Alain Dautant
email: a.dautant@ibgc.cnrs.fr.

Philippe Meyer

Florian Georgescauld
email: georgescauld@ibpc.fr

Hydrogen/Deuterium Exchange Mass Spectrometry reveals Mechanistic Details of Nucleoside Diphosphate Kinases Activation by Oligomerization

Most oligomeric proteins become active only after assembly, but why oligomerization is required to support function is not well understood. Here, we address this question using the WT and a destabilized mutant (D93N) of the hexameric nucleoside diphosphate kinase from the pathogen Mycobacterium tuberculosis (Mt-NDPK). The conformational dynamics and oligomeric states of each were analyzed during unfolding/folding by Hydrogen/Deuterium exchange mass spectrometry (HDX-MS) at peptide resolution and by additional biochemical techniques. We found that WT and D93N native hexamers present a stable core and a flexible periphery, the latter being more flexible for the destabilized mutant. Stable but inactive species formed during unfolding of D93N and folding of WT were characterized. For the first time, we show that both of these species are native-like dimers, each of its monomers having a major subdomain folded, while a minor subdomain (Kpn/α 0 ) remains unfolded. The Kpn/α 0 subdomain, which belongs to the catalytic site, becomes structured only upon hexamerization, explaining why oligomerization is required for NDPK activity. Further HDX-MS studies are necessary to establish the general activation mechanism for other homo-oligomers.

INTRODUCTION

Depending on the organism, 40 to 60% of proteins assemble and become functional after forming oligomeric complexes. [START_REF] Nishi | Evolutionary, physicochemical, and functional mechanisms of protein homooligomerization[END_REF][START_REF] Marsh | Structure, dynamics, assembly, and evolution of protein complexes[END_REF] These proteins must, within a well determined tridimensional complex structure, subtly balance thermodynamic stability with conformational flexibility. [START_REF] Karshikoff | Rigidity versus flexibility: The dilemma of understanding protein thermal stability[END_REF][START_REF] Teilum | Protein stability, flexibility and function[END_REF] Thermodynamic stability is required to maintain a protein's activity throughout its lifetime while preventing proteolysis or aggregation. In parallel, flexibility is necessary for newly synthesized chains to acquire the native conformation, to assemble and afterwards for the protein to perform its function. How oligomerization induces a protein's "functionality" and how a protein's function, thermodynamic stability and conformational flexibility relate to each other are poorly understood, essentially due to lack of experimental information on the dynamic aspects. Since a single analytical method cannot provide information on a large panel of the physico-chemical properties of proteins, multi-technique approaches and development of new time and space resolved techniques appear essential in this understanding. [START_REF] Engen | Analysis of protein conformation and dynamics by hydrogen/deuterium exchange ms[END_REF][START_REF] Lossl | The diverse and expanding role of mass spectrometry in structural and molecular biology[END_REF][START_REF] Marcoux | Towards integrative structural mass spectrometry: Benefits from hybrid approaches[END_REF][START_REF] Mehmood | Mass spectrometry of protein complexes: From origins to applications[END_REF] Hydrogen/Deuterium exchange detected by mass spectrometry (HDX-MS) is a methodology applied to proteins in recent years for investigating their properties, at peptide resolution, such as dynamics, conformational changes, post-translational modifications, viral capsid assembly, spontaneous and assisted folding, and interactions with partners or ligands. [START_REF] Wales | Hydrogen exchange mass spectrometry for the analysis of protein dynamics[END_REF][START_REF] Engen | Analytical aspects of hydrogen exchange mass spectrometry[END_REF][START_REF] Pirrone | Applications of hydrogen/deuterium exchange ms from 2012 to 2014[END_REF][START_REF] Konermann | Mass spectrometry methods for studying structure and dynamics of biological macromolecules[END_REF][START_REF] Guttman | Isotope labeling of biomolecules: Structural analysis of viruses by hdx-ms[END_REF] The method is applied to proteins in solutions mimicking their physiological environment and relies on the natural property that proteins rapidly exchange their solvent accessible backbone amide hydrogens that are not involved in stable H-bonds. Exposure of the protein of interest into a D 2 O-containing solvent environment induces the replacement of labile accessible backbone hydrogen ( 1 H, H) atoms with deuterium ( 2 H, D). The exchange is measured by MS after protein digestion and interpreted in the view of the structure. The advantages of HDX-MS are that it requires only a small amount of material (in the pmol range), that it has great resolution and sensitivity, that it allows for an accessible mass range of up to 10 6 Da, that it allows high speed analysis and that it is a nonensemble technique; each single spectrum contains extractible information about all discrete coexisting states of the protein of interest. By massively increasing its interest in protein therapeutics within these last years, the biopharmaceutical industry became particularly interested in improving and developing spatially resolved analytical techniques like HDX-MS for its needs in both research and quality control. [START_REF] Marciano | Hdx-ms guided drug discovery: Small molecules and biopharmaceuticals[END_REF][START_REF] Leurs | Getting to the core of protein pharmaceuticals--comprehensive structure analysis by mass spectrometry[END_REF] Since so far HDX-MS was mostly implemented on monomeric proteins (16- 22) and only a few studies have been conducted on homo-oligomeric proteins [START_REF] Georgescauld | Groel/es chaperonin modulates the mechanism and accelerates the rate of tim-barrel domain folding[END_REF][START_REF] Singh | Structural effects of multiple pathogenic mutations suggest a model for the initiation of misfolding of the prion protein[END_REF][START_REF] Huang | Hydrogen/deuterium exchange and electron-transfer dissociation mass spectrometry determine the interface and dynamics of apolipoprotein e oligomerization[END_REF][START_REF] Wang | Continuous and pulsed hydrogen-deuterium exchange and mass spectrometry characterize csge oligomerization[END_REF][START_REF] Monroe | Hydrogen/deuterium exchange analysis of hiv-1 capsid assembly and maturation[END_REF] or protein complexes, [START_REF] Faleri | Two cross-reactive monoclonal antibodies recognize overlapping epitopes on neisseria meningitidis factor h binding protein but have different functional properties[END_REF][START_REF] Malito | Defining a protective epitope on factor h binding protein, a key meningococcal virulence factor and vaccine antigen[END_REF][START_REF] Shi | Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome[END_REF][START_REF] Veesler | Architecture of a dsdna viral capsid in complex with its maturation protease[END_REF] efforts have to be continued for extracting the dynamic determinants of complexes.

Nucleoside diphosphate kinase (NDPK) is a multifunctional protein which catalyzes the gammaphosphate transfer from nucleoside triphosphate to nucleoside diphosphate [START_REF] Lascu | The catalytic mechanism of nucleoside diphosphate kinases[END_REF] in order to maintain equilibrium between the pools of nucleoside triphosphates (NDP/NTP). Several isoforms are found in eukaryotes, [START_REF] Boissan | Learning about the functions of nme/nm23: Lessons from knockout mice to silencing strategies[END_REF] isoform 1 (called nm23A) being the first metastasis suppressor described in humans. [START_REF] Steeg | A critical evaluation of biochemical activities reported for the nucleoside diphosphate kinase/nm23/awd family proteins: Opportunities and missteps in understanding their biological functions[END_REF][START_REF] Takacs-Vellai | The metastasis suppressor nm23 as a modulator of ras/erk signaling[END_REF][START_REF] Marino | Insights into the biology and prevention of tumor metastasis provided by the nm23 metastasis suppressor gene[END_REF] Additionally, NDPK also acts as a protein histidine kinase, [START_REF] Attwood | Histidine kinases from bacteria to humans[END_REF][START_REF] Attwood | Nucleoside diphosphate kinase as protein histidine kinase. Naunyn-Schmiedeberg's[END_REF][START_REF] Srivastava | Histidine phosphorylation relieves copper inhibition in the mammalian potassium channel kca3.1[END_REF] and recent studies have shown that by interacting with and providing GTP to dynamins, NDPK is involved in membrane-remodeling events. [START_REF] Boissan | Membrane trafficking. Nucleoside diphosphate kinases fuel dynamin superfamily proteins with gtp for membrane remodeling[END_REF][START_REF] Schlattner | Mitochondrial nm23-h4/ndpk-d: A bifunctional nanoswitch for bioenergetics and lipid signaling[END_REF] Like many enzymes, NDPKs are non-allosteric oligomers, but in vitro and in vivo studies suggest that their assembly is required for kinase activity or metastasis suppression function. [START_REF] Lascu | Quaternary structure of nucleoside diphosphate kinases[END_REF][START_REF] Kim | Point mutations affecting the oligomeric structure of nm23-h1 abrogates its inhibitory activity on colonization and invasion of prostate cancer cells[END_REF][START_REF] Mocan | Protein phosphorylation corrects the folding defect of the neuroblastoma (s120g) mutant of human nucleoside diphosphate kinase a/nm23-h1[END_REF] Many aspects of the kinase activity mechanism are now understood from a structural point of view, [START_REF] Lascu | The catalytic mechanism of nucleoside diphosphate kinases[END_REF] namely, the role of amino acids involved in the catalysis, but the requirement for assembly to become functional is still unclear and remains to be elucidated. [START_REF] Lascu | Quaternary structure of nucleoside diphosphate kinases[END_REF][START_REF] Kim | Point mutations affecting the oligomeric structure of nm23-h1 abrogates its inhibitory activity on colonization and invasion of prostate cancer cells[END_REF] NDPKs contain ferredoxin-like folds [START_REF] Janin | Three-dimensional structure of nucleoside diphosphate kinase[END_REF] which assemble first as dimers and then as tetramers or hexamers. [START_REF] Moynie | The structure of the escherichia coli nucleoside diphosphate kinase reveals a new quaternary architecture for this enzyme family[END_REF][START_REF] Tokunaga | Residue 134 determines the dimer-tetramer assembly of nucleoside diphosphate kinase from moderately halophilic bacteria[END_REF][START_REF] Yonezawa | Dimeric structure of nucleoside diphosphate kinase from moderately halophilic bacterium: Contrast to the tetrameric pseudomonas counterpart[END_REF] Tetramers are present in gram-negative bacteria and archaea, while hexamers are present in gram-positive bacteria and eukaryotes. Hexamers represent the most abundant type of oligomerization, which specifically relies on the well conserved surface loop that carries the site of the natural "Killer of prune" mutation (Kpn-loop) (Figures 1,S1). In addition, the stabilization of hexamers by subunit interactions has been studied. [START_REF] Chen | X-ray structure of mycobacterium tuberculosis nucleoside diphosphate kinase[END_REF][START_REF] Karlsson | Nucleoside diphosphate kinase. Investigation of the intersubunit contacts by site-directed mutagenesis and crystallography[END_REF][START_REF] Mesnildrey | Coupling between catalysis and oligomeric structure in nucleoside diphosphate kinase[END_REF][START_REF] Giartosio | Thermal stability of hexameric and tetrameric nucleoside diphosphate kinases. Effect of subunit interaction[END_REF][START_REF] Georgescauld | Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of mycobacterium tuberculosis[END_REF] Crystallographic structures of several-NDPK mutants provide detailed understanding of the WT stabilization mechanism, [START_REF] Georgescauld | Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of mycobacterium tuberculosis[END_REF][START_REF] Georgescauld | Structure of mycobacterium tuberculosis nucleoside diphosphate kinase r80n mutant in complex with citrate[END_REF] but provide little understanding as to why oligomerization is necessary for function. So, we realized that by combining stability aspects with the study of their dynamic properties, we would improve this understanding.

FIGURE 1

Mycobacterium tuberculosis (Mt), the pathogenic bacteria responsible of tuberculosis, possesses only one NDPK isoform (Mt-NDPK), which is hexameric (Figures 1,S1). [START_REF] Chen | X-ray structure of mycobacterium tuberculosis nucleoside diphosphate kinase[END_REF] In addition to its kinase function, Mt-NDPK binds DNA [START_REF] Kumar | Nucleoside diphosphate kinase from mycobacterium tuberculosis cleaves single strand DNA within the human c-myc promoter in an enzyme-catalyzed reaction[END_REF] and also blocks phagosome maturation in murine macrophages leading to evasion of innate immunity. [START_REF] Sun | Mycobacterial nucleoside diphosphate kinase blocks phagosome maturation in murine raw 264.7 macrophages[END_REF] The escape mechanism is unknown, but efficient intracellular survival of Mt requires a strong thermodynamic stability of proteins. [START_REF] Ganaie | Thermostable hexameric form of eis (rv2416c) protein of m. Tuberculosis plays an important role for enhanced intracellular survival within macrophages[END_REF] We recently showed in vitro that isolated sub-hexameric assemblies of Mt-NDPK are inactive, complete assembly being essential for function. [START_REF] Georgescauld | Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of mycobacterium tuberculosis[END_REF] By combining crystallography and biochemistry, we also demonstrated that hexamer's high thermostability (76°C) is dependent on six inter-subunit R80-D93 salt bridges. [START_REF] Georgescauld | Structure of mycobacterium tuberculosis nucleoside diphosphate kinase r80n mutant in complex with citrate[END_REF] The point mutation D93N reduces the melting point by 28°C, without significant alteration of the hexameric 3D structure or enzymatic properties. [START_REF] Georgescauld | Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of mycobacterium tuberculosis[END_REF] The link between the protein's function, stability and oligomerization is still an open question.

Here, we show why the full oligomeric state is required for and affects protein function, as demonstrated by the study of WT and D93N Mt-NDPK. In addition to the classical biochemistry methods (fluorescence, enzymology, size exclusion chromatography), we use the HDX-MS technique to reveal the overall and local structure and dynamics of NDPK by monitoring amide hydrogen atoms in polypeptides. We show that both native hexamers are stable, without in/out subunit exchange, but that the D93N destabilizing mutation increases the flexibility at the hexamer surface. Inactive dimers which accumulate during folding of WT and D93N, as well as during D93N unfolding were analyzed. Strikingly, differences in conformational flexibility between hexamers and dimers explain why hexamerization is required for kinase activity.

MATERIALS AND METHODS

Reagents. Chemicals were of the highest purity grade from Sigma. Solutions of urea were freshly prepared for each experiment.

Proteins and enzymatic assays. WT and D93N Mt-NDPK were expressed without tag, using a pET24 vector (Novagen) in BL21-CodonPlus ® (DE3)-RIL (Stratagene) strain. Transformed cells were induced at 37°C with 1 mM IPTG for 6 h in the presence of 80 µg/mL kanamycin and the purification steps were carried out at 4°C as previously described. [START_REF] Georgescauld | Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of mycobacterium tuberculosis[END_REF] After sonication and centrifugation, the soluble fraction containing Mt-NDPK was DNase-treated, and the extract was applied to a Q-Sepharose column equilibrated with 100 mM Tris-HCl, pH 7.4. The enzyme was eluted with a linear gradient of 0 to 0.8 M NaCl in the same buffer. Active fractions were precipitated into 80% saturated ammonium sulfate (AS) and further purified by salting-out chromatography on a Sepharose 6B column equilibrated with 80% AS, 100 mM Tris-HCl, pH 7.4.

The protein was eluted by decreasing AS concentration from 80% to 20% in the same buffer. The active fractions were dialyzed against 100 mM Tris-HCl, pH 7.4, and further purified on a Source 15Q column, under the conditions described for the Q-Sepharose chromatography. The enzymes were precipitated by dialysis against a saturated solution of AS, recovered by centrifugation and further purified by size-exclusion chromatography on a Sephacryl S-200 column equilibrated with 0.2 M sodium phosphate buffer, pH 7.0. The final hexameric preparations were kept frozen in 50 mM Tris-HCl, pH 7.4 at -80°C.

The size-exclusion chromatography (SEC) was performed in 20 mM phosphate buffer, 150 mM NaCl using a Superdex 75 analytical column, in conditions described in the supplemental.

Molecular markers (cytochrome c, 12 400 Da; P105G Dd-NDPK, monomeric version from Dictyostelium discoideum, 16 800 Da; myoglobin, 17 000 Da; carbonic anhydrase, 29 000 Da; ovalbumin, 44 000 Da; BSA, 68 000 Da; aldolase, 158 000 Da) from GE Healthcare Life Sciences were used. The P105G Dd-NDPK, monomeric version from Dictyostelium discoideum, was a gift from Pr. Ioan Lascu and has an elution volume of 12.6 mL (data not shown).

NDPK enzymatic activity was measured using a coupled assay. [START_REF] Giartosio | Thermal stability of hexameric and tetrameric nucleoside diphosphate kinases. Effect of subunit interaction[END_REF][START_REF] Georgescauld | Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of mycobacterium tuberculosis[END_REF] The reactions of the test are shown below and the enzymes catalyzing each reaction are indicated in parentheses. In this assay, NDPK catalyzes phosphate transfer from ATP to 8-bromoinosine-5'-diphosphate (8-BrIDP).

The enzyme mix contained in a final volume of 0.8 mL: 20 mM Tris-HCl (pH 7.5), 5 mM MgCl 2 , 100 mM KCl, 1 mg/mL BSA, 1 mM de phosphoenol pyruvate, 0.1 mM NADH, 1 mM ATP, 0.2 mM 8-BrIDP, 2U/mL of pyruvate kinase and lactate dehydrogenase. We measured NADH disappearance at 340 nm and 25°C, using a Perkin-Elmer spectrophotometer. For such assay, the errors associated with the kinetic parameters are under 20%. [START_REF] Lascu | The catalytic mechanism of nucleoside diphosphate kinases[END_REF][START_REF] Georgescauld | Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of mycobacterium tuberculosis[END_REF] ATP + 8-BrIDP → ADP + 8-BrITP

FIGURE 2

For full length experiments, protein samples were immediately injected into an Alltech analytical guard column, packed with POROS 20-R1 reversed-phase media (PerSeptive Biosystems). Each sample was washed within 20 s with 400 µL of 0.1% formic acid, ice cold. The guard column was then switched in line with the HPLC inject port with a flow rate of 200 µL/min of 15% acetonitrile/0.1% formic acid, pH 2.5 for 2 min. The protein was eluted at 40 µL/min using a 1.5 min gradient of 15%-75% acetonitrile directly into a Waters Synapt G1 mass spectrometer with a standard electrospray interface. The injector, column and all associated tubings were kept at 0°C using an ice bath to minimize back exchange. [START_REF] Wales | High-speed and highresolution uplc separation at zero degrees celsius[END_REF] All experiments were conducted under identical conditions to allow comparison of relative deuterium levels. [START_REF] Wales | Hydrogen exchange mass spectrometry for the analysis of protein dynamics[END_REF] Mass accuracy of less than 20 ppm was maintained by infusing horse heart myoglobin into the mass spectrometer at the end of each chromatographic gradient. Intact mass spectra were deconvoluted using MassLynx (Waters) and masses were corrected for back exchange (17%) and dilution effect (10%). As controls, unfolded and native Mt-NDPKs were subjected to D 2 O pulse labeling and LC-MS analysis. The backexchange of 17% was measured by diluting denatured Mt-NDPKs (6 M GuDCl, 1 mM DTT) into 20 mM Tris-HCl, 20 mM KCl buffer prepared with 99.9 D 2 O. After 10 s of deuteration, the reaction was quenched and immediately subjected to LC-MS at 0°C.

To obtain HDX data at peptide resolution, 200 µL of each acid-quenched sample were injected into a loop of 100 µL inside an HDX Waters nanoACQUITY Ultra Performance Liquid Chromatography (UPLC). [START_REF] Wales | High-speed and highresolution uplc separation at zero degrees celsius[END_REF] The sample passed through a Poroszyme-immobilized pepsin cartridge (Applied Biosystems) accommodated within the HDX manager at a flow rate of 100 µL min -1 and temperature of 20°C. Peptic peptides eluting from the pepsin column were trapped and desalted for 3 min at 100 µL min -1 , and then separated in 7 min with 8% to 40% acetonitrile:water gradient at 40 µL min -1 , all steps were carried out in 0.1% formic acid pH 2.5 before MS analysis.

The separation column was a 1.0×100.0 mm ACQUITY UPLC C18 BEH (Waters) containing 1.7 µm particles and the back pressure averaged 8000 psi. All chromatographic elements were held at 0°C for the entire time of the measurements. The average amount of back exchange was around 25% based on analysis of highly deuterated peptide standards. [START_REF] Wales | High-speed and highresolution uplc separation at zero degrees celsius[END_REF] Deuterium levels were corrected for 25% back-exchange and 10% dilution, all comparison experiments being done under identical experimental conditions. The UPLC steps were performed with protonated solvents, thereby allowing deuterium to be replaced with hydrogen from side chains and the amino/carboxyl terminus that exchange much faster than amide linkages. [START_REF] Engen | Analysis of protein conformation and dynamics by hydrogen/deuterium exchange ms[END_REF][START_REF] Wales | Hydrogen exchange mass spectrometry for the analysis of protein dynamics[END_REF] The error in the determination of the deuterium levels was ± 0.20 Da in this experimental setup consistent with previously obtained values. [START_REF] Iacob | Conformational disturbance in abl kinase upon mutation and deregulation[END_REF][START_REF] Georgescauld | Groel/es chaperonin modulates the mechanism and accelerates the rate of tim-barrel domain folding[END_REF][START_REF] Houde | The utility of hydrogen/deuterium exchange mass spectrometry in biopharmaceutical comparability studies[END_REF] Mass spectra were obtained with a Waters Synapt G1 using a standard ESI source (Waters Corp., Milford, MA, USA) over an m/z range of 50-1700. Mass accuracy was ensured by calibration with Glu-fibrinogen peptide, and was less than 10 ppm throughout all experiments. Identification of the peptic fragments was accomplished with at least 4 replicate MS E analyses [START_REF] Georgescauld | Groel/es chaperonin modulates the mechanism and accelerates the rate of tim-barrel domain folding[END_REF] using Identity Software (Waters Corp., Milford, MA, USA). MS E was performed by realizing series of low-high collision energies ramping from 5-30 V, therefore ensuring proper fragmentation of all the peptic peptides eluting from the LC system.

Pulse HDX. To characterize the unfolding pathway of D93N, the purified protein was first pre-incubated at 2 μM in 0-8 M urea, for 16 h at 10°C. After this step, the pulse labeling experiment was performed following the scheme (Figure S3C). 20 µL of each sample were deuterated for 12 s by dilution of the protein stock solution 10-fold into 20 mM Tris-HCl, 20 mM KCl, pD 7.5, 99.9% D 2 O, at 10°C. Deuteration was stopped by quenching the reaction with formic acid, final pH at 2.5, at 0°C. After the quenching step, samples were digested or not and analyzed as described in the previous paragraph concerning the continuous labeling.

The unfolding pathway of the WT was characterized in the same way as for the D93N one, except that we used a higher performance mass spectrometer (Synapt G2 Si, Waters) allowing a 9fold lower working protein concentration. Pre-incubation of WT was performed at about 220 nM final and the pulse labeling experiment followed the scheme (Figure S3C), all the other experimental aspects were identical to the ones explained for unfolding of D93N.

Spontaneous refolding of WT Mt-NDPK was initiated by 400-fold dilution from 7.2 M GuHCl into refolding buffer (20 mM Tris-HCl, pH 7.5, 100 mM KCl) at 10°C and final protein concentration of 220 nM. Residual GuHCl concentration was as low as 18 mM in order to not disturb the refolding process. The reaction scheme shown in Figure S4A was followed. Aliquots of refolded Mt-NDPK were deuterated for 12 seconds, quenched with formic acid at pH 2.5 and analyzed as described above for the continuous labeling experiments. A Synapt G2 Si mass spectrometer was used for these experiments. The unfolded references were arbitrarily given a refolding time of 3 s in these graphs.

Peptide Mass Analysis and Data Processing. Mass spectra were processed using DynamX 2.0 software (Waters) by centroiding the isotopic distribution corresponding to all charge states of each peptide. Automated selection of the isotope distribution was verified manually for all peptides and all charge states. The resulting relative deuterium levels (corrected for 25% back exchange and 10% dilution factor) were plotted versus the exchange-in time with respect to experimental described conditions. All comparison experiments were done under identical experimental conditions. NDPKs unfolded during 1 h in GuDCl 6 M were used as controls for 100% deuterated protein. The error of determining the deuterium levels was ± 0.20 Da for the experimental setup used. [START_REF] Iacob | Conformational disturbance in abl kinase upon mutation and deregulation[END_REF][START_REF] Georgescauld | Groel/es chaperonin modulates the mechanism and accelerates the rate of tim-barrel domain folding[END_REF][START_REF] Houde | The utility of hydrogen/deuterium exchange mass spectrometry in biopharmaceutical comparability studies[END_REF] For the pulse labeling experiments, when the isotope distributions were bimodal, all isotopes of the entire bimodal pattern were selected for processing. [START_REF] Georgescauld | Groel/es chaperonin modulates the mechanism and accelerates the rate of tim-barrel domain folding[END_REF] The relative deuterium incorporation was calculated by subtracting the centroid of the isotopic distribution for peptide ions of the native reference from the centroid of the isotopic distribution for peptide ions from each refolding sample (Figure S4B). All molecular structure figures were prepared using PyMol, [START_REF] Schrodinger | The pymol molecular graphics system[END_REF] and the alignment figure using ESPript (http://espript.ibcp.fr). [START_REF] Robert | Deciphering key features in protein structures with the new endscript server[END_REF] RESULTS AND DISCUSSION

Impact of destabilizing D93N mutation on Mt-NDPK hexamer dynamics

Here we perform continuous labeling HDX coupled to liquid chromatography (LC) and mass theoretically accessible H's, show that more than half of each chain is solvent accessible. At each incubation time, WT incorporates between 5 and 11 D's less than D93N indicating a higher overall rigidity for WT. The WT is thermodynamically more stable than the D93N mutant, clearly supporting the view that increased stability reduces flexibility. [START_REF] Karshikoff | Rigidity versus flexibility: The dilemma of understanding protein thermal stability[END_REF] To gain spatial information, we next performed continuous labeling HDX-MS at peptide level resolution by adding a pepsin digestion step before LC-MS (Figure S2A). 27 identical polypeptides covering 95.6% of the sequence were analyzed in both proteins (Figure S2B). 17 peptides had identical or almost identical behavior while 10 overlapping peptides (gray shaded panels) covering 27% of the total sequence, start and end with different exchange levels of at least 0.50 Da, representing more than 15% when normalized to the maximal incorporation value (Figure S2C).

The highest flexibility is noticed in both hexamers for surface regions 39-62 (hairpin α A /α 2 ), 108-113 (helix α3'), and 132-136 (C-term end), which interestingly correspond to the most divergent sequences in NDPKs (Figures 2C-D, S2D). For the C-terminal end, the deuteration is already maximal at the first experimental time point after 10 s, as indicated by the corresponding flat plots.

Because of its biological relevance, original search for flexible regions in NDPKs started two decades ago using 1 H NMR. [START_REF] Xu | Nmr studies on the flexibility of nucleoside diphosphate kinase[END_REF] Here, our HDX-MS results show that as previously suggested, [START_REF] Karlsson | Nucleoside diphosphate kinase. Investigation of the intersubunit contacts by site-directed mutagenesis and crystallography[END_REF][START_REF] Xu | Nmr studies on the flexibility of nucleoside diphosphate kinase[END_REF] the hairpin α A /α 2 which is a part of the nucleotide binding site, is highly flexible and could undergo large movement in order to accommodate DNA, explaining the DNA binding function. [START_REF] Kumar | Nucleoside diphosphate kinase from mycobacterium tuberculosis cleaves single strand DNA within the human c-myc promoter in an enzyme-catalyzed reaction[END_REF] Both WT and D93N hexamers present with full deuteration after 10 min, with three quarters of their sequence being already deuterated at 10 s. The regions specifically affected by the D93N mutation are contiguously located at the hexamer surface (9-15: helix α 0 ) and at the trimer interface (85-113: 2C-D, left columns showing the surface). The region 108-113 is fully solvent accessible in the mutant, while in WT it becomes fully deuterated after 3 min exposure to solvent.

Kpn-loop) (Figures
The peptides covering region 114-130 present about 1 Da difference in deuterium incorporation, which corresponds to less than 15% difference indicating that mutation did not substantially affect this part of the structure. The strand β 2 (32-38) and the helix α 1 (18-27), which form the dimer interface, remain inaccessible to solvent indicating high stability for dimers (Figure 2D, right column showing the interface). In conclusion, differences in dynamics for D93N and WT Mt-NDPK hexamers correlate only with their difference in stability, while their 3D structures are very similar with an RMSD of 0.23 Å. Importantly, the decrease of thermostability of D93N is accompanied by the increase of the conformational flexibility at the hexamer surface, without affecting the stable core of the protein (Figure S2D).

D93N and WT unfolding pathways comparison

The next questions we addressed were whether the conformational flexibility of NDPK varies with the oligomeric state and whether this potential variation is the reason why oligomerization is required to support function. To find sub-hexameric species, unfolding and refolding pathways of WT and D93N were analyzed in the presence of urea. First, enzyme activity was measured after incubation of native and unfolded NDPK for 16 h in 0 to 7.2 M urea (Figure S3A). In both cases a hysteresis phenomenon was observed, since unfolding and refolding curves did not overlap, indicating that WT and D93N proteins were not at equilibrium. Second, the hydrodynamic radii of NDPK were measured by SEC in 0 to 7.2 M urea, after the same unfolding/refolding procedure (Figure S3B). Again, a hysteresis phenomenon was observed, since for the same urea concentration, the elution volume of NDPK differed between folding and unfolding. Taken together, these results

show an absence of equilibrium for WT and D93N during unfolding/folding experiments. They are in full agreement with our previous data on Mt-NDPK as well as with other similar studies on NDPK which show hysteresis after about 1 day of unfolding/refolding in presence of denaturing agents. [START_REF] Lascu | Quaternary structure of nucleoside diphosphate kinases[END_REF][START_REF] Georgescauld | Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of mycobacterium tuberculosis[END_REF] Having established that unfolding and refolding pathways were different for both WT and D93N

NDPKs, we next characterized the potential sub-hexameric species formed during unfolding. WT hexamers were incubated in 0 to 7.2 M urea during 16 hours and the oligomeric state was established by SEC for the different urea concentrations (Figure S3B). Two elution volumes of 9.65 and 10.2 mL were noticed, indicating respectively a direct passage from native hexamers (0 to 4.0 M urea) to unfolded monomers (>4 M urea). The WT unfolding pathway was next characterized by enzyme activity, which reflects integrality of quaternary structure and by the fluorescence of the unique tryptophan (W132), which reflects integrity of tertiary structure. Both curves overlapped, indicating again concomitant dissociation and unfolding (Figure 3B).

FIGURE 3

To obtain mechanistic details, pulse labeling HDX-MS at the peptide level was performed, following the scheme described in (Figure S3C). The level of deuterium which is detected in the peptides is a signature of the NDPK conformations during unfolding, providing a snapshot of the different coexisting populations. The 31 peptides analyzed, covering 98.5% of the sequence (Figure S2B), all showed bimodal behavior with one transition between the folded/assembled hexamers and unfolded monomers around 4.5 M urea (Figure S3D). Two representative peptides are shown in Figure 4 (left panels). This result clearly shows concomitant dissociation and unfolding of WT hexamers (Figure 3B), in full agreement with SEC, tryptophan fluorescence and activity data.

FIGURE 4

When the same strategy of characterization by SEC, enzyme activity and tryptophan fluorescence, followed by HDX-MS analysis was applied to the D93N mutant, a different unfolding pathway was highlighted. By SEC, in addition to hexamers and unfolded monomers, a new species was detected at low denaturant concentrations (1 and 2 M urea). The column calibration indicates that the elution volume of 11.7 mL corresponds to native dimers (Figure S3B). At 4 M or higher urea concentrations, this species disappears and only one new peak corresponding to unfolded monomers appears (10.2 mL), indicating that dimers dissociate and unfold concomitantly.

Confirmation of this unfolding pathway was obtained using two additional methods: enzyme activity is lost for urea concentrations higher that 0.5 M (Figure 3C, empty red squares), while from

Trp fluorescence, only one transition between native and unfolded species was observed around 2.7

M urea (Figure 3C, empty blue circles). Taken together, these results indicate that D93N hexamers disassemble into dimers before further concomitant dissociation and unfolding into monomers.

Pulse labeling HDX-MS at full-length protein level was performed to further characterization. In the absence of urea, hexameric full length D93N incorporates 36 D, while between 0.5 and 2.0 M urea, dimers incorporate additional 19 D (Figure 3C, green diamonds). From 3 to 8 M urea, progressive incorporation from 111 to 125 D over the 129 available amide protons reveals monomers with a slightly different level of unfolding, in agreement with SEC, fluorescence and activity data (Figure 3C).

To structurally determine why such dimers are enzymatically inactive, the D93N unfolding pathway was characterized by pulse labeling HDX-MS at peptide level, as described in the scheme of Figure S3C. 30 peptides covering 100% of the sequence (Figure S2C) were analyzed (Figures 3D, S3E-F). All spectra show bimodal isotope distribution, which corresponds to native or fully unfolded peptide, except peptides 108-113 and 132-136, which are permanently unfolded. Bimodal peptides had transitions between their folded and unfolded states at the same urea concentrations as the ones noticed for the loss of activity (0.5 M) or tertiary structure (2.7 M). The specific behavior of these two categories of peptide is illustrated by their mass spectra of peptides 9-21 and 25-35, respectively (Figure 4, right panels). Remarkably, at 0.5 M urea, the loss of enzyme activity is due to unfolding of the regions 85-107 (Kpn-loop) at the trimer interface and 9-16 (helix α 0 ). These regions are far in the sequence but close in the structure and constitute a separate subdomain of the rest of the monomer, further called Kpn/α 0 subdomain. The rest of the sequence including the dimer interface (region 17-40: helix α 1 , and strand β 2 ) is not altered up to urea concentrations higher than 2.7 M, when the dimers simultaneously dissociate and unfold. Taken together, these data show that the D93N inactive intermediate species correspond to native-like folded dimers, instead of monomers, as previously proposed. [START_REF] Georgescauld | Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of mycobacterium tuberculosis[END_REF] These dimers are well folded except for the Kpn/α 0 subdomain which is fully solvent accessible and by consequence lacks stable structure.

WT and D93N hexamers exhibit similar enzyme properties but different thermodynamic stabilities, which are responsible for their different unfolding pathways (Figure S3G). WT hexamers dissociate and unfold concomitantly indicating high cooperativity between all six subunits. D93N hexamers first dissociate into dimers before further concomitant dissociation and unfolding, indicating a partial loss of cooperativity between subunits, and so demonstrate the important role played by ionic bonds for cooperativity inside complexes. D93N dimers are inactive and much less stable compared to WT-hexamers (Figures 3A and3C, blue curves). Importantly, the lack of activity is only due to the lack of stable structure of the Kpn/α 0 subdomain, indicating that even a single-domain protein can have its dynamics uncoupled between its structural subdomains.

Characterization of refolding and assembly pathway of WT Mt-NDPK

Stable but inactive species accumulate for WT Mt-NDPK only during refolding at low protein concentration (Figure 5A, blue circles), [START_REF] Georgescauld | Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of mycobacterium tuberculosis[END_REF] and their characterization by size exclusion chromatography indicates they are dimers (Figure S3B).

FIGURE 5

Next, we took advantage of the HDX-MS sensitivity to analyze the WT refolding and assembly pathway at peptide resolution. Unfolded WT protein in 7.2 M GuHCl final concentration was diluted 400-fold into refolding buffer to a final concentration of 220 nM for 0-3 h. Aliquots were deuterated, quenched and digested, and the peptide masses were immediately measured (Figure S4A). It is important to note that the residual 18 mM GuHCl in the refolding buffer is insufficient to unfold any peptide and did not affect the folding/assembly pathway. Strikingly, after 10-30 seconds refolding, all peptides became native, except for the previously described Kpn/α 0 subdomain (84-107 and 9-21), which stays fully unfolded during at least 3 h (Figure S4B). The weak hydrophobicity of the Kpn/α 0 subdomain, when compared to the rest of the NDPK sequence, is probably not sufficient to allow its burying/folding by hydrophobic collapse. We conclude that, in agreement with stopped-flow data, [START_REF] Georgescauld | Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of mycobacterium tuberculosis[END_REF] WT monomers are rapidly folded and assembled into inactive dimers (Figures 5B,S4C) similar to the ones described for D93N unfolding. WT and D93N dimers are native-like except for the Kpn/α 0 subdomain, which gets buried and structured only during assembly into hexamers, inducing enzyme activation.

During two decades and based on spectroscopic methods, it has been proposed that structurally single-domain proteins -like NDPKs -present only two conformations (folded or unfolded), the interconversion between them being performed within one single step, without formation of stable intermediates. [START_REF] Englander | Protein folding-how and why: By hydrogen exchange, fragment separation, and mass spectrometry[END_REF] Here we show that such methods are insufficient to fully characterize properties of folding/unfolding intermediates, mostly when such intermediates present many but not all physical properties the native protein possess. We propose that as for a few proteins, [START_REF] Hu | Cytochrome c folds through foldon-dependent native-like intermediates in an ordered pathway[END_REF] Mt-NDPK folding/unfolding appears by blocks or subdomains, which are called foldons in the literature. [START_REF] Englander | Protein folding-how and why: By hydrogen exchange, fragment separation, and mass spectrometry[END_REF] Clearly in our study the Kpn/α 0 subdomain is indeed such a foldon. We could demonstrate that the Kpn/α 0 foldon is unstructured in monomers/dimers and acquires stable conformation only upon full hexameric assembly. Since the Kpn/α 0 foldon is part of the catalytic site, this explains why full hexamerisation is required for enzyme function acquisition. Finally, our study demonstrates that during the phenomena of folding/unfolding of NDPK, the Kpn/α 0 foldon is stabilized/destabilized in a cooperative sequential order.

CONCLUSIONS

In this study we have applied a combination of biochemical and biophysical methods to Mt-NDPK including the emerging technique of structural mass spectrometry to investigate the link between a protein's function and several of its physical parameters: conformational dynamics, stability and oligomerization. Even though NDPK research today comprises more than 2500 publications, the HDX-MS methodology has only marginally been applied to study this important class of proteins. [START_REF] Kim | Structure of nm23-h1 under oxidative conditions[END_REF] Here, for the first time, its application provides an explanation of why oligomerization is required for the kinase function. We show that in order to be active, hexameric NDPK requires a structured Kpn/α 0 foldon, while to be structured the foldon requires the assembly of the NDPK. NDPKs are oligomeric enzymes devoid of allosteric regulation. Many structures of NDPKs have been solved in the presence of different ligands (ADP, non hydrolysable ATP analogues, small molecules, …), but the apo and in complex structures are identical and do not show conformational changes. Here, we show that the Kpn/α 0 foldon is an activation region and a critical control switch, through its shifting between the structured or unstructured conformation.

Since not all NDPKs are hexameric, the question arises as to how bacterial tetrameric and dimeric ones are active. The analysis of crystal structures shows that in the type 2 tetramer, [START_REF] Moynie | The structure of the escherichia coli nucleoside diphosphate kinase reveals a new quaternary architecture for this enzyme family[END_REF] the Kpn/α 0 foldon is located at the dimer-dimer interface and has the same control switch role. On the contrary, in type 1 tetramer [START_REF] Boissier | An intersubunit disulfide bridge stabilizes the tetrameric nucleoside diphosphate kinase of aquifex aeolicus[END_REF] and dimer, [START_REF] Tokunaga | Residue 134 determines the dimer-tetramer assembly of nucleoside diphosphate kinase from moderately halophilic bacteria[END_REF][START_REF] Arai | A structural mechanism for dimeric to tetrameric oligomer conversion in halomonas sp. Nucleoside diphosphate kinase[END_REF][START_REF] Yonezawa | Nucleoside diphosphate kinase from psychrophilic pseudoalteromonas sp. As-131 isolated from antarctic ocean[END_REF] the Kpn/α 0 foldon is fully exposed and could not have such a regulatory role. High eukaryotes have several isoforms of NDPK with additional functions: metastasis suppression in humans [START_REF] Takacs-Vellai | The metastasis suppressor nm23 as a modulator of ras/erk signaling[END_REF][START_REF] Marino | Insights into the biology and prevention of tumor metastasis provided by the nm23 metastasis suppressor gene[END_REF][START_REF] Rosengard | Reduced nm23/awd protein in tumour metastasis and aberrant drosophila development[END_REF][START_REF] Giraud | Crystal structures of s120g mutant and wild type of human nucleoside diphosphate kinase a in complex with adp[END_REF] and involvement in animal development. (71- 73) Interestingly, natural mutants for these two hexameric NDPKs present folding or assembly defects. [START_REF] Lascu | A pro/ser substitution in nucleoside diphosphate kinase of drosophila melanogaster (mutation killer of prune) affects stability but not catalytic efficiency of the enzyme[END_REF] Since our present study on Mt-NDPK shows that flexibility is increased by the hexamer's destabilization, we suggest these mutants are inactive because of aberrant conformational dynamics. Potentially, their stabilization could be enhanced by rational design of their most dynamic parts, a promising but actually unexplored strategy in cancer or developmental research. In conclusion, our results illustrate the power of MS-based technologies to uncover new concepts in studying structure-activity relationships, in complement to biochemical techniques, X-ray crystallography and NMR.

ASSOCIATED INFORMATION

Sequence of Mt-NDPK (136 aa) with the secondary structure elements, the Kpn-loop, the active site residues, the inter-subunit salt bridge, the solvent accessibility of each residue (Figure S1A), scheme of continuous labeling HDX-MS experiment at peptide level (Figure S2A), peptide maps (Fig S2B ), kinetics of deuterium incorporation for peptic peptides of WT and D93N (Figure S2C), local dynamics of WT and D93N (Fig S2D ), unfolding and refolding of WT and D93N followed by activity (Figure S3A) and size-exclusion chromatography (Figure S3B), scheme of the pulse labeling HDX-MS experiment for the D93N and WT after incubation in urea (Figure S3C), deuterium incorporation for peptic peptides of WT (Figure S3D) and D93N (Figure S3E), pulse labeling HDX data for D93N in 0 and 1 M urea mapped onto the WT structure (Figure S3F), pulsed labeling HDX-MS data for WT and D93N in 0-6 M urea were mapped onto the monomer WT structure (Figure S3G), scheme of the refolding/assembly experiment followed by pulsed labeling HDX-MS at peptide level (Figure S4A), kinetics of deuterium incorporation for peptic peptides of WT (Figure S4B), pulsed labeling HDX-MS data for refolding and assembly pathway of WT mapped onto the monomer WT structure. Unfolding was monitored by intrinsic fluorescence (blue circles), residual enzymatic activity (red squares) [START_REF] Georgescauld | Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of mycobacterium tuberculosis[END_REF] (A, C). The values are normalized to the experiment in the absence of urea. D93N unfolding was also monitored by a 12 sec pulse labeling deuterium incorporation at full-length level (green diamond) (C). The relative percentage of deuterium incorporation measured at peptide level with a 12 sec D 2 O pulse for WT in 0, 3, 4, and 5 M urea (B) and D93N in 0, 1, 2 and 3 M urea (D) was mapped onto the crystal structure of Mt-NDPK using a blue (0%) to red (100%) scale (Figure S3). A top view (left) and a split dimer interface (right) were shown. When the hexamer was dissociated or unfolded, it was split into three dimers or six randomly oriented monomers, respectively. The white contour on columns two and four outlines the fingerprint of the dimer interface. [START_REF] Georgescauld | Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of mycobacterium tuberculosis[END_REF] The lines do not represent theoretical models but were drawn to help the reader. (B) Refolding and assembly pathway of WT Mt-NDPK.

LEGENDS OF FIGURES

The percentage of deuterium incorporation after 12 seconds D 2 O pulse at different refolding time is plotted onto the structure (Figure S4B). Six monomers in random orientation are drawn when unfolded. For each time point, a top view (left) and a split dimer interface of three split dimers (right) are shown. Residues with no data are colored in black. Note for refolding experiments that at this low protein concentration, only dimers are formed. Hexamers are formed at ten time higher concentrations. [START_REF] Georgescauld | Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of mycobacterium tuberculosis[END_REF]

  Unfolding and refolding. The unfolding/refolding experiments were performed as previously described.[START_REF] Georgescauld | Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of mycobacterium tuberculosis[END_REF] 10 µg/mL final concentration of native or unfolded Mt-NDPK were incubated for 16 h in 0-8 M urea or 0-5 M GuHCl, and 20 mM phosphate buffer, pH 7.0 at 25°C. Fluorescence intensities of the single tryptophan residue Trp132 were measured at 335 nm with an excitation at 295 nm. Continuous HDX. Experiments were performed at full length and peptide resolution, by following the schemes (Figures 2A and S2A). Stock solutions of 2.0 µM native Mt-NDPKs (WT and D93N) were prepared in 20 mM Tris-HCl pH 7.5, 100 mM KCl, H 2 O. Deuterium exchange was initiated by dilution of the Mt-NDPK stock solution 10-fold into 20 mM Tris-HCl, 20 mM KCl, pD 7.5, 99.9% D 2 O, 10°C. The KCl salt concentration was reduced from 100 mM to 20 mM in order to obtain better MS spectra quality. At different times of incubation, a 200 μL aliquot from the exchange reaction was removed (from 10 s to 90 min) and labeling was quenched by adjusting the pH to 2.5 with formic acid, at 0°C.

  spectrometry (MS) to characterize the overall conformational dynamics of purified hexameric WT and D93N Mt-NDPKs (Figure2A). Samples kept at 10°C are incubated for 10 s to 90 min into 90% D 2 O final concentration and acid quenched on ice to stop the H/D exchange. On line injection of full length protein to a LC-MS system allows immediate mass measurement. Deuterium incorporation in WT and D93N hexamers follows apparent first-order exponential kinetics from 10 s to 40 and 60 min respectively, when two maximum values are reached (Figure2B). These values of 67 and 75 D's (after correction for back exchange (17%) and dilution effect (10%)), on 129

Figure 2 .

 2 Figure 2. Overall and local dynamics of WT and D93N Mt-NDPK. (A) Scheme of full length continuous labeling HDX-MS experiment. (B) Plot of deuterium incorporation over time, after correction for back-exchange and dilution. On top, arrows refer to analysis at peptide level on panels C and D. (C, D) Relative percent of deuterium incorporation into each peptide (Figure S2C-D) mapped onto the protein surface using a blue (0%) to red (100%) scale. For WT (C) and D93N (D), a top view along the 3-fold axis of the 6-mer (first column) and the dimer interface after splitting the two trimers (second column) are shown. The white contour outlines the fingerprint of the dimer interface. Residues with no data are coloured in black.

Figure 3 .

 3 Figure 3. Unfolding pathway at full length and peptide level of D93N (A-B) and WT (C-D) Mt-NDPK. Each sample was pre-incubated during 16 hours at different concentrations of urea.Unfolding was monitored by intrinsic fluorescence (blue circles), residual enzymatic activity (red squares)[START_REF] Georgescauld | Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of mycobacterium tuberculosis[END_REF] (A, C). The values are normalized to the experiment in the absence of urea. D93N unfolding was also monitored by a 12 sec pulse labeling deuterium incorporation at full-length level (green diamond) (C). The relative percentage of deuterium incorporation measured at peptide level with a 12 sec D 2 O pulse for WT in 0, 3, 4, and 5 M urea (B) and D93N in 0, 1, 2 and 3 M urea (D) was mapped onto the crystal structure of Mt-NDPK using a blue (0%) to red (100%) scale (FigureS3). A top view (left) and a split dimer interface (right) were shown. When the hexamer was dissociated or unfolded, it was split into three dimers or six randomly oriented monomers, respectively. The white contour on columns two and four outlines the fingerprint of the dimer interface.

Figure 4 .

 4 Figure 4. Representative HD-XMS spectra, obtained during peptic peptide analysis for WT and D93N Mt-NDPK after a 12 second pulse at different urea concentrations. The detailed experimental scheme is shown in the figure S3B. Mass spectra of the +2 charge state of peptides 9-21 and 25-35

Figure 5 .

 5 Figure 5. Unfolding and refolding pathways of WT Mt-NDPK. (A) Refolding (blue lines) versus unfolding (red lines) pathways measured by intrinsic fluorescence (full circles) and enzyme activity (empty squares) after 16 hours pre-incubation in GuHCl.[START_REF] Georgescauld | Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of mycobacterium tuberculosis[END_REF] The lines do not represent theoretical models but were drawn to help the reader. (B) Refolding and assembly pathway of WT Mt-NDPK. The percentage of deuterium incorporation after 12 seconds D 2 O pulse at different refolding time is plotted onto the structure (FigureS4B). Six monomers in random orientation are drawn when unfolded. For each time point, a top view (left) and a split dimer interface of three split dimers (right) are shown. Residues with no data are colored in black. Note for refolding experiments that at this low protein concentration, only dimers are formed. Hexamers are formed at ten time higher concentrations.[START_REF] Georgescauld | Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of mycobacterium tuberculosis[END_REF]

.

  Structure of Mt-NDPK (A) View of a Mt-NDPK monomer with labelled secondary structural elements. (B) Side view of a dimer showing the dimer interface (residues 17,19-21, 23- 24, 27, 33-38, and 72) and the active site pocket (K10, Y50, R104, N114, H117, S119, E128). (C) Top view of a trimer. At the trimer interface (residues 16, 25, 28-31, 79-80, 83-84, 87-88, 93-96, 98-102, and 105-110) the Kpn/α 0 foldon (the Kpn-loop and the α 0 helix are colored in magenta and pink, respectively), and the R80-D93 salt bridge (sticks) are involved in hexamer assembly. The trimers stack 'head-to-head' and not 'head-to-tail' such that the Kpn/α 0 is exposed on either side of the hexamer. (D) Side view of the surface of the 6-color hexamer Mt-NDPK. The active site is colored in yellow. In (B) and (C), the chains are colored as in (D).

ACKNOWLEDGEMENTS

The authors wish to thank Pr Anna Giartosio, Pr Ioan Lascu, Dr Erin Henninger and the European Synchrotron Radiation Facility (ESRF, Grenoble, France). This work was supported by the Fondation ARC pour la Recherche sur le Cancer (FR) to F.G and P.M (Grant SFI20101201793) and by Agence Nationale de la Recherche to A.D. (Grant ANR-12-BSV8-024) and P.M. (Grant ANR-11-BSV8-015-03 and ANR-16-CE11-0032). P.M. was supported by the LABEX DYNAMO (Grant ANR-11-LABX-0011) and EQUIPEX CACSICE (ANR-11-EQPX-0008CACSICE). Keywords: structure-activity relationship · protein folding · hydrogen deuterium exchangemass spectrometry · nucleoside diphosphate kinase · oligomerization


D Benedikovic
email: daniel.benedikovic@u-psud.fr

L Vivien

P Cheben

J H Schmid

S Wang

D.-X Xu

B Lamontagne

J Lapointe

S Janz

C Alonso-Ramos

R Halir

A Ortega-Moñux

J.-G Wangüemert-Pérez

I Molina-Fernández

J.-M Fédéli

M Dado

High-efficiency single etch

Efficient optical coupling interfaces are one of the key building blocks in photonics integrated circuitry. Coupling of light to and from microphotonic waveguides has been recognized as a major practical challenge since the early years of integrated optics. The coupling is particularly difficult for silicon-on-insulator technology, since the cross-sectional area of silicon waveguides is more than two orders of magnitude smaller than that of a standard single-mode fiber. The surface grating couplers are an effective approach to circumvent this issue [START_REF] Halir | Waveguide sub-wavelength structures: a review of principles and applications[END_REF][START_REF] Benedikovic | High-efficiency single etch step apodized surface grating coupler using subwavelength structure[END_REF][START_REF] Benedikovic | High-efficiency fully etched fiber-chip grating couplers with subwavelength structures for datacom and telecom applications[END_REF][START_REF] Benedikovic | Single-etch subwavelength engineered fiber-chip grating couplers for 1.3 µm datacom wavelength band[END_REF][START_REF] Benedikovic | Subwavelength index engineered surface grating coupler with sub-decibel efficiency for 220-nm silicon-on-insulator waveguides[END_REF].

We have demonstrated by theory and experiments a series of subwavelength refractive index engineered surface grating couplers that require only a one fabrication step of lithography patterning and reactive ion etching, while providing the state-of-the-art coupling performance [START_REF] Benedikovic | High-efficiency single etch step apodized surface grating coupler using subwavelength structure[END_REF][START_REF] Benedikovic | High-efficiency fully etched fiber-chip grating couplers with subwavelength structures for datacom and telecom applications[END_REF][START_REF] Benedikovic | Single-etch subwavelength engineered fiber-chip grating couplers for 1.3 µm datacom wavelength band[END_REF][START_REF] Benedikovic | Subwavelength index engineered surface grating coupler with sub-decibel efficiency for 220-nm silicon-on-insulator waveguides[END_REF]. Specifically, we have reported various apodized grating couplers with an optimized directionality, with a coupling efficiency of up to -2.2 dB and -2.5 dB for wavelengths near 1.55 and 1.3 μm, respectively [START_REF] Benedikovic | High-efficiency single etch step apodized surface grating coupler using subwavelength structure[END_REF][START_REF] Benedikovic | High-efficiency fully etched fiber-chip grating couplers with subwavelength structures for datacom and telecom applications[END_REF][START_REF] Benedikovic | Single-etch subwavelength engineered fiber-chip grating couplers for 1.3 µm datacom wavelength band[END_REF]. The grating couplers were implemented in a standard 220-nm-thick silicon-on-insulator substrates, as typically offered by silicon photonics foundries. In addition, a sub-decibel fiber-chip coupling efficiency of -0.7 dB has been experimentally realized for the first time in 220-nm silicon-on-insulator waveguides by utilizing the complementary metal-oxide-semiconductor-compatible manufacturing via backside processing [START_REF] Benedikovic | Subwavelength index engineered surface grating coupler with sub-decibel efficiency for 220-nm silicon-on-insulator waveguides[END_REF].

Figure :

 : Figure: Single-etch apodized surface grating couplers in (a) linearly-tapered and (b) curved layout configurations, and subwavelength grating nanostructure inside the trenches. (c) Optical micrograph image of the surface grating couplers layout with metal reflector underneath. (d) Detail view of the surface grating coupler with metal reflector.


Zane Bicevska
email: zane.bicevska@di.lv

Applying of Smart Technologies: Evaluation of Effectiveness

Keywords:

This paper is dedicated to various aspects and perspectives of applying the smart technologies in software solutions. Based on experience acquired during software development projects, the author proposes criteria to evaluate the effectiveness of enhancing of existing products with smart technologies. The evaluation of effectiveness can be used to support decision making on implementing the smart technologies in existing products or products in development.

Introduction

The main goal in development of an information system is to ensure conformity of the software with the initial system requirements. But it also should build a solid base for further information system's development, improvement and maintenance. One of the possibilities to establish long-term reliable software is to include additional support mechanisms for better usage, maintenance and enhancement already during the initial design and development phases of the information system.

The idea of smart technologies [START_REF] Bicevska | Smart Technologies in Software Life Cycle[END_REF] bases on the vision about "clever" software that like living beings is able to "self-management". Hence the smart software is able to handle unpredictable events in unknown environments. Unlike external solutions providing specific supporting features from outside a smart technology conform software is able to react on external and internal events adequately by itself. The typical external events are changes in the infrastructure or changes in external systems; the typical internal events are changes in database structures or new versions of source code.

Smart technology oriented approach implies that the core functionality of software solution should be enhanced and integrated with several additional features supporting the usage, maintenance and further development of software.

The identified components of smart technologies are following:

-Business model-based system's functionality -the functionality of information system changes according to business processes described in the configuration. -Built-in version management and data synchronization -automatic updating of software versions from the central server, including the conversion of data structures and data. -Built-in self-testing -ability to check the internal integrity by automatic execution of test cases in the productive environment and to inform users and developers about detected inconsistencies.

-Environment testing -ability to check the external environment (options of operating system, data base management system etc.), to adapt itself to the specific environment and/ or to inform developers about detected inconsistencies. -Data quality monitoring -ability to check the completeness and integrity of data stored in the database. -Availability testing -monitoring of system availability using agent technologies; ability to inform developers remote about the status of the software and additional components needed for a correct functioning. -Security and load testing -monitoring of system security using agent technologies; ability to provide monitoring of performance and load balancing.

The smart technologies include already practically verified components like selftesting [START_REF] Bicevska | Self-testing: A New Testing Approach[END_REF], external environment testing [START_REF] Rauhvargers | Environment Testing Enabled Software -a Step Towards Execution Context Awareness[END_REF], intelligent version management [START_REF] Bicevska | Applying of Smart Technologies in Software Development: Automated Version Updating[END_REF] but also other features that are topicality for further research.

The proposed concept of smart technologies is related to the concept of autonomous systems announced 2001 by IBM [START_REF] Ganek | The Dawning of the Autonomic Computing Era[END_REF][START_REF] Sterritt | Towards an Autonomic Computing Environment[END_REF][START_REF] Lightstone | Foundations of Autonomic Computing Development[END_REF]. Both concepts set as a goal the increasing of software "intellect" to achieve a wide range of non-functional features -an ability to adapt itself to variable external events, self-recovering, self-optimizing etc. -, but the proposed technical solutions are different.

The decision about applying of smart technologies can be made not only during the initial design and development of an information system but also in later phases of the development or maintenance, e.g. within the new release of the software or as an separate project. But there should be a SWOT-analysis made to settle on the applying of smart technologies.

A number of criteria could be evaluated within the decision making to prove or to disapprove the usage of smart technologies in the concrete case. The goal of this paper is to present empirically indentified criteria for evaluation the effectiveness of applying smart technologies.

2 Criteria for Evaluation of Effectiveness

Effectiveness vs. Efficiency

The notion of effectiveness is explained as a "power to be effective; the quality of being able to bring about an effect" [START_REF]The Free Dictionary -Effectiveness[END_REF] or "the extent to which a program or service is meeting its stated goals and objectives" [9].

In contradiction to efficiency that describes the degree of achievement of definite parameter values and in fact is close to the notion of productivity, the effectiveness is an ability of an object or a process to fulfill predefined goals under definite conditions.

The evaluation is described as a "systematic determination of merit, worth, and significance of something or someone using criteria against a set of standards" [10].

The most precise form of evaluation is measurement, but it is not the only way to evaluate something. The measurement is "the process of assigning a number to an attribute (or phenomenon) according to a rule or set of rules" [11].

So there can be identified the following preconditions to evaluate the effectiveness:

-Choice of criteria characteristic for effectiveness;

-Identifying of optimal values for criteria (etalon values); -Description of evaluation (measurement) process.

The next chapters outline a proposal for different criteria inferred from the practical evidence collected in real software development projects. Identifying of optimal values for criteria and description of an exact evaluation process to have a basis for decision making for or against usage of smart technologies in information systems are a subject for further research.

Economical Criterion: Return on Investment

The prime task in every commercial project is to maximize the potential return on investment. Every rational market participant will be ready to invest in a project only in that case if the investments in closer or further future could bring a profit [START_REF] Sullivan | Economics: Principles in action[END_REF].

Hence the first criterion proposed for the evaluation of effectiveness is a prospective return on investment (ROI). An estimation of ROI gives an approach to measure the investment potential by comparing the expected gains to investment costs. The value of this variable can be calculated on the basis of an investment return plan containing a comparison of an initial investment and discounted future cash flows: ROI is the ratio of average prospective cash flow over the period of the project divided by the initial investment outlay.

The commonly used effort, cost and schedule estimating methods (e.g. COCOMO [START_REF] Boehm | Software Cost Estimation with COCOMO II (with CD-ROM)[END_REF]) can be employed for the estimation of an initial investment. But the following adjustments should be taken into account:

-Previous experience and efforts in implementing of smart technologies can reduce the necessary initial investments; it is rather possible that already once developed smart technology components will be adaptable for similar tasks than if the components are to be developed from scratch. -Modularity of the product to be prepared with smart technologies: the more modular is the software product the easier is to add to it "just" one more module. -The available time schedule should be comfortable enough to implement and/or integrate smart technology features besides the basic functionality desired by the customer explicitly. -Feedback and level of user tolerance show whether the concrete users will be able and ready to tolerate short-term inconveniences if they would arise and take an active part in the improvement of the product features. Not all customers are happy to be involved in "experiments". Doubtless more open for innovations in their projects are long-standing customers with high allegiance to the supplier.

For estimation of prospective cash flows can be used both bottom-up and top-down approaches. The bottom-up approach estimates the minimal savings of costs/ expenses (could be achieved by enhancing the software product (total projected savings). The typical examples are transport and personnel costs that were spent for travelling to end users as well as installation materials, hardcopy of documentation, partly user support resources etc. It is some kind of "production-oriented" way to find the break-even-point where the initial investment becomes profitable.

The top-down approach is based on the estimation of the market opportunities, i.e. the achievable prices and sales volume is evaluated to have a view of feasible gains of smart technologies.

The simultaneous usage of both approaches returns the gap where the prospective cash flows should fit in to be realistic.

When both components -the initial investment and prospective cash flows -are obtained, they should be made comparable. The usual way to do it is a discounting of future cash flows with the rate of interest.

The discounted present value (for one cash flow in one future period) is expressed as:

DPV = FV (1 + i) n = FV(1 d) n , (1)
where -DPV is the discounted present value of the future cash flow (FV), or FV adjusted for the delay in receipt; -FV is the nominal value of a cash flow amount in a future period; i is the interest rate, which reflects the cost of tying up capital and may also allow for the risk that the payment may not be received in full; d is the discount rate, which is i/(1+ i), ie the interest rate expressed as a deduction at the beginning of the year instead of an addition at the end of the year; n is the time in years before the future cash flow occurs. [14] Incorporation of smart technologies into software products is beneficial if the initial investment is less than the present net value of prospective cash flows.

But there should never be forgotten that the calculations are based on the estimated (not real!) values. In particular the effort estimation methods can be very inaccurate in some cases [START_REF] Schach | Object-oriented and Classical Software Engineering[END_REF].

Quality Criteria

The quality of a product or service is intrinsically linked with reputation and hence is crucial to organisation's sustainability. Considerations on quality assurance could enhance integration of smart technologies into strategically important products, even if economic returns on such investments can be expected only in long-term.

The quality achieved by implementing smart technologies can hardly be measured. Prevailing practice in quality evaluation is based on experience values acquired during similar projects.

One of the possible quality criteria is an efficiency of customer support. This variable shows in what extent the level of client support could be improved if the software product is equipped with smart technologies by the same number of support staff. The efficiency of customer support can be qualitatively measured by interviewing clients and analyzing selected users' messages, etc. The quantitative indicators can include number of supported users (number of processed cases) in a certain period of time or average proceeding time of problem processing: Either more clients should receive support, or service quality for existing clients should be increased by the given number of support resources.

Alternatively there could be estimated if the existing customer support level can be assured by reduced amount of support staff. If implementation of smart technologies has not resulted in increased number of clients, still the effectiveness is positive due to fewer resources used to provide more qualitative support to the same number of clients.

Another possible criterion for evaluation of the effectiveness of smart technologies is efficiency of maintenance. As the developers consider software maintenance as a very significant area for applying smart technologies this criterion would show if the quality of software equipped with smart technologies is increased. Quick problem detecting, short problem solving time, low number of user reported errors -all these are quantifiable variables indicating high quality of maintenance and resulting in clients' satisfaction.

Organizational and Marketing Criteria

Unlike the previously described criteria targeted at profit and reputation, the organizational criteria deal with developers' organizational and technical ability to carry out the development and the necessary integration of smart technologies into end-products.

The development of smart technologies is not possible without highly professional system architect and skilful (able) developers. On the one hand, the development of smart technologies requires good academic knowledge and professional background, but on the other hand, such development also requires profound understanding of product's architecture and functionality. During the development issues such as workdiscipline, rigorous following the guidelines and avoiding the use of specific techniques that could cumber maintenance should be considered.

These criteria are hardly quantifiable; therefore evaluation of these criteria is preferably done by experienced architects and developers.

Since implementation of smart technologies contribute to significant changes in software construction, potential error probability is not excluded; therefore a welldesigned crisis management plan should exist from organizational point of view. Strict and prudent managers possessing power of persuasion are the best support in such cases. The developed end-product should undergo scrupulous testing, therefore appropriate technical support including virtual machines and powerful central server should be available.

The main goal of applying smart technologies is maximizing of profits expected in long-term. Reduced expenses or improved quality of software -the customer should have a clear concept of an added value. The task of marketing activities is to promote the emotional and practical adjustment of the customer to the improved product, to motivate the user to be familiar with the new features of the product. So, the decision about (for or against) smart technologies means also the answering the question if there is a marketing power in the enterprise that is able to "make money" from the technological innovation.

Empirical Data Sources

Principles of smart technologies are partially implemented and approved in software development and maintenance projects. Although, in neither of software products smart technologies were implemented to the full extent, the gathered experience shows that usage of even only several features or narrowed version of features can bring an additional effectiveness and economical benefit.

The criteria for evaluation of effectiveness briefly described in the previous chapter are all identified in concrete development projects where particular smart technology features were added to existing information systems supplementary.

One of the future research tasks is the gathering of statistics to demonstrate whether and how the usage of smart technologies improves the quality and maintainability of software. The development projects serving as a source for the actual research are shortly described in the next chapters.

Version Management and Environment Checking Components

The Project A was started 2002, and the main task of it was to create a finance management information system according to system requirements formulated by a ministry in Latvia. The supplier had to develop software for structured gathering of financial information on several hierarchal levels.

Already shortly after the first release of the solution new customers accrued from the importance of the solution's functionality in the public sector. The number of users grew up from ca 100 in January 2003 to ca 500 in January 2004. In the face of continuous increasing demand the information system was transferred to a standardised software product purchasable in three different versions (lite, standard, advanced) and two separately usable functional models.

At the end of 2006 the solution in its different completions was used by ca. 1500 users. The system was supposed to run in over than 600 public offices located throughout the territory of Latvia ensuring regular gathering of information by different time periods. Furthermore, the specific requirements dictated that in certain periods the most users will use the system simultaneously. Since it was required that the system should be able to run not only in offices provided with internet connection but also in offices with irregular and instable internet connection or even in offices without it, the system architecture was based on client-server application principles with central data base and local desktop applications that were able to run either in online mode if internet was available, or offline in cases of internet absence.

In view of a large amount of users and their geographical location as well as very restrictive agreements with customers and limited resources allocated for installation and maintenance services, developers made a decision to enhance the software product with one of the smart technology features -the automated version management including both installing and updating. In approximately a year the developers added to the software product one more smart technology component -the automated environment checking including a comprehensive messaging mechanism.

As a result an improved version of the software product was obtained ensuring completely automated remote installing and updating of software versions in different target environments (different computer systems with various operating systems, office packages etc.).

The automated feature for checking of external environment was developed as an independent but configurable module providing the "spying" (pre-checking of target environment) and informing functions necessary for detecting the adequacy of the target environment's parameters to the minimal technical values.

The acquired result exceeded the expectancy: The features of automated version updating and automated environment checking have been significantly lightened the processes of software installation and maintenance as well as reduced the consultant resources necessary for the user support.

The following conclusions were captured within the project:

1. Adding of smart technologies to the software after the development is useful though requires more resources than including smart technology already in the software architecture design phase. 2. Implementation of smart technology principle in software takes fewer resources than full-range configuration support. In the same time smart technology places fewer constraints on the acceptable means of expression. 3. Smart technologies allow reducing the efforts (time, resources) for software testing and setting up, thus increasing the client service level significantly. 4. Smart technologies assist to provide software performance in a changing environment and environment containing heterogeneous platforms and infrastructure. Nevertheless mechanisms of smart technologies need regular adaptation to the environment changes. 5. It is very important to provide in-depth messaging mechanism to inform the developers about indicated problems in time. 6. Even the mechanisms making the users work easier should be appropriate explained to end users. The absence of a human in the set-up process of software is in discrepancy with users' former experience.

Version Management and Data Synchronisation Components

The Project B was started 2008, and the main task was to create a completely new version of an over 10 years old software product (to rewrite it). As the product had over 50 installations in various places of Latvia, there was already during the design phase decided to use the successful experience from the Project A and to include features of smart technologies into the new version of the product. As enhancement was automated version management planed. During the detailed analysis there was an additional important smart technology feature -advanced data synchronising -identified. This feature was necessary for the effective distribution of configuring and classifying information from the central database to local bases and for collecting of data entered by users from local data bases to the central base.

The modernisation, including the complete recoding of the core functionality, took approximately eight man-months. About 50% of the effort was spent for adaptation of data synchronisation module (a sketch of the feature was already there) to the specific requirements. The product was successfully completed, and it is being offered in the market now.

The implemented solution of automated initial installation of the product was not as successful as originally desired. Due to specifics of the used commercial database management system there was quite often assistance by the developers necessary to ensure, the initial installation will be performed successfully.

However the automated updating of version including the data synchronisation worked outstanding; it saved resources spent for user support and reduced effort necessary for software maintenance indeed.

The project results analysis passed after the project's end showed the main reason for the waste of resources -the chosen technical solution of data synchronisation.

The version installing and updating in the Project A was managed according to a specific configuration file containing statements for controls and setup and the software solution was able to interpret these statements adequately. The Project B had another technical strategy for data synchronization management -the control flow statements were stored in the database.

The following conclusions were captured within the project:

1. The moment of decision-making about enhancing the information system with the features of smart technology is less important than the concrete technical solution. 2. Implementing the functions of smart technologies it is advisable to use proven and robust architectural and technical solutions -it will reduce the number of problems to be solved simultaneously and improve the controllability of the development process. 3. Although the clients approve opportunities provided by the smart technologies, usually, they are not willing to provide additional financial means to ensure them. 4. The smart technologies are certainly cost-effective and should pay-off in long-term business projects and long-term cooperation with notable number of users and differently configured users' workstations.

Conclusions

The following conclusions result from the practical experience described above:

-The prospective effect from the applying of smart technologies is considerable; it ranges over client service quality up to increasing of staff qualification. -Even usage of single or several smart technology features can be profitable.

-Economical aspects are the main barrier for wider spread of smart technologies.

-Only very qualified and skilled suppliers are able to fulfil the set of preconditions necessary to ensure the successful usage of smart technologies in commercial projects. -Other hard evaluable factors like reputation of enterprise, marketing/sales capacity, user support level etc. also impact the final decision about the applying of smart technologies.