wild-	Q86A	T87S	T87A	T87G	Q86A	T92S	T87A	E236A
		type					T87A		T92S
	koff (s -1 )	0.034	0.14	0.32	2.77	4.8	3.5	0.33	19	1.43
		±0.001	±0.001	±0.002	±0.037	±0.13	±0.065	±0.005	±0.97	±0.017

  [START_REF] Hackney | Evidence for alternating head catalysis by kinesin during microtubule-stimulated ATP hydrolysis[END_REF]

	B factors
	Protein	29.6	33.7	49.5	57.7	58.3
	Ligand/ion	68.6	99.5	90.6	117	103
	Waters	32.2	49.2	37.7	45.2	47.5
	Coordinate error (Å)	0.26	0.22	0.43	0.40	0.48
	R.m.s.d.
	Bond lengths (Å)	0.010	0.007	0.009	0.009	0.010
	Bond angles ()	1.09	1.08	1.41	1.40	1.50
	Ramachandran (%)
	Favored region	100	98.64	95.94	95.40	93.26
	Allowed region	0	1.36	3.17	33.6	5.15
	Outliers	0	0	0.89	1.3	1.59
	Rmeas	0.20 (1.38)	0.17 (1.38)	0.29 (1.15)	0.23 (1.61)	0.32 (1.68)
	I / I	4.8 (1.2)	9.2 (2.0)	3.8 (1.3)	6.8 (1.9)	4.5 (1.2)
	CC1/2	98.6 (40.6)	99.4 (67.1)	96.7 (49.0)	98.5 (60.3)	96.4 (30.8)
	Completeness (%)	99.7 (95.9)	99.9 (99.9)	97.3 (91.0)	98.0 (97.6)	96.5 (80.1)
	Multiplicity	3.7 (3.2)	6.4 (6.5)	2.9 (2.6)	3.5 (3.6)	3.4 (3.3)
	Refinement
	Resolution (Å)	28.5-1.95	44.9-2.0	33.2-2.59	38.4-2.60	49.28-2.88
	No. reflections	53434	26062	59275	59440	43981
	Rwork / Rfree	0.214 / 0.234 0.170/0.213 0.197/0.274 0.187/0.258 0.188/0.285
	No. atoms
	Protein	4874	2337	13771	13860	13923
	Ligand/ion	53	44	40	55	60
	Water	278	219	200	319	250
			92 93

(a)

Data were collected on a single crystal. Values in parentheses are for the highest-resolution shell.

Table 6 .1 DSC results of wild type kinesin and kinesin mutants

 6

		T m 1 (°C)	H1 (Kcal/mol)	T m 2 (°C)	H2 (Kcal/mol)
	ADP-WT	43	64	44.9	45
	Apo-WT	39.4	46	41.8	31
	Apo-T92V	34.3	38	36.4	22
	Apo-T87G	36.1	39	38.1	25
	Apo-T87A	35.7	39	37.7	30

The structural switch of nucleotide-free kinesin Kinesin-1 is a dimeric ATP-dependent motor protein that moves towards microtubules (+)-ends. Whereas structures of ADP-kinesin and of two forms (nucleotide-free and complexed to an ATP analog) of a monomeric construct bound to tubulin have been determined, structures of apo-kinesin-1 in the absence of tubulin are still missing. Therefore, the role of nucleotide release in the structural cycle of kinesin-1 is left unsettled. Here we identified mutations in the kinesin P-loop nucleotide-binding motif that led to kinesin unable to bind ADP (T92V mutant) or releasing nucleotide 100-fold faster than the parental construct (T87A mutant). The structures of these two single amino acid mutants in their apo form were either isomorphous to ADP-kinesin-1 or to tubulin-bound apo-kinesin-1. Remarkably, both structures were also obtained from the nucleotide-depleted wild-type protein. Our results lead to a model in which, detached from microtubules, apo-kinesin alternates between the two conformations we characterized, whereas microtubule binding stabilizes the tubulin-bound apo-kinesin one. This conformation is primed to bind ATP and, therefore, to run through the natural nucleotide cycle of kinesin-1.

Keywords: Intracellular transport/Kinesin/Nucleotide release/P-loop/Structure Apo-kinesins in an ADP-kinesin like conformation.

We first determined the 2.0 Å resolution structure of apo-T92V in a P21 space group crystal form (Supplementary Table 1). There were two similar molecules in the asymmetric unit, with a root mean square deviation (r.m.s.d.) after superposition of 0.47 Å (305 Cαs compared). The overall conformation of the protein is like that of ADP-kinesin-1 (r.m.s.d. = 0.72 Å over 300 Cαs compared to the 1BG2 pdb data set (Kull et al. 1996), averaged value for the two molecules of the T92V asymmetric unit) (Fig. 2A) and much more different from the conformation of tubulin-bound apo-kinesin-1 (r.m.s.d. = 1.37 Å over 296 Cαs compared to kinesin in the 4LNU pdb data set (Cao et al. 2014)) (Supplementary Fig. 2).

Compared to ADP-kinesin-1, the three nucleotide binding motifs are conserved, as expected from the small deviation between the two structures: the Switch 1 and P-loop motifs superimpose very well, whereas a part of the L11 loop, where the Switch 2 motif is embedded is disordered in both structures (Fig. 2A). However, in apo-T92V, helix α4, which is C-terminal to L11, is longer than that of ADP-kinesin-1 (pdb id 1BG2). Whereas in ADPkinesin-1, the N-terminal part of α4 is disordered up to residue K256, in apo-T92V, residues are ordered since D249 resulting in a two helix turns longer α4 (Fig. 2A). In this case, the elongation of α4 is likely to be due to a crystal contact. α4 is nevertheless shorter than in tubulin-bound kinesin-1, whatever the nucleotide state, where it starts at position 246 (Cao et al. 2014;Gigant et al. 2013). Finally, whereas T92 interacts with D231 in wildtype ADP-kinesin-1 (Kull et al. 1996) (Supplementary Fig. 1), this hydrogen bond is not formed in the T92V mutant, as expected (Fig. 2B). Supplementary Fig. 3). Whereas most of the following analysis was performed taking the apo-T87A structure as a reference, identical conclusions could be reached from these wildtype apo-kinesin and T92V isomorphous structures.

The r.m.s.d. of Cαs of the apo-T87A kinesin-1 compared to ADP-kinesin-1 (pdb id 1BG2) varies between 1.2 Å and 1.5 Å. By contrast, the conformation of apo-T87A is much more similar to that of apo wild-type kinesin-1 bound to tubulin (pdb id 4LNU), the r.m.s.d. of Cαs being close to 0.8 Å whatever the apo-T87A molecule considered. Consistently, the secondary structure elements of both proteins superimpose very well (Fig. 3A) and very similar subdomain rearrangements are seen in both proteins as compared, for instance, to ADP-kinesin (Supplementary Fig. 2). The conformations of the three nucleotide-binding motifs are consistent with this scheme. In particular, the structure of the P-loop motif, which is part of the P-loop subdomain, does not change significantly between ADP-kinesin and apo-T87A or upon binding of kinesin-1 to tubulin. The structures of the Switch 1 and Switch 2 motifs, the N-terminal part of the former and the C-terminal part of the later not belonging to any of the subdomains (Cao et al. 2014), may differ in the kinesin-1 structures that have been determined. This is the case of a small portion of the L11 loop, a loop that comprises the Switch 2 motif, which is disordered in apo-T87A whereas interactions with tubulin stabilize it in the complex of apo-kinesin-1 with tubulin (Fig. 3B). By contrast, the tip of the L9 loop (the loop to which Switch 1 belongs to) is disordered both in T87A and in the initially determined apo-kinesin-1 structure (Cao et al. 2014).

There are two main additional differences between the structures of apo-T87A and that of apo-kinesin-1 bound to tubulin. The first one concerns residue E236, the last residue of the Supplementary Table 1. Figures of structural models were generated with PyMOL (DeLano 2010).

Nucleotide content analysis

T92V (60 µl at about 350 µM) and wild-type (100 µl at about 200 µM) kinesins were denatured with 1 µl Trifluoroacetic acid. After removal of the denatured protein by centrifugation, the pH of the supernatant containing released nucleotide was neutralized and the supernatant was loaded on a Mono Q column (GE Healthcare) equilibrated with 20 mM Tris-HCl pH 8.0. The nucleotide was eluted using an NaCl gradient. ADP and ATP solutions were injected as references to calibrate the column.

Mant-ADP release rate measurement

As a means to evaluate the ADP release rate [START_REF] Cheng | Interaction of mant-adenosine nucleotides and magnesium with kinesin[END_REF], kinesin constructs were loaded with mant-ADP and the fluorescence decrease associated with mant-ADP dissociation from kinesin was monitored using a SX20 stopped-flow spectrometer (Applied Photophysics) at 25°C. Specifically, kinesin was first desalted using a Micro Bio-spin 6 column (BioRad) equilibrated with 25 mM Pipes pH 6.8, 2 mM MgCl2, 1 mM EGTA and 1 mM DTT to remove excess nucleotide. Then kinesin was incubated with a fourfold excess of mant-ADP on ice for about one hour. In the case of the mutants, because the release of ADP could be very fast, the excess mant-ADP was not removed and 0.5 μM kinesin was mixed with an equal volume of a 1 mM ADP-containing buffer in the stoppedflow cell, resulting in an ADP:mant-ADP ratio of 500:1. The fluorescence intensity (excitation at 355 nm) was recorded using a 400 nm long-pass filter. The experimental data were fitted to an exponential decay to yield the dissociation rate constant koff. remains flexible, in tubulin-bound apo-kinesin, this stretch interacts with tubulin and L11 is fully ordered. (C) The side chain of residue E236 is disordered in apo-T87A. In the complex with tubulin of apo-kinesin-1, E236 is ordered and interacts with the Switch 1 residue R203. In T87A, R203 stays at a position similar to that in tubulin-bound apo-kinesin-1 and establishes a hydrogen bond with E250 in α4, but the E236 side-chain is disordered beyond C. (D) The Q86 interaction changes in apo-kinesin-1. The structures of nucleotide-depleted wild-type kinesin-1 in the ADP-kinesin like conformation (pink) and in the tubulin-bound apo-kinesin like conformation (green) have been superimposed (taking the P-loop subdomain as a reference).

Supplementary Figure 1. View of the ADP-kinesin nucleotide-binding site (pdb id 1BG2).

Supplementary Table 1. Data collection and refinement statistics for apo-kinesin structures.

ADP-kinesin like form

Tubulin-bound apo-kinesin like form T92V Wild-type T87A Wild-type T92V

Data collection (a) Space (a) Averaged over the 6 molecules of the asymmetric unit.

(b) Averaged over the 2 molecules of the asymmetric unit.

(c) Residues 84 to 93.

Crystallization of kinesin-14 in complex with tubulin-DARPin 3.1 Objectives

To study the motility of kinesin-14, my objective is to obtain the crystal structure of a kinesin-14 in different nucleotide states complexed with a tubulin heterodimer, as presented in the introduction. I have worked with several constructs based on two wellstudied kinesin-14 (Ncd from Drosophila and the yeast Kar3).

Ncd, as a representative of kinesin-14 has already been presented in the introduction of this these. Here, I will give a brief introduction on Kar3.

Kar3 is the only kinesin-14 member found in Saccharomyces cerevisiae. During karyogamy and mitosis, as Ncd, Kar3 is able to crosslink microtubules with its C-terminal motor domain bound to one microtubule and its N-terminal ATP-independent tail bound to another one.

Although in vitro Kar3 can form a homodimer as Ncd [START_REF] Chu | Kar3 interaction with Cik1 alters motor structure and function[END_REF], in vivo it heterodimerizes with either Vik1 or Cik1 selectively to achieve distinct functions [START_REF] Gonzalez | Common mechanistic themes for the powerstroke of kinesin-14 motors[END_REF]). Vik1 and Cik1 are two kinesin-related proteins (they share at least 25% amino acid sequence identity [START_REF] Manning | Differential regulation of the Kar3p kinesin-related protein by two associated proteins, Cik1p and Vik1p[END_REF])) but they are unable to bind a nucleotide. Kar3/Vik1 localizes at the poles of the mitotic spindle, crosslinking parallel microtubules, involved in spindle stabilization, whereas Kar3/Cik1 is capable of crosslinking antiparallel microtubules in the overlap zone, contributing to the spindle mid region geometry stabilization.

Previous studies indicated that both Kar3/Vik1 and Kar3/Cik1 go through a similar power stroke as the Ncd homodimer. The non-motor partner, Vik1 or Cik1 participates in the motile cycle directly by modulating the binding of the Kar3 motor domain to microtubules. ADP-Kar3 motor alone has a relatively low affinity for microtubules (KD = 233 nM), while Vik1 head domain has a much higher affinity (KD = 43 nM), similar to that of ADP-Kar3/Vik1 heterodimer (KD = 38 nM). This suggests that the binding of ADP-Kar3/Vik1 to microtubules is first guided by the non-motor partner [START_REF] Allingham | Vik1 modulates microtubule-Kar3 interactions through a motor domain that lacks an active site[END_REF]. A kinetic study on Kar3/Cik1 leads to the same conclusion [START_REF] Chen | Kinesin Kar3Cik1 ATPase pathway for microtubule cross-linking[END_REF]. A model of Kar3/non-motor partner motility cycle is summarized in the following figure (Fig. 7.1).

The heterodimer was constructed by adding at the N-terminal end of the monomers one or the other of the two following SHD sequences:

Chain A: EIAALEKEIAALEKEIAALEKEI (also called E3)

Chain B: KIAALKEKIAALKEKIAALKEKI (also called K3)

They are able to dimerize by hydrophobic interaction (Fig. 7.5) whereas the homodimer formation is prevented because of the presence of charged residues. The same SHD was used to dimerize Kar3 and Vik1 to generate a Kar3-Vik1 dimer whose structure has been determined [START_REF] Rank | Kar3Vik1, a member of the kinesin-14 superfamily, shows a novel kinesin microtubule binding pattern[END_REF].

Fusion with AC2

As in the case of tubulin-kinesin-1 complexes, tubulin in complex with a kinesin-14 should be stabilized for crystallization experiments. Making ternary tubulin-kinesin-DARPin complexes is a suitable approach that has worked in the case of kinesin-1. In addition, in the case of kinesin-14 kinesins, I have designed DARPin-kinesin covalent constructs as a means to produce more stable complexes with tubulin. To this end, repeats of G4S (Gly-Gly-Gly-Gly-Ser) were used to link the C-terminus of the Kinesin-14 motor domain to the Nterminus of the DARPin named AC2, which has a high affinity for tubulin (KD in the nanomolar range) [START_REF] Ahmad | Destabilizing an interacting motif strengthens the association of a designed ankyrin repeat protein with tubulin[END_REF]). In addition, because the tubulin-AC2 structure has been determined, I am able to model the ternary complex in order to optimize the length of the G4S linker.

Summary

The following table lists all the kinesin-14 constructs I have tested.

Name

Characterization

Expression and purification of Kinesin-14

Vik1 motor-homology domain was purified by a His-trap column then the His-tag was removed using TEV protease. This purification was polished by a gel filtration column. The molecular weight of Vik1 motor-homology domain was checked by mass spectrometry. The measured molecular weight (34688 ± 2 Daltons) matches the theoretical value deduced from the sequence (34687 Daltons).

In the case of kinesin-14 heterodimers, care should be exercised that both chains are present. In particular the detection of the motor-less SHD moiety is not conveniently recorded by SDS-PAGE analysis because of its small size. The first metal-affinity column is not a problem because this peptide comprises the His-tag. Then, after proteolysis of the TEV cleavage site and the subsequent gel filtration chromatography step, the presence of the SHD peptide in the case of the Hetero-Ncd 700 construct was checked by mass spectrometry. Mass spectrometry analysis confirmed the presence of two chains of molecular mass 44823 Daltons and 5203 Daltons, respectively. The experimental data match the theoretical mass of both chains of the heterodimer, indicating that proper kinesin-14 heterodimers can form and be purified. Although the stoichiometry of two chains has not been checked by experiment, a similar method was used to purify Kar3/Vik1

Conclusions and Perspectives

During my thesis, I have been interested in three aspects of kinesins motility. The first one is the structural study of a kinesin-1-tubulin-DARPin complex in which kinesin is in its nucleotide-free state. This structure was the main missing one in the kinesin mechanochemical cycle. The second one is, through a mutagenesis approach, the study of the mechanism of ADP release by kinesin-1. In the course of this study, I have also determined the structure of apo-kinesins (both WT kinesin and mutants) in two different conformations. Finally I have tried to obtain the crystal structure of kinesin-14 in complex with tubulin-DARPin. This last part has not met success so far.

A summary on the structural basis of kinesin-1 motility

Together with previous studies of kinesin motility, my work has allowed me to summerize the kinesin-1 homodimer's movement corresponding to the mechanochemical cycle of its two motor domains (Fig. 8.1).

Vik1

Vik1 is the motor-homology partner of Kar3. It is supposed to form a heterodimer with Kar3 in cells and modulate the binding of the Kar3 motor domain to microtubules. Its motor-homology domain alone, whose crystal structure has been determined [START_REF] Allingham | Vik1 modulates microtubule-Kar3 interactions through a motor domain that lacks an active site[END_REF], is able to bind microtubules itself with a high affinity (KD= 43 nM). But there is neither an EM structure nor a crystal structure of Vik1 bound to a microtubule or to tubulin. How the motor-homology domain interacts with microtubules is unclear. Therefore, I also tried to obtain the crystal structure of Vik1 motor-homology domain (353-647) in complex with tubulin-DARPin.

Plasmid construction

Monomer

Ncd-GFP was a gift from Dr. Ron Vale (Addgene plasmid # 24507). Plasmids containing full length Vik1 and Kar3 gene are gifts from Dr. Stefan Westermann.

Ncd 325-700, Kar3 353-729 and Vik1 353-647 were amplified by PCR; restriction sites were added to both sides of the sequence. The amplified genes were then inserted into pET-M11 plasmid, right after a His-tag and a TEV cleavage site.

Ncd 325-679 plasmid was generated by inserting a stop codon after that of residue 679.

The sequences of inserting fragments were checked with T7 (TAATACGACTCACTATAGGG) and pET-RP (CTAGTTATTGCTCAGCGG) as sequencing primers.

Heterodimer

In order to express the two chains of the heterodimer in a same cell, pET-Duet1 with two T7 promoters is used as a vector. It has two ribosome binding sites, each of which is followed by an independent open reading frame (Fig.

Figure 9.2 pETDuet-1 cloning/expression regions

There are two open reading frames in the pETDuet-1 plasmid, each of which is following an independent pair of T7 promoter and ribosome binding site. As a consequence, both inserted constructs can be expressed independently in the same system at the same time.

Production of Kinesin-Tubulin-DARPin complex 3.1 DARPin purification

Several DARPins that bind tubulin with high affinity, KD ranges from 100 nM (or lower) to 200 nM, without interfering with kinesin binding were available in the laboratory.

Transformation

cf. Transformation of kinesin-14. To express DARPin, XL1 blue (Invitrogen TM ) are used as competent cells. 50 g/mL Ampicillin is used as antibiotics to select positive colonies.

Pre-culture

Inoculate 30 mL LB containing 50 g/mL Ampicillin and 1% Glucose with a single colony and incubate the pre-culture overnight at 37 o C in a shaker.

Culture

Inoculate 1 L LB containing 50 g/mL Ampicillin with the overnight pre-culture to make the initial OD600 around 0.1. The culture is then incubated at 37 o C under shaking until its OD600 reaches 0.6 to 0.8. 0.5 mM IPTG is added to induce the expression of the DARPin. The culture is kept in the same conditions as pre-culture for four hours. Finally, the cells are harvested by centrifugation at 6000 g for 15 minutes at 4 °C. If the protein is not extracted immediately, the harvested cells are stored at -80 °C.

Extraction

Use 50 mL Lysis buffer containing Tris pH 8 50 mM, NaCl 300 mM, MgCl2 5 mM, imidazole 10 mM, lysozyme 0.5 mg/mL, beta-mercaptoethanol 10 mM which is added extemporaneously together with a complete pill of EDTA free anti-protease inhibitor cocktail (Roche TM ) to resuspend the pellet. Cells are kept on ice and sonicated 6 times for 30 seconds at 40 W with 30 seconds intervals. Then, the supernatant and pellet are separated by centrifugation at 20000 g at 4 °C for 20 minutes. The pellet is discarded. The supernatant is filtered with a 0.2 m filter right before injected into a column.

Purification

DARPin is purified in two steps: the first one is by affinity chromatography on a Ni-column (HisTrap HP 5 ml, GE Healthcare TM ). Then a gel filtration chromatography on a Superdex 75 column (GE Healthcare TM ) is performed.

For the HisTrap column, the loading buffer contains Tris pH 8 50 mM, NaCl 300 mM, Imidazole 15 mM and beta-mercaptoethanol 1 mM; the high salt buffer contains Tris pH 8 J'ai étudié plus particulièrement deux aspects du cycle mécano-chimique de la kinésine-1, en combinant des approches de biologie structurale et l'étude de mutants. Les deux aspects concernent l'étude de la fixation de la kinésine-ADP au microtubule, conduisant à l'éjection du nucléotide et à une liaison forte de la kinésine au microtubule. Dans un premier temps, j'ai déterminé la structure du domaine moteur de la kinésine-1, dépourvue de nucléotide, et sous forme d'un complexe avec la tubuline. La tubuline est la protéine constitutive des microtubules. Cette structure était la donnée principale qui nous manquait dans le cycle structural de la kinésine.

En comparant cette structure avec celle de la kinésine dans un état ATP, on peut rendre compte des changements de conformation de la kinésine selon le mouvement de trois sous-domaines du domaine moteur. Cette analyse explique notamment le lien entre la fixation de l'ATP et l'ouverture d'une poche hydrophobe distante de 28 Å du site du nucléotide. Cette cavité va accommoder le premier résidu du neck linker, conduisant à la stabilisation de ce peptide situé en partie C-terminale du domaine moteur. En s'ordonnant, le neck linker va faire avancer la charge ainsi que l'autre domaine moteur de la kinésine dimérique. Il lie ainsi la fixation de l'ATP au mouvement. L'étude de l'effet de mutations du neck linker montre aussi comment, réciproquement, le neck linker bloque la kinésine dans la conformation active pour l'hydrolyse de l'ATP. Ceci diminue la probabilité que l'ATP soit hydrolysé avant que l'étape mécanique se soit produite; cet aspect est essentiel pour rendre compte de la processivité de la kinésine-1.

Ces données structurales suggèrent également comment la fixation de la kinésine-ADP au microtubule accélère l'éjection de l'ADP. Pour étudier cet aspect plus en détail, j'ai étudié l'effet de mutations sur la vitesse de largage de l'ADP. L'idée était de mimer à l'aide de mutations la fixation au microtubule. J'ai identifié ainsi deux séries de mutants qui présentent une vitesse accélérée de largage spontané de l'ADP, ce qui suggère deux voies pour interférer avec la fixation du nucléotide. J'ai ensuite déterminé la structure de deux de ces mutants dépourvus de nucléotide, ainsi que celle de la kinésine de départ également dans une forme apo, obtenue par digestion de l'ADP. En absence de microtubule, la kinésine dépourvue de nucléotide adopte une conformation soit à l'image de celle de la kinésine-ADP, ou proche de celle de la kinésine-apo liée à la tubuline. Dans un contexte naturel, seule la deuxième conformation est compatible avec la fixation au microtubule. L'ensemble de ces résultats suggère que le microtubule accélère l'éjection du nucléotide par un double mécanisme : en interférant avec la liaison du magnésium et en déstabilisant le motif P-loop de liaison du nucléotide. I have studied two aspects of the kinesin mechanochemical cycle, by combining structural and mutational approaches. Both aspects rely on the binding of ADP-kinesin to a microtubule, which leads to the release of the nucleotide and to a tight kinesin-microtubule association. First I determined the crystal structure of nucleotidefree kinesin-1 motor domain in complex with a tubulin heterodimer, which is the building block of microtubule. This structure represented the main missing piece of the structural cycle of kinesin. Three subdomains in the kinesin motor domain were identified through the comparison of this complex structure with ATP-analog kinesin-1-tubulin structure. The relative movements of these subdomains explain how ATP binding to apokinesin bound to microtubule triggers the opening of a hydrophobic cavity, 28 Å distant from the nucleotidebinding site. This cavity accommodates the first residue of the "neck linker", a short peptide that is C-terminal to the motor domain, allowing the neck linker to dock on the motor domain. The docking of the neck linker is proposed to trigger the mechanical step, i.e. the displacement of the cargo and the stepping of the dimeric kinesin. By studying mutants of the neck linker, I have shown that, reciprocally, this peptide locks kinesin in the ATP state, which is also the conformation efficient for ATP hydrolysis. Doing so, it prevents the motor domain from switching back to the apo-state. It prevents also an untimely hydrolysis of ATP, before the mechanical step has occurred. These features are required for movement and processivity.

Second, these structural data also suggest how the binding of ADP-kinesin to tubulin enhances nucleotide release from kinesin. To further study this step of the kinesin cycle, I studied the effect of kinesin-1 mutations. These mutations were designed in isolated kinesin to mimic the state when kinesin is bound to a microtubule. I identified two groups of mutations leading to a high spontaneous ADP dissociation rate, suggesting that there are two ways to interfere with ADP binding. Then I determined the crystal structures of the apo form of two mutants as well as that of the nucleotide-depleted wild type kinesin. It showed that apo-kinesin adopts either and ADP-like conformation or a tubulin-bound apo-like one. In the natural context, the second one is stabilized upon microtubule binding. Overall, the mutational and structural data suggest that microtubules accelerate ADP dissociation in kinesin by two main paths, by interfering with magnesium binding and by destabilizing the nucleotide-binding P-loop motif.