Philippe Delepelaire
email: philippe.delepelaire@ibpc.fr

Keywords: Iron, siderophore, heme, substrate-binding protein

Iron acquisition is an essential aspect of cell physiology for most bacteria. Although much is known about how bacteria initially recognize the various iron sources they can encounter, whether siderophore, heme, host iron/heme binding proteins, much less is known about how the iron containing compounds (Fe 2+ , Fe 3+ , Fe 3+ -siderophore complex or heme) are transported across the cytoplasmic membrane. This last transport step is powered by specific ABC (ATP-Binding-Cassette) transporters, made up of a substrate binding protein (SBP) that delivers its cargo to the TMD (TransMembrane Domain) of the ABC transporter triggering the entry of the substrate inside the cytoplasm upon catalytic activity of the ABC module. This review focuses on structural aspects of the functioning of such ABC transporters with the most part devoted to the substrate binding proteins.

Introduction

This review deals with the structure and function of ATP-binding cassette (ABC) transporters specifically involved in heme/iron transport across membranes. Whether monoderm or diderm, bacteria devote a substantial part of their genomes to the acquisition of iron containing compounds. Iron is needed as an essential component of many key enzymes and also takes part under the heme form to several constituents of respiratory chains. Each Escherichia coli cell contains ca. 200000-800000 iron atoms [START_REF] Hartmann | Iron Uptake and Iron Limited Growth of Escherichia coli K12[END_REF], which it must acquire from its environment during a cell division time. Iron being also toxic under its Fe 2+ form (due to the Fenton reaction), its homeostasis is strictly controlled at several levels [START_REF] Dukan | Hypochlorous Acid Stress in Escherichia coli: Resistance, DNA Damage, and Comparison with Hydrogen Peroxide Stress[END_REF][START_REF] Touati | Lethal Oxidative Damage and Mutagenesis Are Generated by Iron in delta-fur Mutants of Escherichia coli: Protective Role of Superoxide Dismutase[END_REF]. At near-neutral and basic pH, and in the presence of oxygen, Fe is essentially found under the form of highly insoluble ferri-hydroxy compounds, and although iron is an important component of terrestrial media, its soluble concentration is extremely low, well below the needs of the bacteria. Under acidic and/or reducing conditions iron is mostly found under the readily soluble form Fe 2+ , that is acquired by many bacteria. When developing in mammalian hosts, bacteria face the so-called nutritional immunity imposed by the host, sequestering Fe in very high affinity components [START_REF] Hood | Nutritional immunity: transition metals at the pathogen-host interface[END_REF], that has to be overcome by the bacterium to ensure a successful colonization. Under iron starvation, most bacteria synthesize organic compounds with very high affinity for Fe 3+ , named siderophores and able to displace Fe 3+ from its initial sources [START_REF] Ratledge | Iron Metabolism in Pathogenic Bacteria[END_REF].

In the case of Gram-negative bacteria, the Fe 3+ -siderophore complex is internalized in the periplasmic space by specialized outer membrane (OM) transporters. They are called TonB-Dependent Transporters (TBDT), and the TonB complex that sits in the cytoplasmic membrane conveys the energy of the proton-motive force (pmf) to the TBDT to allow entry of the substrate bound to the extracellular site of the receptor into the periplasm [START_REF] Noinaj | TonB-Dependent Transporters: Regulation, Structure, and Function[END_REF][START_REF] Wandersman | Bacterial Iron Sources: From Siderophores to Hemophores[END_REF]. Most bacterial species also express receptors for so-called xenosiderophores, synthesized by other microorganisms. Among the most potent siderophores is enterobactin (the E. coli siderophore, also named enterochelin) with an affinity constant for Fe 3+ over 10 50 M -1 [START_REF] Carrano | Ferric ion sequestering agents. 2. Kinetics and mechanism of iron removal from transferrin by enterobactin and synthetic tricatechols[END_REF].

Transport of the Fe 3+ containing compound across the cytoplasmic membrane is ensured in most cases by specific ABC transporters consisting of at least one soluble substrate binding protein (SBP, also named periplasmic binding protein for Gram-negative bacteria, or PBP [START_REF] Kalckar | The Periplasmic Galactose Binding Protein of Escherichia coli[END_REF]) and a membrane bound ABC complex, which in turn in most cases contains two integral transmembrane domains (TMD) and two peripheral ABC modules [START_REF] Davidson | Structure, Function, and Evolution of Bacterial ATP-Binding Cassette Systems[END_REF]. In the case of Gram-positive bacteria, the Fe 3+ -siderophore complex is recognized by substrate binding proteins that are in most cases retained in the cytoplasmic membrane by a lipid anchor. Although heme is also ultimately bound to a SBP in the case of Gram-positive bacteria, heme/heme source recognition may involve several previous steps occuring at the cell surface by proteins either secreted in the milieu and/or anchored at different levels of the cell wall [START_REF] Hood | Nutritional immunity: transition metals at the pathogen-host interface[END_REF][START_REF] Choby | Heme Synthesis and Acquisition in Bacterial Pathogens[END_REF][START_REF] Sheldon | Iron Acquisition Strategies of Bacterial Pathogens[END_REF]. The siderophore/heme loaded SBP is recognized by the TMD subunit of the ABC complex, that internalizes the substrate during its cycle, powered by ATP binding and hydrolysis, by the ABC subunit. The TMD, as well as the ABC might be encoded by either one or two genes. These ABC transporters belong to the larger class of ABC importers that have been subdivided at least into two subfamilies [START_REF] Beis | Structural basis for the mechanism of ABC transporters[END_REF][START_REF] Locher | Mechanistic diversity in ATP-binding cassette (ABC) transporters[END_REF]. The type I ABC importer is exemplified by the Escherichia coli Maltose transporter (MalEFGK2, MalF and MalG being the TMD, MalK the ABC and MalE the SBP) whereas the type II is exemplified by the BtuC2D2F from E. coli that transports the B12 vitamin (BtuC being the TMD, BtuD the ABC, and BtuF the SBP). Most often the TMD's of type II ABC importers have 10 TM helices, whereas their number is smaller for type I ABC importers. Differences are also found in the mode of action of the two families, where in the « resting state » of the transporter, the cavity at the interface of the TMD's is open toward to the cytoplasm in type I importers and towards the periplasm in type II importers [START_REF] Beis | Structural basis for the mechanism of ABC transporters[END_REF][START_REF] Locher | Mechanistic diversity in ATP-binding cassette (ABC) transporters[END_REF][START_REF] Chen | Molecular mechanism of the Escherichia coli maltose transporter[END_REF]. This review will specifically deal with some recent results concerning the structure and function of Fe/siderophores/heme transport via ABC transporters. Fig. 1 shows the general architecture of those systems across the Gram-negative envelope.

I. Substrate binding proteins: structural aspects

This part will mostly deal with the promiscuity displayed by binding proteins, that can bind several substrates, as opposed to the rather strict specificity of the siderophore outer membrane receptors that often bind only one substrate with quite high affinity. Escherichia coli K12 synthesizes one siderophore (enterobactin or enterochelin, of the catecholate type), whose OM receptor is FepA. It synthesizes several other outer membrane receptors, that recognize various siderophores CirA (Fe-DHBS) (DHBS, dihydroxybenzoylserine, a breakdown product of enterobactin), YoeA (the coding region is interrupted by an IS2), YncD (the substrate is unknown), YbiL or Fiu (DHBS3Fe), FhuA (ferrichrome), FecA (Fe-dicitrate), FhuE (Fe-rhodotorulic acid/ coprogen). Substrate binding proteins are bilobate proteins that bind their substrate at the boundary between the two lobes before transferring it to the ABC TMD domain of the ABC transporter. An initial classification of substrate-binding proteins recognized three classes, according to the number of links between the two domains. Class I SBP, exemplified by the MBP (Maltose Binding Protein) has three links between the two lobes.

Class II SBP exemplified by Histidine-Binding Protein has two links between the two lobes, and class III SBP, exemplified by BtuF, the vitamin B12 binding protein has only one link between the two lobes. A more recent classification based on structural alignments of SBP's enlarged the number of classes/clusters up to 6, the initial class II being subdivided in four structurally distinct clusters [START_REF] Berntsson | A structural classification of substrate-binding proteins[END_REF]. Up to now, no class I SBP has been identified as involved in iron/iron complex transport in bacteria, and all of them belong either to class III (structural class A from [START_REF] Berntsson | A structural classification of substrate-binding proteins[END_REF]) or class II (structural classes C and D from [START_REF] Berntsson | A structural classification of substrate-binding proteins[END_REF]).

Most structural data related to SBP's came from crystallographic studies of the free and substrate-bound forms, whereby they are most likely trapped in low-energy states that do not reflect their dynamic properties.

The binding protein has to fulfill several functions, first of efficiently binding the substrate when it arrives the periplasm, then to bind to the ABC transporter in order to release its cargo, and finally to cycle back to its initial free state in the periplasmic space. There is the common belief that binding proteins are in stoichiometric excess as compared to the ABC transporters [START_REF] Schmidt | The quantitative and condition-dependent Escherichia coli proteome[END_REF].

As compared to class I or class II SBP's, the main characteristics of the class III SBP's are the much more modest, or even absent, large movements of one domain with respect to the other upon substrate binding. However, it is also well possible that the free forms of the binding proteins undergo larger relative movements of the two domains than the bound forms, this being a basis for the discrimination by the TMD domains.

Siderophores have been divided in at least four classes according to the metal chelating groups, catecholates, carboxylates, hydroxamates, or mixed type. Despite this rather simple classification, there is a huge structural diversity of siderophores even in a single class, arising from the modifications added on the siderophore backbone during its synthesis [START_REF] Hider | Chemistry and biology of siderophores[END_REF][START_REF] Neilands | Siderophores: Structure and Function of Microbial Iron Transport Compounds[END_REF]. As a consequence the binding proteins have adapted to this huge diversity, and the following chapter will underline this diversity. The situation is somewhat different for heme binding proteins, as the iron atom is bound by the four pyrroles of the heme molecule, leaving two potential valences available for binding.

IA Class III (structural class A) siderophore/heme binding proteins (supplementary tables S1 and S2)

Up to now all siderophore binding proteins identified belong to the class III, with only one alpha-helix connecting the two domains, that are each made of a distorted beta-sheet surrounded by alpha-helices. The binding site for the siderophore lies at the intersection of the two domains. The Table SI presents a list of PDB entries corresponding to siderophore binding proteins, as complete as possible at the present time. The class III was initially defined by the structures of both PsaA and TroA, two divalent metal binding proteins of Streptococcus pneumoniae and Treponema pallidum respectively [START_REF] Lawrence | The crystal structure of pneumococcal surface antigen PsaA reveals a metal-binding site and a novel structure for a putative ABC-type binding protein[END_REF][START_REF] Lee | Treponema pallidum TroA is a periplasmic zinc-binding protein with a helical backbone[END_REF]. PsaA, which has been identified as an essential virulence factor in an animal model is likely a Mn 2+ binding protein, whereas TroA is a Zn 2+ binding protein.

IA1 Catecholate binding proteins

-Escherichia coli enterobactin binding protein (PDB codes 2M6K, 2M6L, 3TLK).

Enterobactin (Ent) is the main siderophore synthesized by E. coli. It belongs to the catecholate family (it is essentially a cyclic siderophore made up of three molecules of dihydroxybenzoylserine, DHBS), and the complex Fe 3+ -Ent is transported across the OM by the TBDT FepA, and then bound to the FepB binding protein, before its transport across the IM by the FepCDG complex. Surprisingly structural studies on FepB either in its free form or complexed to enterobactin, have been scant and only appeared recently one by X-ray crystallography [START_REF] Li | An unusual crystal structure of ferricenterobactin bound FepB suggests novel functions of FepB in microbial iron uptake[END_REF] the other one by NMR in solution [START_REF] Chu | The solution structure, binding properties, and dynamics of the bacterial siderophore-binding protein FepB[END_REF]. In the X-ray study FepB was crystallized in the presence of enterobactin, and an unusual form was observed, with three FepB-FeEnt molecules in the asymetric unit with their binding sites facing each other, together with another FeEnt molecule shared between the three FepB molecules. Each subunit has ca. 1100 Å 2 buried upon interaction. It is not completely clear whether the supplementary FeEnt molecule is necessary for complex formation. This crystal (PDB 3TLK) form was obtained with a 3-fold excess of FeEnt as compared to FepB (30 vs 10 µM). Although there is no doubt that this form might exist, its biological meaning is questionable, as there was no study as a function of concentration of the appearance of this FepB3FeEnt4 complex. However a very crude estimation of the number of FepB molecules (under full induction, a few thousand) given the periplasmic space volume, is actually within the 10 µM range (assuming a periplasmic volume of 0,1 fL) used in this study. The use of a FepDG mutant would clarify this point.

A similar observation has been made with a linear siderophore shared by two binding protein molecules is the case of CeuE of Campylobacter jejuni in complex with FeMECAM (1,3,5-N,N',N″-tris-(2,3-dihydroxybenzoyl)-triaminomethylbenzene, a tri-catecholate synthetic siderophore) [START_REF] Muller | An [{Fe(mecam)}2]6-bridge in the crystal structure of a ferric enterobactin binding protein[END_REF]. However in this last case, there is no extensive contact between the two polypeptide chains of the asymetric unit, that are linked together via the linear FeMECAM, each MECAM molecule respectively contributing 4 and 2 valences for holding the iron atom

(see below).

There is no crystal structure of the apo form of E. coli FepB. The NMR study (which, by the way was quite a tour de force, given the size of the protein) has provided many interesting insights in particular into the dynamic of the protein. The alpha-helices are well defined, whereas this is not so true for the beta-sheets (perdeuteration effect). The comparison of the chemical shift perturbations between GaEnt-FepB and FepB allows to see three zones (145-150, 225-245 and 290-310) where GaEnt binding affects the structure of the protein (see Fig. S1); GaEnt was used instead of FeEnt, as FeEnt perturbs too much the NMR spectra ; GaEnt binds to FepB with a similar affinity compared to FeEnt. There is no large effect on overall structure of FepB upon binding of GaEnt, however several residues in the 225-242 loop region, as well as 77-81 and 293-303 appear more rigid upon ligand binding. HoloFepB also appears slightly more compact than apoFepB. Contrarily to class I and II SBP's where domain contacts vary greatly between the unbound and bound forms of the SBP, this is not the case for the class III SBP : contacts between the N-and the C-domains already exist in the apo form, and they are slightly rearranged upon ligand binding. Also the ligand is much more accessible in the class III SBP's than in the class I or II, where it is deeply buried at the domain interface.

-Bacillus subtilis FeuA [START_REF] Peuckert | Structural basis and stereochemistry of triscatecholate siderophore binding by FeuA[END_REF][START_REF] Peuckert | The siderophore binding protein FeuA shows limited promiscuity toward exogenous triscatecholates[END_REF] (PDB codes 2WHY, 2WI8, 2XUZ, 2XV1)

In this case, the protein is held on the cytoplasmic membrane via a lipid anchor, as it is the case for the Gram-positive BP's [START_REF] Tam | Structural, functional, and evolutionary relationships among extracellular solute-binding receptors of bacteria[END_REF]. Upon binding of the natural substrate, bacillibactin, there is a slight closing and rotation movement of the two domains and the substrate adopts a single conformation constrained by the binding site (Fig. 2,I); other substrates (enterobactin and Fe-MECAM are bound by mostly the same residues, with a few exceptions. Bacillibactin binds with a high affinity to FeuA (Kd ca 27 nM), as well as enterobactin (kd 40 nM), Fe-MECAM (Kd 1030 nM). Vibriobactin, the tris catecholate siderophore from Vibrio cholerae does not bind specifically. In the same study, the two surface exposed residues E90 and E220 were proposed as interacting with the membrane components FeuBC. A double mutant FeuAE90A-E220A, although still able to bind bacillibactin, showed impaired iron acquisition. Both Febacillibactin and Fe-enterobactin promote efficient growth of B. subtilis. This is not the case with Fe-MECAM ; this might be due to the difficulty in hydrolysing the MECAM backbone. The binding of MECAM is not the same as for the C. jejuni CeuE binding protein (see below).

-Campylobacter jejuni CeuE (PDB codes 2CHU, 3ZKW, 5A1J, 5A5D, 5A5V, 5AD1, 5ADV, 5ADW, 5LWH, 5LWQ, 5MBQ, 5MBT, 5MBU, 5OAH, 5OD5, 5TCY) Sixteen structures of CeuE have appeared, making it particularly interesting [START_REF] Muller | An [{Fe(mecam)}2]6-bridge in the crystal structure of a ferric enterobactin binding protein[END_REF][START_REF] Raines | Bacteria in an intense competition for iron: Key component of the Campylobacter jejuni iron uptake system scavenges enterobactin hydrolysis product[END_REF][START_REF] Raines | Interactions of a periplasmic binding protein with a tetradentate siderophore mimic[END_REF][START_REF] Wilde | Interactions of the periplasmic binding protein CeuE with Fe(III) n-LICAM(4-) siderophore analogues of varied linker length[END_REF]. There is a very slight closure movement upon substrate binding (3ZKW, apo vs 5ADV two DBS molecules, Fig. S2). This is a particularly interesting case, as most strains of C. jejuni do not appear to synthesize their own siderophores and therefore rely on xenosiderophores. Feenterobactin is a source of iron for C. jejuni. It appears that the trilactone ring of enterobactin can only be hydrolyzed in the periplasm of C. jejuni, as there is only one esterase, Cee residing in this compartment [START_REF] Zeng | Identification and characterization of a periplasmic trilactone esterase, Cee, revealed unique features of ferric enterobactin acquisition in Campylobacter[END_REF]. This points to the fact that the substrate transported across the inner membrane might be different from the one transported across the outer membrane (see Pseudomonas case below).

The first structure of a hexadentate siderophore bound to CeuE was that of Fe-MECAM. In this particular case, a dimer of Fe-MECAM binds two molecules of CeuE. In this case the Fe 3+ coordinations are completely fulfilled by the siderophore catecholate groups, two coming from one MECAM molecule, the others from the other molecule. Such an arrangment is actually incompatible with further transport. It happens that Fe-MECAM is poor iron source for C. jejuni. Whether or not this is due to this arrangement is not known. No details were given in the purification protocol. Several other crystals were obtained in the presence of tetradentate synthetic siderophores as well as with dihydroxybenzoylserine (DHBS). In all those forms, besides the Arg and Lys residues that contribute to bonds with the catecholate, two residues, His227 and Tyr288 make direct contacts with the Fe 3+ to fulfill its coordination requirements (Fig. S2). A very interesting observation was made, as it was shown that the Kd of CeuE for Fe-ENT was much higher than for Fe-bisDHBS (400 vs 10 nM). The 10 nM Kd is in the same range as that observed for FepB-Fe-ENT, for example. The double mutant His227Leu-Tyr288Phe has a Kd of ca 500 nM, indicating that the presence of the two residues H227 and Y288 are crucial for the high affinity towards the Fe-bisDHBS.

-Escherichia coli FitE [START_REF] Shi | Trapping open and closed forms of FitE: a group III periplasmic binding protein[END_REF] (PDB codes 3BE5, 3BE6)

This represents an interesting case : FitE was identified as a putative ferrichrome binding protein from E. coli O157 :H7 [START_REF] Ouyang | Identification and characterization of a novel ABC iron transport system, fit, in Escherichia coli[END_REF], it behaves as a dimer in solution, which is already unusual for a binding protein. The natural substrate is unknown, but given its very hydrophilic nature and its lining with mostly positive residues, a catecholate derivative is more likely to represent the natural substrate. Its sequence is actually closer to E. coli K12 FepB. However, enterobactin (and Fe-citrate) are not substrates of the fit system. Two crystal forms were obtained and for each, the asymetric unit contained two dimers and the four monomers do not share the same conformation, mimicking the open and closed conformations (Fig. 2,II). This indicates that, in the absence of substrate, the binding protein can undergo quite substantial conformational changes, and that the structure is not as rigid as previously thought.

-Vibrio cholerae ViuP [START_REF] Li | Unique iron coordination in ironchelating molecule vibriobactin helps Vibrio cholerae evade mammalian siderocalinmediated immune response[END_REF] (PDB codes 3R5S, 3R5T) This represents also an interesting case. Vibriobactin is a catecholate siderophore having, besides the three catechols, also two oxazolines, and questions have arisen whether the oxazoline nitrogen could coordinate the iron. The structure of the Fe-vibriobactin-ViuP complex shows that one of the oxazoline nitrogen coordinates the iron atom, leading to the rotation and the exposure of one of the catecholate hydroxyl groups towards the solvent (Fig. S3). The structure of the binding site is quite polar with many residues establishing bonds with the siderophore backbone (Table S2). The structure of the vibriobactin also, with one free catechol hydroxyl group, is also in line with the fact that siderocalin-2 (a component of the innate immunity system of the host, able to bind enterobactin) [START_REF] Fischbach | How pathogenic bacteria evade mammalian sabotage in the battle for iron[END_REF] is not able to bind it with high affinity, most likely due to steric hindrance (Kd of 1.4 µM for Fe-Vib2-, compared to 2.6 nM for Fe-Ent3-). This might contribute to the escape of the innate immunity provided by siderocalin-2. In this case, there is also quite a sizeable movement of one lobe with respect to the other upon vibriobactin binding. ViuP shows low (20%) level of primary sequence conservation, as compared to the other Fe-catecholate binding proteins.

-Vibrio cholerae VctP [START_REF] Liu | Crystal structure of periplasmic catecholate-siderophore binding protein VctP from Vibrio cholerae at 1.7 A resolution[END_REF] (3TEF) VctP is a SBP from V. cholerae that binds both vibriobactin and enterobactin. Based on structural alignments and docking studies of both siderophores, the residues potentially involved in siderophore binding have been identified. The surface potential shows a binding site surrounded by positive residues (Fig. S4).

IA2 Hydroxamate binding proteins

-Bacillus subtilis Bsu3320 (FhuD) [START_REF] Schneider | Iron-hydroxamate uptake systems in Bacillus subtilis: identification of a lipoprotein as part of a binding protein-dependent transport system[END_REF] ferrichrome (PDB code 3G9Q) B. subtilis is able to use several hydroxamate siderophores (shizokinen, ferrioxamine B and ferrichrome) and mutants were isolated, that allowed to identify the genes involved. It was shown that the cloned gene could complement an E. coli mutant defective in FhuD, and that the protein was a lipoprotein. The binding site is not surrounded by positives charges, as is the case for the catecholate siderophores (Fig. S5).

-Escherichia coli hydroxamate binding protein FhuD [START_REF] Clarke | X-ray crystallographic structures of the Escherichia coli periplasmic protein FhuD bound to hydroxamate-type siderophores and the antibiotic albomycin[END_REF][START_REF] Clarke | The structure of the ferric siderophore binding protein FhuD complexed with gallichrome[END_REF] (PDB codes 1EFD, 1ESZ, 1K2V, 1K7S).

There is no structure for the apoprotein, so the movements upon siderophore binding are not known. The siderophore binding site is globally more hydrophobic than for catecholates and is lined with several Trp/Phe residues. This was the first structure of a siderophore binding protein. Contrarily to Fe-enterobactin which is negatively charged, Feferrichrome is not charged, and it has been hypothesized that the negative charges around the binding site would repel catechol siderophores (Fig. S6). In the case of albomycin (a mixed ferrichrome-like/uracil analog antibiotic that enters the periplasm via the FhuA receptor [START_REF] Braun | Functional Organization of the Outer Membrane of Escherichia Coli: Phage and Colicin Receptors as Components of Iron Uptake Systems[END_REF]), the antibiotics moiety was not seen in the electron density. As can be seen from the Fig. S6, although the Fe atom occupies almost exactly the same place (not exactly the same as Ga in gallichrome), the backbone of the siderophore is not always in the same position, and it is clear that more space is still available in the binding site.

-Staphylococcus aureus FhuD2 [START_REF] Mariotti | Structural and functional characterization of the Staphylococcus aureus virulence factor and vaccine candidate FhuD2[END_REF][START_REF] Podkowa | Crystal and solution structure analysis of FhuD2 from Staphylococcus aureus in multiple unliganded conformations and bound to ferrioxamine-B[END_REF] (PDB codes 4B8Y, 4FIL, 4FKM, 4FNA) Four structures have appeared, two in the apo form (one with methylated lysines residues, which shows a slightly more closed form than the non methylated one (green)), one with Fe-Ferrioxamine B (magenta), the other one with Fe-Ferrichrome (cyan) (Fig. S7), the two of them more closed than the apo forms, and the one with Fe-Ferrichrome being the most closed form.

The Kd of FhuD2 for ferrichrome (derived from SPR experiment) is ca. 10 nM, and this interaction is stricly dependent upon the presence of iron. FhuD2 is a potential vaccine candidate. As compared to the E. coli FhuD protein, the main residues that establish H-bonds with the siderophore are found on different lobes of the protein, on the N-terminal lobe in E. coli (see above), on the C-terminal lobe in S. aureus ; in the B. cereus case (3TNY) residues from both lobes establish bonds with the siderophore backbone (in this case shizokinen).

FhuD2 can bind ferrichrome, as well as ferrioxamine B ; as can be seen on the Fig. S7, the siderophore backbone does not occupy the same position being closer to the C-terminal lobe in the ferrioxamine case. Two residues E97 and E231 have been identified as potentially important for the interaction with the ABC transporter ; FhuD2 with mutations in either E97 and/or E231, still allow efficient ferrichrome transport, but not of ferrioxamine B [START_REF] Vines | Demonstration of the functional role of conserved Glu-Arg residues in the Staphylococcus aureus ferrichrome transporter. Biometals : an international journal on the role of metal ions in biology[END_REF].

-Streptococcus pneumoniae PiaA [START_REF] Cao | The mechanism of ironcompensation for manganese deficiency of Streptococcus pneumoniae[END_REF][START_REF] Cheng | Structures of Streptococcus pneumoniae PiaA and its complex with ferrichrome reveal insights into the substrate binding and release of high affinity iron transporters[END_REF][START_REF] Zhang | Crucial residue Trp158 of lipoprotein PiaA stabilizes the ferrichrome-PiaA complex in Streptococcus pneumoniae[END_REF] (PDB codes 4H59, 4HMO, 4HMP, 4HMQ, 5JJ5) PiaA recognizes ferrichrome with a higher affinity than E. coli FhuD (Kd of 5 vs 100 nM, respectively) [START_REF] Cheng | Structures of Streptococcus pneumoniae PiaA and its complex with ferrichrome reveal insights into the substrate binding and release of high affinity iron transporters[END_REF], and together with PiaB and PiaC imports ferrichrome inside the cell [START_REF] Cao | The mechanism of ironcompensation for manganese deficiency of Streptococcus pneumoniae[END_REF]. In the crystal structures, the conformational changes upon substrate binding appear quite modest (Fig. S8). The binding pocket is rather hydrophobic, with few polar residues. The Trp63 residue contributes to the binding pocket, without establishing a H-bond with the siderophore. Its mutation for Ala leads to a 6-fold increase in Kd. As in FhuD2 compared to E. coli FhuD, the residues stabilizing the backbone of the siderophore are in the C-terminal lobe of PiaA. Somewhat conflicting results regarding the Kd's of PiaA (and its mutants) have been obtained depending upon the technique used for the measure, with much higher Kd's obtained by fluorescence quenching, as opposed to ITC (Isothermal Titration Calorimetry).

Two other hydrophobic residues (Trp158 and Phe255) have been mutated to Ala and also affect the binding of Ferrichrome, the Trp158 mutation having a marked effect on the thermal stability of the protein, whether in the absence or presence of the substrate [START_REF] Zhang | Crucial residue Trp158 of lipoprotein PiaA stabilizes the ferrichrome-PiaA complex in Streptococcus pneumoniae[END_REF].

-Staphylococcus pseudointermedius FhuD [START_REF] Abate | Crystal structure of FhuD at 1.6 A resolution: a ferrichrome-binding protein from the animal and human pathogen Staphylococcus pseudintermedius[END_REF] (PDB code 5FLY) This binding protein was crystallized in the presence of PEG400, that sits in the binding site of the protein and makes many contacts with residues of the binding sites, which appears to be somewhat hydrophobic/negatively charged (Fig. S9). The protein is quite stable in its apo form and is even more stabilized by its substrate.

-Vibrio cholerae FhuD (PDB codes 5GGX, 5GGY)

The structure of V. cholerae FhuD has been solved in the apo and Fe-desferal (ferrioxamine) bound forms (Fig. 2, III). There is virtually no relative movement of the two lobes upon ligand binding. The binding site contains two residues making polar bonds with the siderophore and several hydrophobic residues.

IA3 Mixed type, polycarboxylate, siderophore binding proteins -Acinetobacter baumanii: BauB (PDB code 6MFL) A. baumannii is among the « ESKAPE » WHO (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. World Health Organization) list, and as such has received deep attention, in particular for its many iron acquisition systems. Three classes of siderophore have been identified in clinical isolates of A. baumannii: acinetobactin, fimsbactin, both mixed type siderophores and baumannoferrin, hydroxamate. Acinetobactin [START_REF] Yamamoto | Isolation and structure elucidation of acinetobactin., a novel siderophore from Acinetobacter baumannii[END_REF], synthesized by the vast majority of clinical isolates is the most studied one. Acinetobactin is synthesized (on NRPS) as a preacinetobactin that spontaneously isomerizes to acinetobactin. Both acinetobactin and preacinetobactin bind iron (under the form of Acb2Fe or PreAcb2Fe) and can serve as iron source for the bacterium, via the TBDT BauA. Each siderophore contributes 3 iron ligands (two O from the catechol, one N from the imidazole ring). The periplasmic binding protein is BauB.

The binding site of the siderophore is relatively neutral, with positive charges on the C-terminal half (Fig. S10); only one residue (Y301) makes a direct H-bond with the siderophore moiety. Whereas both PreAcb and Acb have similar Kd's for BauB (380 vs 300 nM), BauB strongly prefers FeAcb2 over FePreAcb2 (160 vs 750 nM). BauB can also bind Fimsbactin (a mixed bis catechol-hydoxamate siderophore), both in its free and Fe bound forms with similar affinities (360 vs 240 nM). It has also been shown in vivo that Fim inhibits growth promotion of FeAcb2, whereas the converse (Acb inhibition of growth promotion by FeFim) is much less true. Whereas the ABC transporter associated associated with BauB is likely to be BauCDE, much less is known about the Fim pathway ; it is hypothesized that FbsP, a putative periplasmic reductase, encoded in the fim locus might release the iron from the FeFim (to be then taken up by a Feo type transporter). Whether all these observations are relevant in a clinical setting is not known, but they point to a potential role of the BauB protein in the regulation of iron acquisition. By analogy with Pseudomonas, it is proposed that Fimsbactin is equivalent to pyoverdin, that never enters the cytoplasm, whereas Acinetobactin is equivalent to pyochelin, being transported to the cytoplasm [START_REF] Bohac | Fimsbactin and Acinetobactin Compete for the Periplasmic Siderophore Binding Protein BauB in Pathogenic Acinetobacter baumannii[END_REF]. This is also a case where the substrate transported across the OM is not the same as across the IM as the one transported across the OM by BauA is a preAcb-AcbFe complex, not having the same configurations of the iron binding sites.

-Bacillus subtilis YclQ (PDB code 3GFV) [START_REF] Zawadzka | Characterization of a Bacillus subtilis transporter for petrobactin, an anthrax stealth siderophore[END_REF] One of the siderophores transported by YclQ is petrobactin, a mixed bis-catechol hydroxyacid siderophore synthesized by B. anthracis for example. The transporter is YclNOPQ.

The binding protein presents a binding site surrounded by positive charges (Fig. S11).

-Thermobifida fusca, fuscachelin binding protein FscJ [START_REF] Li | Structure and functional analysis of the siderophore periplasmic binding protein from the fuscachelin gene cluster of Thermobifida fusca[END_REF] (PDB codes 5DH0, 5DH1, 5DH2) Fuscachelin is a mixed catechol/hydroxamate type of siderophore. Only the apo structure has been solved, on different crystal forms at different pH's, and, as for previous examples there is some conformational variability, with a slight closing of the two domains on each other. The Kd of fuscachelin for FscJ is in the µM range. The binding site is also quite hydrophobic and mostly surrounded by a rather negatively charged surface (Fig. S12).

-Staphylococcus aureus HtsA [START_REF] Beasley | Characterization of staphyloferrin A biosynthetic and transport mutants in Staphylococcus aureus[END_REF][START_REF] Grigg | The Staphylococcus aureus siderophore receptor HtsA undergoes localized conformational changes to enclose staphyloferrin A in an arginine-rich binding pocket[END_REF] (PDB codes 3EIW, 3EIX, 3LHS, 3LI2) S. aureus synthesizes two siderophores, named staphyloferrin A and B, which are of the polycarboxylate type. They are respectively synthesized by the sfa and sfn loci, encoding NIS synthases (NRPS Independent Siderophore synthases). A double sfa sfn mutant is no longer able to grow in serum [START_REF] Beasley | Characterization of staphyloferrin A biosynthetic and transport mutants in Staphylococcus aureus[END_REF]. Contiguous to the sfa locus is the htsABC locus that encodes the HtsA substrate-binding protein, and HtsB and HtsC two membrane subunits of an ABC transporter. No ABC subunit is encoded in this locus. The ABC subunit, common to several siderophore ABC transporters is FhuC, encoded in the fhuCBG operon. HtsA was initially supposed to be the binding protein for heme, as a htsA mutant is slightly affected in growth on heme as an iron source. It was later conclusively shown that HtsA is in fact the staphyloferrin A binding protein and not a heme-binding protein. The structure of HtsA, without or with Fe-staphyloferrin A has been determined (Fig. S13). Two forms have been obtained in the presence of the siderophore, one closely resembling the apo form, the other one slightly more closed on the substrate. The movements between the two forms are minor, and the highly polar nature of the binding site and the absence of a potential heme iron binding residue are not in favour of heme binding ability. HtsA has a very high affinity for Staphyloferrin A (subnanomolar Kd) and does not bind Staphyloferrin B, although it is a hydroxy-carboxylate type siderophore.

-Staphylococcus aureus SirA (PDB codes 3MWF, 3MWG) [START_REF] Grigg | Specificity of Staphyloferrin B recognition by the SirA receptor from Staphylococcus aureus[END_REF] Like HtsA for staphyloferrin A, SirA is the specific lipoprotein for staphyloferrin B. Like for HtsA, there is little difference between the free and the siderophore loaded forms (Fig. S14). SirA binds Fe-staphyloferrin B with a high affinity (subnanomolar range Kd), and high specificity. This is opposite for example to the FhuD case of E. coli that binds several hydroxamate type siderophores. Although the overall structure of SirA is highly similar to HtsA (top hit in DALI server), the residues involved in the siderophore binding and the position of the siderophore in its binding pocket are not the same for both proteins, explaining their high specificity.

IA4 Heme binding proteins [START_REF] Ho | Holo-and apo-bound structures of bacterial periplasmic heme-binding proteins[END_REF][START_REF] Mattle | Two stacked heme molecules in the binding pocket of the periplasmic heme-binding protein HmuT from Yersinia pestis[END_REF][START_REF] Muraki | Structural Characterization of Heme Environmental Mutants of CgHmuT that Shuttles Heme Molecules to Heme Transporters[END_REF][START_REF] Naoe | Structural basis for binding and transfer of heme in bacterial heme-acquisition systems[END_REF][START_REF] Vergauwen | Glutathione import in Haemophilus influenzae Rd is primed by the periplasmic heme-binding protein HbpA[END_REF] (supplementary tables S3 and S4)

-Pseudomonas aeruginosa and Shigella dysenteriae PhuT/ShuT (PDB codes 2R79, 2RG7, 2RTA)

The first structures of two periplasmic heme-binding proteins were solved in 2007, in the group of T. Poulos, namely PhuT from Pseudomonas aeruginosa and ShuT from Shigella dysenteriae (Fig. S15) [START_REF] Ho | Holo-and apo-bound structures of bacterial periplasmic heme-binding proteins[END_REF]. They show the typical class III SBP fold, with one heme molecule bound inbetween the two lobes of the protein. The heme molecule has opposite orientations in the two structures, with the propionates respectively pointing inward and outward in the PhuT and ShuT structures. Otherwise, a single Tyr residue coordinates the iron atom leading to a pentacoordinated heme iron. In the case of the ShuT protein, which also crystallized in the apo form, there is a small closing movement upon ligand binding. In both cases, the binding site is lined with a few apolar/hydrophobic residues, and the heme is quite accessible from the medium.

-Yersinia pestis HmuT (PDB codes 3MD9, 3NU1)

HmuT was the first heme-SBP to be crystallized with two heme molecules in the binding pocket [START_REF] Mattle | Two stacked heme molecules in the binding pocket of the periplasmic heme-binding protein HmuT from Yersinia pestis[END_REF]. The two molecules are in an anti-parallel arrangement, with the propionates of one heme facing outward, whereas those from the other facing inwards (Fig. S16). One heme iron is coordinated via a Tyr70 residue, whereas the other one is coordinated by a His167 residue. ITC titrations ruled out this resulting from a potential crystallization artifact. Two successive binding sites were putatively identified with Kd's of respectively 0.3 and 30 nM, the second Kd, being also quite low. This was also confirmed by UV-visible absorption spectroscopy, since the second heme does not absorb (Soret band) at the same wavelength as the first (375 vs 404 nm). Whether this is physiologically relevant will be discussed in the transport section.

-Corynebacterium glutamicum HmuT [START_REF] Muraki | Structural Characterization of Heme Environmental Mutants of CgHmuT that Shuttles Heme Molecules to Heme Transporters[END_REF] (PDB codes 5AZ3, 5B4Z, 5B50, 5B51)

The WT HmuT protein from C. glutamicum was crystallized in the presence of heme as well as three mutants, two of the axial ligands (His141 and Tyr240), and one of the Arg242 establishing an H-bond with Tyr240, to stabilize its tyrosinate form, able to bind the iron atom.

The WT protein presents the classical class III SBP fold, with heme at the junction of the two lobes. The binding site is lined with non polar residues, and His141 and Tyr240 constitute the heme iron axial ligands. The heme adopts two preferential orientations (rotation around the two heme iron axial ligands) positioning the propionates in two distinct positions, besides also rotation along the alpha-gamma meso axis. In the WT protein, the heme cannot be reduced by dithionite. There are only marginal conformation changes upon the mutations, and the heme is held in the same position (Fig. S17). However, there is only one orientation of the heme in the His140Ala and Arg242Ala mutants. There were unfortunately no measure of the affinities of the various HmuT mutants for heme, precluding further in vivo characterization.

-Burkholderia cenocepacia BhuT [START_REF] Naoe | Structural basis for binding and transfer of heme in bacterial heme-acquisition systems[END_REF] (PDB codes 5GIZ, 5Y89, 5Y8A) Roseiflexus sp. RhuT (PDB codes 5GJ3, 5Y8B) BhuT represents a very interesting case, where, as for Y. pestis HmuT, two heme molecules can bind to a BhuT monomer and where an in-depth characterization of the binding was made. The first heme binds to BhuT with a Kd of 18 nM, mostly driven by entropic gain, whereas the second heme binds with a lower Kd (360 nM), and driven by enthalpic gain. The two species can also be distinguished by their UV-visible (as well as resonance Raman) spectral properties. The crystal structures show that heme 1 iron is bound to Tyr87 residue (in interaction with Arg89 to stabilize the tyrosinate), and that upon binding of the second heme, the first one reorients along the Fe-Tyr87 axis so that its propionates are now pointing outwards. The propionates of the second heme point towards the binding cleft. In this form, the second heme iron is not coordinated to a protein residue, the second heme molecule is parallel to the first one. The propionates of the second molecule are in interaction with several residues of the C-lobe. The structure also shows that both apo and two-heme bound forms are in a more open conformation than the one-heme bound form (Fig. 3). This is evident when This will be discussed further.

During the same work the authors also explored another heme-SBP from Roseiflexus sp. which is also able to bind two hemes per monomer. ITC graphs yielded complex patterns that were difficult to fit using a simple two-site model, but contrarily to the BhuT case, the first binding step was clearly driven by an enthalpic gain, indicating very diverse modes of binding. As compared to the apo form, the holo2 form was more open ; the two hemes are in the same orientation with the propionates pointing towards the solvent. Both heme irons are liganded by Tyr residues that are stabilized in the tyrosinate form by Arg residues and a Gln residue for heme 1. The propionates of the first heme are also in interaction with an Arg residue.

The HutB protein from V. cholerae was also crystallized in the apo form [START_REF] Agarwal | Structure and dynamics of Type III periplasmic proteins VcFhuD and VcHutB reveal molecular basis of their distinctive ligand binding properties[END_REF] (PDB code 5KHL) and shows a quite wide cleft putatively able to harbour two heme molecules. It was actually shown by UV-visible heme titration that Vc HutB could fix successively two heme molecules and potential heme iron ligands were identified by comparing the structure of Y.

pestis HmuT with that of V. cholerae (Fig. S18). The residues Tyr65 and His164 are the putative heme iron ligands. A mutant Tyr65Ala showed altered UV-visible absorption spectrum as compared to the WT. Molecular dynamics studies also showed that HutB could explore a large range of twisting motions of one lobe versus the other, with correspondingly very high bfactors.

IA5 Unidentified substrate

-Mycobacterium tuberculosis Mtb RV0265c (also called FecB2, structure 4PM4) [START_REF] Chao | Iron Acquisition in Mycobacterium tuberculosis[END_REF][START_REF] Mitra | PPE Surface Proteins Are Required for Heme Utilization by Mycobacterium tuberculosis[END_REF] The substrate is unknown ; it is possible it to be heme, as Rv0265c has been shown to bind heme, although with rather low affinity (detected by SPR on immobilized his-tagged protein), and to be involved in heme acquisition. In this respect there is a conspicuous Tyr residue that could potentially bind the heme iron atom (Tyr39). Rv0265c is most likely a lipoprotein anchored in the inner leaflet of the mycomembrane (Fig. S19).

-Saccharomonospora viridis (PDB code 5CR9)

The substrate is unknown and there is one Mn atom in the binding site.

-Yersinia pestis YiuA [START_REF] Radka | The crystal structure of the Yersinia pestis iron chaperone YiuA reveals a basic triad binding motif for the chelated metal[END_REF] (PDB codes 6B2X, 6B2Y)

This putative SBP has been crystallized in its apo form, in two crystal forms that show some variations, one being slightly more « open » than the other. The substrate has not been identified, although from the surface potential it likely appears to be a catecholate, or a polycarboxylate, given its highly positive charge near the binding site (Fig. S20).

IB Class II iron/heme binding proteins

IB1 Ferric ion binding proteins [START_REF] Abdizadeh | Computational approaches for deciphering the equilibrium and kinetic properties of iron transport proteins[END_REF][START_REF] Khambati | The role of vicinal tyrosine residues in the function of Haemophilus influenzae ferric-binding protein A[END_REF][START_REF] Siburt | FbpA--a bacterial transferrin with more to offer[END_REF] These proteins (named FbpA) were amongst the first SBP's identified, in Haemophilus influenzae or Neisseria gonnorhoeae (see for [START_REF] Siburt | FbpA--a bacterial transferrin with more to offer[END_REF] a review); the initial substrate recognized by the OM TBDT is transferrin or lactoferrin [START_REF] Noinaj | Structural basis for iron piracy by pathogenic Neisseria[END_REF]. They belong to class II SBP's. The iron binding site resembles that of transferrin [START_REF] Abdizadeh | Computational approaches for deciphering the equilibrium and kinetic properties of iron transport proteins[END_REF]; however there is at one point a change in the co-ion, from carbonate to phosphate or sulphate. As for the siderophore SBP's there is some movement of the two domains relative to each other, in the iron bound forms (3ODB/3OD7, or mutants vs WT, Fig. S21). Likewise the apo form is in a more open conformation. Several mutants of the iron binding site have been made without affecting too much in some cases, the affinity of the mutant for iron. For example Tyr195Ala and Tyr196Ala mutants, have barely one log difference of Fe3+ binding, as measured by citrate competition (logKa from19,62 to 19,2) [START_REF] Khambati | The role of vicinal tyrosine residues in the function of Haemophilus influenzae ferric-binding protein A[END_REF]. Those mutants are inactive in H. influenzae, yet they are active in a reconstituted system in E. coli. The reason why they are inactive in H. influenzae is far from being clear.

There are also two other types of ferric ion binding proteins, that can be distinguished on the basis of the Fe 3+ coordination in the binding site. A first type is exemplified by Mannheimia hemolytica FbpA, that displays two Fe 3+ binding modes, one without a synergistic anion, in an open conformation of the two lobes, the other with a synergistic anion, in the closed conformation, different from the one found in the case of H. influenzae : the synergistic anion is carbonate, providing two coordinations to the Fe 3+ and three Tyr residues (structures 1Q35, 1SI1 and 1SI0 in the PDB). A second variation is found in the YfuA protein from Y. enterocolitica or SfuA from Serratia marcescens (structures 1XVY and 1XVX in the PDB) in which there is no longer a synergistic anion (replaced by an Asp residue), but all the other coordinationg residues are the same as in H. influenzae FpbA (two Tyr oxygen, une Glu oxygen, one water molecule oxygen, and one His nitrogen) [START_REF] Shouldice | Novel anionindependent iron coordination by members of a third class of bacterial periplasmic ferric ion-binding proteins[END_REF][START_REF] Shouldice | Structural basis for iron binding and release by a novel class of periplasmic iron-binding proteins found in gram-negative pathogens[END_REF].

IB2 Heme-binding proteins -Escherichia coli

In E. coli K12 that has no heme outer membrane TBDT, extracellular heme can still reach the cytoplasm and be used as an iron source, provided the expression of a functional OM heme TBDT. Two SBP's were shown to bind heme respectively DppA and MppA (the binding proteins for dipeptides and tripeptides derived from murein respectively) [START_REF] Letoffe | The housekeeping dipeptide permease is the Escherichia coli heme transporter and functions with two optional peptide binding proteins[END_REF] and donate heme to the Dpp ABC transporter, showing the promiscuity of these SBP's. Heme is, roughly speaking, the condensation product of 8 molecules of delta-amino-levulinic acid, an amino-acid that is also transported by the Dpp ABC transporter in E. coli [START_REF] Verkamp | The periplasmic dipeptide permease system transports 5-aminolevulinic acid in Escherichia coli[END_REF]. This might explain the promiscuity of peptides binding protein for this compound.

-Haemophilus influenzae Haemophilus influenzae (Hi), is a strict heme auxotroph with a reduced genome. The RdKW20 strain (an avirulent d serotype strain, devoid of capsule) was the first completely sequenced bacterial genome and has been used in many studies. It expresses many different heme/hemoprotein receptors, and there are several reports of periplasmic heme-binding proteins [START_REF] Vergauwen | Glutathione import in Haemophilus influenzae Rd is primed by the periplasmic heme-binding protein HbpA[END_REF][START_REF] Tanaka | Oligopeptide-binding protein from nontypeable Haemophilus influenzae has ligand-specific sites to accommodate peptides and heme in the binding pocket[END_REF]. Whereas several Pasteurellacae species do contain class III heme SBP's, this is not the case for H. influenzae. Instead, four SBP's with other functions have been reported as potential heme SBP's ; they are all belonging to class II SBP's. HbpA (heme binding protein A, also named HI0853 in RdKW20) was the first periplasmic heme binding protein reported in this organism [START_REF] Hanson | Molecular cloning, partial purification, and characterization of a haemin-binding lipoprotein from Haemophilus influenzae type b[END_REF][START_REF] Hanson | The hbpA gene of Haemophilus influenzae type b encodes a heme-binding lipoprotein conserved among heme-dependent Haemophilus species[END_REF]. It was identified in the Hib strain DL42 (a virulent b serotype strain) as a lipoprotein, most likely anchored in the outer leaflet of the cytoplasmic membrane. Its main function might be to transport glutathione [START_REF] Vergauwen | Glutathione import in Haemophilus influenzae Rd is primed by the periplasmic heme-binding protein HbpA[END_REF]. The gene encoding HI0853 was not identified to be induced under heme/iron starvation [START_REF] Whitby | Transcriptional profile of Haemophilus influenzae: effects of iron and heme[END_REF]. Three other SBP's were also identified as heme binding proteins : HI0213 (also described as OppA, and subject in some strains to heme/iron starvation regulation, also a lipoprotein, most likely anchored to the outer leaflet of the cytoplasmic membrane) [START_REF] Morton | The dppBCDF gene cluster of Haemophilus influenzae: Role in heme utilization[END_REF], HI1124 (identified as OppA) and finally SapA [START_REF] Mason | Heme utilization by nontypeable Haemophilus influenzae is essential and dependent on Sap transporter function[END_REF] (HI1638) that also binds cationic peptides. In this last case, the authors have assigned an essential role to SapA in heme transport, based on the observation, that, upon prolonged heme-iron starvation, a ∆sapA mutant is unable to grow on heme, whereas the WT and the complemented strains are able to grow. Yet it appears that this mutant has been isolated on sBHI, under aerobic condtions, where heme is strictly necessary. It is well possible that, given the apparent redundancy of the heme binding proteins in Hi, this effect might not be direct. Furthermore, as in the E. coli case, where peptides compete with heme for binding to DppA or MppA, there is also a competition between heme and cationic peptides for binding to SapA.

The binding affinity of heme for these various proteins has been estimated between 50 and 500 µM. HbpA has also been shown to have a role in heme acquisition in Hi [START_REF] Morton | The heme-binding lipoprotein (HbpA) of Haemophilus influenzae: role in heme utilization[END_REF]. On the contrary, SapF (HI1642) appears essential for growth on sBHI, and might be part of the conduit through which porphyrins enter the cells.

Although many structures of periplasmic di-, tri-, oligo-peptide binding proteins have appeared, there is not yet a structure of such a protein cocrystallized in the presence of heme.

Many peptides bind to OppA, and the binding occurs through the backbone of the peptide. However, model building has shown that it is possible to accomodate a heme molecule in these binding sites [START_REF] Tanaka | Oligopeptide-binding protein from nontypeable Haemophilus influenzae has ligand-specific sites to accommodate peptides and heme in the binding pocket[END_REF]. There is no structure of apoOppAHi. However, E. coli OppA is 50,4% identical to H. influenzae OppA and has been crystallized in both apo and peptide bound form. This allows to see the large movement of one part of the molecule with respect to the other (Fig. S22). Further studies also showed that both halves of the molecule have specific role regarding either heme or peptide binding, the first domain (residues 21-290+ 510-541) having preference for heme over peptides (Kd's of 0.6 vs 12 µM respectively) whereas the second domain (residues 291-509) having a preference for peptides over heme (Kd's of 0.15 vs 2.5 µM respectively).

II Substrate binding proteins: in vivo aspects

A special mention should be made for cases where the substrate transported across the inner membrane is not the same as the one transported across the inner membrane. This is relevant for several bacteria, C. jejuni, P. aeruginosa and E. coli [START_REF] Zeng | Identification and characterization of a periplasmic trilactone esterase, Cee, revealed unique features of ferric enterobactin acquisition in Campylobacter[END_REF][START_REF] Perraud | A Key Role for the Periplasmic PfeE Esterase in Iron Acquisition via the Siderophore Enterobactin in Pseudomonas aeruginosa[END_REF][START_REF] Zhu | Functions of the siderophore esterases IroD and IroE in iron-salmochelin utilization[END_REF]. In those three cases either Fe-enterobactin or Fe-salmochelin are taken up across the OM, and degraded by periplasmic esterases (Cee for C. jejuni, PfeE for P. aeruginosa, and IroE for E. coli), before import of a Fe containing compound in the cytoplasm. In the Pseudomonas case, the import system once enterochelin has been degraded by the periplasmic esterase is not identified ; in the E. coli case, it is very likely to be the Fep ABC transporter, and eventually the IroC cytoplasmic membrane protein ; whereas there is no apparent enterobactin esterase in Pseudomonas and Campylobacter, this is not the case in E. coli, where Fes is the cytoplasmic esterase, that degrades enterobactin. In Campylobacter that does not synthesize any siderophore and yet can use Fe-enterobactin as an iron source, the import system across the inner membrane relies on the CeuE SBP (see above), which has better affinity for linear siderophores than enterobactin. This would provide a competitive advantage to Campylobacter over other bacterial species.

Pyoverdin (PVDI) is one of the two siderophores synthesized by P. aeruginosa (the other being pyochelin), a mixed siderophore coupled to a fluorescent moiety. Fe-PVDI is transported across the OM by FpvA, a TBDT receptor. The fpv locus contains an operon of four genes fpvCDEF, FpvE being an ABC-ATPase, FpvD a TMD, and FpvC and FpvF being two SBP's with different specificities [START_REF] Brillet | An ABC transporter with two periplasmic binding proteins involved in iron acquisition in Pseudomonas aeruginosa[END_REF][START_REF] Gao | The Two-Operon-Coded ABC Transporter Complex FpvWXYZCDEF is Required for Pseudomonas aeruginosa Growth and Virulence Under Iron-Limiting Conditions[END_REF]. FpvC belongs to the PsaA/TroA family of SBP's (metal binding) whereas FpvF belongs to the siderophore family of SBP's. In solution, FpvC is a monomer that has no affinity for PVDI and low affinity for Fe-PVDI. On the other hand, PvdF is able to form dimers, that are the only form able to bind either PVDI or Fe-PVDI. Furthermore FpvC-FpvF2 and FpvC2-FpvF2 oligomers have been observed both in vitro and in vivo, that are able to bind both PVDI and Fe-PVDI [START_REF] Brillet | An ABC transporter with two periplasmic binding proteins involved in iron acquisition in Pseudomonas aeruginosa[END_REF]. A deletion of the fpvCDEF operon leads to accumulation of Fe-PVDI in the periplasm, indicating that this ABC transporter is required for Fe-PVDI derived iron import in the cytoplasm. It was also shown that PVDI-Fe dissociates in the periplasm, most likely by a reduction step, allowing the recycling of PVDI in the extracellular medium [START_REF] Imperi | Molecular basis of pyoverdine siderophore recycling in Pseudomonas aeruginosa[END_REF]. It is therefore possible that FpvC would bind Fe 2+ .

In the pvd locus and just upstream of the fpvCDEF operon, is found another operon of four genes fpvGHJK [START_REF] Ganne | Iron Release from the Siderophore Pyoverdine in Pseudomonas aeruginosa Involves Three New Actors: FpvC, FpvG, and FpvH[END_REF]. It was found that deletion of fpvG or fpvH or fpvJ but not fpvK had an effect on iron uptake across the inner membrane and that the strongest effect was observed with fpvG. In vitro Fe-PVDI can be dissociated upon reduction by DTT and this effect is enhanced upon addition of ferrozine, an Fe2+ chelator. Like ferrozine, FpvC, the metalbinding SBP encoded in the nearby operon, can enhance the dissociation of Fe-PVDI by DTT. FpvG, FpvH, FpvJ and FpvK are predicted to be cytoplasmic membrane proteins with large periplasmic domains. It was also shown that E. coli membrane vesicles isolated from a FpvG producing strain could reduce Fe-PVDI. This strongly suggests that FpvG is the reductase that binds Fe-PVDI and dissociates it. It was however difficult to recapitulate the whole process as addition of FpvC and FpvF slowed down the process [START_REF] Ganne | Iron Release from the Siderophore Pyoverdine in Pseudomonas aeruginosa Involves Three New Actors: FpvC, FpvG, and FpvH[END_REF]. All this work highlights the potentially more complex role of siderophore SBP's in the context of iron release in the periplasm. In PAO1 genome, there are three homologs of fpvG all three being contiguous to genes coding for OM siderophore receptors (PA1909, PA2465, and PA4513).

Having presented the first player of the siderophore/heme ABC transporter, remains to present the membrane components and the ABC-ATPase, for which much less functional, structural and biochemical data are known. Before doing so, it is however worth mentioning the YbtP-YbtQ case from Y. pestis which is involved in iron acquisition [START_REF] Fetherston | YbtP and YbtQ: two ABC transporters required for iron uptake in Yersinia pestis[END_REF] by an unknown mechanism. Both YbtP and YbtQ represent fused TMD-ABC modules, and are closer to exporters than to importers, yet are required for iron entry in Y. pestis and for virulence in a mice model. It is not known at present whether a SBP is associated with this system. It is not known either whether those gene products are directly or indirectly involved in iron transport. changes leading to opening of MalFG towards the periplasmic space and to the transfer of maltose from its MalE site to a binding site exposed on MalFG, with a mM affinity. Transfer of maltose is possible due to the relative opening of the two lobes of MalE, and to the presence of the so-called scoop loop of MalG that comes in steric clash with maltose in its binding site on MalE. Following further rearrangements that position the two MalK monomers together with two ATP molecules in a correct orientation so that the ATP hydrolysis can take place. A high energy ADP-bound state is generated with the MalFG complex open to the cytoplasm (leading to release of Maltose in the cytoplasm), and MalE still bound to MalFG. Release of ADP and MalE restores the resting state (see [START_REF] Chen | Molecular mechanism of the Escherichia coli maltose transporter[END_REF] for a review).

III Heme/iron siderophore ABC transporters cycle

The situation is somewhat different in the BtuCDF B12 importer (BtuF is the class III SBP, BtuC is the membrane domain, and BtuD the ABC ATPase), where in the resting state, BtuCD is open towards the periplasm, and two « gates » have been identified on the cytoplasmic side. Somewhat conflicting results have been obtained on the association of BtuF to BtuCD and its modulation by ATP or a non-hydrolyzable analog like AMP-PNP and the B12 vitamin substrate [START_REF] Fiorentino | The Different Effects of Substrates and Nucleotides on the Complex Formation of ABC Transporters[END_REF][START_REF] Goudsmits | Single-molecule visualization of conformational changes and substrate transport in the vitamin B12 ABC importer BtuCD-F[END_REF][START_REF] Korkhov | Structure of AMP-PNP-bound BtuCD and mechanism of ATP-powered vitamin B12 transport by BtuCD-F[END_REF], whereby ATP or B12 have been found to have a destabilizing effect and ATP has also been shown as accelerating the association of B12-BtuF to BtuCD. These apparent discrepancies might result from the various conformations of the « resting BtuCD » complex and its ability to bind B12 vitamin. The BtuF-BtuCD interaction is modified by ATP [START_REF] Lewinson | A distinct mechanism for the ABC transporter BtuCD-BtuF revealed by the dynamics of complex formation[END_REF], and as in the Maltose transport system, there is a transfer from the B12 vitamin from its binding site on BtuF into a cavity formed by BtuC (subsequently closed on the periplasmic side); ATP hydrolysis leads to opening of the cytoplasmic gates and release of the substrate in the cytoplasm [START_REF] Locher | Mechanistic diversity in ATP-binding cassette (ABC) transporters[END_REF]. There is apparently no specific binding of the B12 molecule to the BtuC cavity. Whereas there is apparently a tight coupling of ATP hydrolysis with transport in type I importers, this appears to be less the case in the Btu system where futile cycles of ATP hydrolysis occur.

Although iron acquisition is a important topic for bacterial physiology, there is a paucity of data on the functional, biochemical and structural characterization of the ABC importers of either siderophores or heme. Some metal ABC importers have been biochemically and structurally characterized, but no purification and reconstitution of iron/siderophore membrane transport system has been carried out at the present time. Several studies have however appeared on a few heme transport systems that will now be described.

-Shigella dysenteriae ShuTUV

The first one concerned the ShuTUV system from Shigella dysenteriae that was reconstituted in artificial membrane vesicles [START_REF] Burkhard | Functional Characterization of the Shigella dysenteriae Heme ABC Transporter[END_REF], with the SBP ShuT trapped inside the vesicle lumen, and the presence of an heme trap, ShuS in the medium. The extent of heme binding to apo ShuS was measured, as well as the ATP hydrolysis. Two types of mutants were also used, one with mutations in the two conserved glutamate residues on ShuT that serve to anchor ShuT to ShuU, and also on two histidine residues on ShuU, that were tentatively assigned a heme binding function. All mutants and the WT apo ShuT displayed a basal level of ATPase activity and no heme accumulation on ShuS, whereas the holo-ShuT led to an increase in ATPase activity, coupled to a transfer from the heme from ShuT to ShuS. Although this study provided an interesting insight into the functioning of of a type II heme importer, no further studies have appeared, to precisely measure stoichiometries, coupling parameters, and relevant kinetics.

-Yersinia pestis HmuTUV

A second study on the HmuTUV heme acquisition system from Y. pestis did use a slightly different way to measure heme uptake across the vesicle membrane, with the binding protein on the extracellular side of the vesicle and, trapped inside the vesicle an ATP regenerating system. Heme was measured based on its peroxidase activity, after binding to apo-HRP [START_REF] Woo | X-ray structure of the Yersinia pestis heme transporter HmuUV[END_REF]. As in the previous case, the transport of heme was strictly dependent upon the ATP hydrolysis, and of the correct association of the SBP with the transporter. Transport rates of ca. 0.14 molecule of heme transported per min and per HmuUV transporter, were measured, comparable to those observed previously. If these rates truly represent in vivo activity they are clearly not as efficient as the maltose transporter (ca 20 maltose molecules transported by transporter by minute), which they don't need to be. The same HmuT mutant was used, unable to dock to HmuUV, and a Walker-B mutant in HmuU (E173Q) also inhibited transport. The HmuUV structure was solved by X-ray crystallography and showed the typical arrangement of type II ABC importers. There is a two fold symetry axis, perpendicular to the membrane plane. Each HmuU subunit presents 10 TM segments, the coupling helix between TM 6 and 7 establishes contacts with the ABC subunits. At the interface of the HmuU monomers there is a cavity open to the extracellular side and delimited by helices 5, 5a and 10 of both monomers (Fig. 4). This cavity is rather apolar and large enough to accomodate a heme molecule. Neither HmuUV or HmuTUV purified in detergent displays a detectable heme affinity. Furthermore, the two histidines residues identified in the work on ShuTUV are clearly facing the cytoplasm, and given their position on the structure, their potential role in heme binding is questionable ; they could be in contact with the ABC subunits at some point of the reaction cycle. Two residues Arg176, and Gly164 completely conserved in the HmuU family, were mutated for Ala. Arg176 is exposed on the periplasmic side, at the entrance of the putative heme binding cavity and Gly 164 is lower in the cavity, at a narrowing of the cavity.

A strong effect was observed with the R176A mutant, that displayed the same basal ATPase activity as the WT, but almost no stimulation by HmuT ; this correlated with a weak binding of HmuT to HmuUV. The Gly164Tyr mutant showed increased basal ATPase activity, with very little stimulation by HmuT, and a very reduced transport activity, indicating uncoupling of the ATPase from the transport activities. Although rather unlikely, it would have been interesting to test whether this mutation might have provided a binding site for heme in the cavity.

Another study using surface plasmon resonance with immobilized HmuUV and HmuT in the running buffer showed that HmuT binds with nanomolar affinity to HmuUV, and that both ATP and an excess of heme lead to the dissociation of the HmuT-HmuUV complex [START_REF] Qasem-Abdullah | ATP binding and hydrolysis disrupt the high-affinity interaction between the heme ABC transporter HmuUV and its cognate substrate-binding protein[END_REF], strengthening thesimilarities between the heme and B12 transporters.

Contrarily to MalFGK2 or Sav1866, that are outward facing in the presence of ATP, and inward facing in absence of nucleotide, the opposite orientation is found in the case of BtuCD or HmuUV, consistent with their classification as type II importers, where the complex is outward facing in the absence of ATP. There are at least two other characterisitics of this type II ABC importers that are worth mentioning : one which is related to the number of molecules that are transported that are orders of magnitude lower in the case of either siderophore/heme or B12 vitamin as compared to a carbon source as maltose; an E. coli cell contains ca. a few hundred thousand iron atoms that it must acquire during a cell division time. Unfortunately, the number of transporters per cell is not known for all those systems; however the observed in vitro rate of ATPase hydrolysis is ca. 10 times higher in the case of the maltose transporter as compared to the Hmu transporter. Whether this is related to the catalytic activity of those various transporters remains to be determined. The other characteristics concerns the amplitude of the movements of the binding protein in the bound and free states in the two systems where it is much smaller in the siderophore/heme case, as compared to the maltose case. Finally, there is also a difference concerning the ligand accessibility in the SBP's of the two classs, whereby class III SBP's have their substrate at least partially exposed to the outside, which is not the case in class I or II SBP's.

-Burkholderia cenocepacia BhuUV Another heme importer BhuUV from Burkholderia cenocepacia has been crystallized, in an ATP-free form, and also in the presence of the heme-SBP BhuT, in the absence of heme [START_REF] Naoe | Structural basis for binding and transfer of heme in bacterial heme-acquisition systems[END_REF][START_REF] Naoe | Crystal structure of bacterial haem importer complex in the inward-facing conformation[END_REF]. There are several differences as compared to the HmuUV structure of Y. pestis. First of all, the BhuUV structure is open on the cytoplasmic side, contrarily to the HmuUV case (Fig. S23). As in the HmuUV case, there is a dimerization of BhuU along TM 5 and 10 of each BhuU monomer. The channel that extends throughout the BhuU dimer interface is blocked on the periplasmic side by the two helices 5a, that form the periplasmic gate mentioned above. On the contrary, the channel is open on the cytoplasmic side, as it can be clearly seen on either Figure . As in the HmuUV case, the cavity is largely apolar with the exception of a conserved aspartate residue (Asp 112 in BhuU, Asp86 in HmuU), that, given its position could fulfil a specific function. Actually mutants of this residue (D112A or D112V) still display basal ATPase activity, similar to the WT protein, but are no longer able to transport heme. In the presence of the BhuT SBP, the ABC domains are slightly more close to each other.

When the BhuTUV complex is formed from holo-BhuT and ATP free BhuUV, the complex is no longer coloured, and heme is not found on the complex. This is understandable, as the periplasmic gating loops from both BhuU, insert into the heme binding pocket and come into steric clash with the heme, if it were to stay on its binding site. Finally, upon BhuT fixation the heme binding pocket resembles more the open apo form than the closed form (Fig. 5).

From a physiological point of view, such a situation, whereby heme would be likely ejected into the periplasmic space, upon BhuT-heme fixation on BhuUV, in the absence of ATP is highly unlikely. Two Glu residues from BhuT (E94, E231) make ion bridge with residues Arg84 from the two BhuU subunits.

In detergent solution, BhuUV has a basal ATPase activity, similar to the one observed in the HmuUV case, that is stimulated upon binding of the BhuT. Both ATP and AMP-PNP, but not ADP binding to the BhuTUV complex lead to the dissociation of BhuT from BhuUV.

The comparison of the BhuU and HmuU structures allows to see two potential states of the transporter in the inward-open and outward open states, in the absence of ATP (Fig. S24). In the absence of a defined heme binding site inside the cavity defined by the HmuU-BhuU dimer, it is extremely dificult to envision the heme transfer from its binding site on the SBP to the cavity, even though the heme binding pocket on the SBP is distorted upon interaction with the TMD subunit, lowering the affinity for heme. It is well possible that even though there might be no « true » binding site for heme inside the cavity, its rather apolar environment might drive heme inside. It is unlikely that, at one step of the transport cycle, the periplasmic loop of BhuU/HmuU joining TM's 5 and 5a might extend deep enough into the BhuT binding pocket. This could be feasible only if helix 5a might unwind and insert inside the binding pocket to act as a scoop loop.

-Sinorhizobium meliloti HmuUV Finally, there is also promiscuity among heme ABC transporters: in Sinorhizobium meliloti, the heme transporter HmuUV is also able to work with two distinct binding proteins, HmuT for heme transport and FhuP for ferrichrome/ferrioxamine transport [START_REF] Cuiv | 85 20628015 6DQQ Haemophilus influenzae OppA-peptide 11/06/2018 1,85 30455346 6DQR Haemophilus influenzae OppA-MGG 11/06/2018 2,08 30455346 6DQT Haemophilus influenzae OppA-LGG 11/06/2018 1,95 30455346 6DQU Haemophilus influenzae OppA-GIINTL 11/06/2018 1,65 30455346 6DTF Haemophilus influenzae OppA-KKK 16/06/2018 1,75 30455346 6DTG Haemophilus influenzae OppA-YLGANGRGGGS 16/06/2018 1,9 30455346 6DTH Haemophilus influenzae OppA-RPPGFSPFR 16/06/2018 1,96 30455346 2R79 Pseudomonas aeruginosa PhuT 07/09[END_REF]. This is reminiscent of the Dpp transporter that can operate with two distinct binding proteins in the case of heme transport, DppA and MppA [START_REF] Letoffe | The housekeeping dipeptide permease is the Escherichia coli heme transporter and functions with two optional peptide binding proteins[END_REF].

Concluding remarks

The past years have seen a wealth of structural data accumulating either on the periplasmic/extracellular siderophore/heme binding proteins, that have essentially clarified the binding specificity of those proteins, but are far from explaining the determinants for selectivity for the binding to the membrane components. There is also an urgent need to get quantitative data on the actual number of the two components of those systems (binding proteins and TMD-ABC's), in living cells under various iron stress conditions. As the reconstitution of the heme importers HmuTUV has been made, one might expect that more biochemical and biophysical data dissecting the different steps of association of the components in terms of affinity and kinetics, with regard to the nucleotide state of the ABC component should bring important data to understand these specific mechanisms. One may regret that the spectroscopic properties of heme have not been more exploited to assess its environment during the transport cycle, and the absence of a structure of a type II importer with the substrate in the transporter is a serious drawback to further characterization of this process. the outer membrane together with potential helper proteins, the TonB complex, and the siderophore/heme/iron ABC transporters in the periplasm/cytoplasmic membrane, made up of the periplasmic binding protein (SBP, bright green) and the ABC complex (TMD in two shades of magenta, ABC modules in two shades of cyan). The peptidoglycane mesh (PG) is represented as a discontinuous line in the periplasm. The various iron/heme sources are represented (Tf: transferrin, Hb: hemoglobin, Hpx: hemopexin, Hb-Hp: hemoglobinhaptoglobin, He: heme). Uptake of the iron complex (red circle) across the outer membrane (OM) is powered by the pmf, and by ATP hydrolysis across the inner membrane (IM). G30, G31, S32, E51, T52, Y67 (Fe), W87, R111 (Pr), A169, G170, G171, S172, S181, V182, A183, Y201, Yersinia pestis

Figure legends

HmuT 3MD9 apo 3NU1 bis-heme S54, T55, Y70 (Fe1), M165, H167(Fe2), L170, M173, T179, A180, A181, R199, Y200,M255, L258 Corynebacterium glutamicum HmuT 5AZ3 WT, heme L101, Y102, V123, S124, T126, G140, H141(Fe), Y240(Fe), R242 (>Y240), V247, F249, L251, Y255, L278, M297, E299, D332, G333, L336 5B4Z H141A heme L101, V123, S124, G139, G140, Y240(Fe), R242 (>Y240), F249, L251, Y255, M297, D332, L336 5B50 Y240A heme L101, Y102, V123, S124, T126, G140, H141(Fe), R242(Pr), L251, Y255, M297, E299, D332, G333, L401

5B51 R242A heme L101, Y102, V123, S124, G140, Y240(Fe), A242, F249, L251, Y255, M297, E299, D332, L336 Burkholderia cenocepacia BhuT 5GIZ apo

5Y89

WT heme G47, T71, T72(Pr1), Y87(>Fe), Q88(Pr1), R89(Pr1+Y87), H133, L190, H192, T193, L198, T204, A205(Pr2), A206(Pr2), Y225(Pr2), A279, A280, L283

5Y8A bis-heme T71, T72, Y87(>Fe1), Q88(Y87), R89(Y87), L190, H192, T195, T204, A205 (Pr He2), A206(Pr He2), H224, Y225(Pr He2), L283

Roseiflexus sp

RhuT 5Y8B apo 5GJ3 bis-heme S124, Y140 (>Fe1), Q141(>Y140), R142 (>Y140), R143 (>Pr 1-2He1), Y239(>Fe2), R241(>Y239), F276, L295, D330, L334

  one measures the distance between the Ca of Glu96 and Glu231 that point towards the TMD extracellular loops. Each protein crystallized with two monomers per asymetric unit and distances vary from 45.7-47.4 Å (apo form) to 40.2-40.7 Å (holo1) and 46.8-46.5 Å (holo2).

I

  will first briefly present an outline of the mechanism of action for type I and type II ABC importers, exemplified by the E. coli maltose and Archaeoglobus fulgidus or H. influenzae molybdenum transporters (respectively MalEFGK2 and ModAB2C2) for type I and the B12 vitamin transporter from E. coli BtuFC2D2 for type II. One archetype of the SBP-ABC transporter of type I is the Maltose transporter MalEFGK2 (MalE is the class II SBP, with micromolar affinity for maltose, the two membrane proteins MalF and MalG, and MalK the ABC ATPase) on which a lot of structural, functional, genetic and biochemical data have been accumulated over the years by several groups. In the maltose system, in the resting state of the system (with very low basal ATPase activity) MalFG is open on the cytoplasmic side, and binding on the one hand of Maltose-MalE on the periplasmic side and ATP on the MalK subunits trigger conformational

Figure 1 :

 1 Figure 1: general organization of the TonB-dependent transporters (TBDT, orange), in

Figure 2 :

 2 Figure 2 : Structures of B. subtilis FeuA (I), FitE from E. coliO157 :H7 (II) of V. cholerae FhuD (III). I, top: surface potential of FeuA showing the highly positive region around bacillibactin (magenta, iron atom as an orange sphere). I, Top bottom : structure of apo (yellow, 2WHY) and Fe-bacillibactin-FeuA complex (cyan, 2WI8), showing the closing of the two lobes upon substrate binding. II, top: surface potential showing the FitE binding site surrounded by positive charges. II, bottom: Structure of two monomers of FitE, in the absence of substrate (chain C of 3BE5, closed form, red, chain B of 3BE5, open form yellow). III, top: surface potential showing the FhuD binding site with Fe-Desferal. III, bottom: superposition of apo (cyan) and Fe-Desferal-FhuD structures in ribbon representation.

Figure 3 :

 3 Figure 3: Structures of BhuT from B. cenocepacia (I) and RhuT from Roseiflexus sp. (II).I, top : superposition of the structures of apo-BhuT (cyan), heme-BhuT (green) and bis-heme-BhuT (magenta). I, middle and bottom: surface potential of heme-BhuT and bis-heme-BhuT with heme in the binding pocket. II, top: structures of apo-RhuT (green) and bis-heme RhuT (cyan). II, bottom: surface potential of RhuT showing the two hemes in the binding site.

Figure 4 :

 4 Figure 4 : Structure of HmuUV from Y. pestis. Top left : surface potential of HmuU, from the cytoplasmic side : the two negatively charged coupling helices are clearly seen. Top right : surface potential of HmuU, from the periplasmic side, with the cavity open on the periplasm. Bottom left : Ribbon structure of HmuUV, with the two transmembrane HmuU (blue and pale

Figure 5 :

 5 Figure 5 : Structure of BhuTUV from B. cenocepacia. Top left : close up on the BhuT-BhuU interface. BhuT is shown as a cyan surface, the left monomer of BhuU is shown as a deep blue surface, the right BhuU monomer as pale blue helices. Heme is represented in stick, clashing with the 5-5a helices. Top right : same as left, in cartoon representation with apo-BhuT (yellow) and heme-BhuT (magenta) superimposed to BhuT from BhuTUV (cyan). Bottom left : surface potential of BhuU dimer from BhuTUV complex, from the periplasmic side, showing the closed cavity, and the potential localisation of heme. Bottom right : surface potential of BhuT, « viewed from BhuU », with the heme placed also in its binding site.

Figure 1

 1 Figure1

Figure 2 Figure 3 Figure 4 Figure 5

 2345 Figure 2

Figure S4 :

 S4 Figure S4 : Surface potential of VctP from V. cholerae, in the absence of siderophore)

Figure S7 :

 S7 Figure S7 : Structure of FhuD2 from S. aureus. Left : superimposition of apo-FhuD2 (green), of Fe-ferrioxamine B-FhuD2 (magenta) and Fe-ferrichrome-FhuD2 (cyan). Right : surface potential of FhuD2 with Fe-ferricrome in the binding site.

Figure S8 :

 S8 Figure S8 : Structure of S. pneumoniae PiaA. Left : surface potential of PiaA with ferrioxamine E in the binding site. Right : PiaA apo (green), in complex with Fe-ferrichrome (cyan), and with Fe-ferrioxamine E (magenta).

  Figure S9 : Structure of S. pseudointermedius FhuD : surface potential ; the binding site is occupied by a PEG400 molecule.

Figure S10 :

 S10 Figure S10 : Structure of A. baumannii BauB. Surface potential of the protein with Fe-Acb2 in the binding site.

Figure S11 :

 S11 Figure S11 : Structure of B. subtilis YclQ. Surface potential of apo YclQ

  Figure S13 : Structure of S. aureus HtsA. Top left : structures of HtsA complexed to Fe-Staphyloferrin A, in the open (green) or closed form (blue). The siderophore is represented in the binding site with the iron atom as a sphere, green or blue. Top right and bottom : two views of the surface potential, with the siderophore in the binding pocket surrounded by highly positive charges.

  Figure S15. Structures of ShuT and PhuT from S. dysenteriae and P. aeruginosa. Top left : surface potential of ShuT with the heme in its binding site. Top right : structures of apo (green) and holo-ShuT (cyan). Bottom left : surface potential of PhuT with heme in the binding site. Bottom right : structures of holo-ShuT (cyan) and holo-PhuT (magenta) showing the heme orientation in their respective pockets.

Figure S17 :

 S17 Figure S17 : Structure of HmuT from C. glutamicum. Left : superposition of the structures of the WT and three mutants of HmuT in the presence of heme. Right : surface potential of HmuT showing heme in the binding site.

  FigureS18: comparison of apo-HutB from V. cholerae (green) and bis-heme HmuT from Y. pestis in the bis-heme conformation (magenta).

Figure S19 :

 S19 Figure S19 : Structure of Rv0265c from M. tuberculosis. Surface potential, showing also the putative binding site.

  Figure S20 : Structure of Y. pestis YiuA. Surface potential with the putative binding site surrounded by positive charges.

  Figure S22 : Structure of E. coli and H. influenzae OppA's, in the unbound (orange, E. coli) and bound (green, H. influenzae) forms.

  Figure S23 : Structure of BhuUV from B. cenocepacia. Top left : surface potential of HmuU, from the cytoplasmic side : the two negatively charged coupling helices are clearly seen, as well as the cavity open on the cytoplasm. Top right : surface potential of HmuU, from the periplasmic side. Bottom left : Ribbon structure of BhuUV, with the two transmembrane BhuU (blue and pale blue) and the two ABC BhuV modules (tan and orange). Bottom right : same as bottom left, rotated 90° along the vertical symetry axis, with one BhuU in thin ribbon and the other as surface with conserved residues across the family coloured labeled (magenta : Pro, yellow : Gly, green : Trp, Phe, deep green : Arg, red : Leu, Ala, cyan : Gln, Asn, Brown : Asp112).

  Figure S24 : Superposition of BhuTU from BhuTUV (green, BhuT as a light ribbon, BhuU as a cartoon) with HmuU from HmuUV (red cartoon). The right view is rotated ca. 90° along the vertical symetry axis.

Acknowledgements

I thank Elie Dassa for helpful advice and discussion during the preparation of this manuscript and the anonymous reviewer for wise comments. This work has been supported by LABEX DYNAMO (ANR-11-LABEX-0011-01).

None.

Conflict of interest

Supplementary figures

Figure S1 : Structures of E. coli FepB. Green : X-ray structure (3TLK), in complex with Fe-Ent (magenta, iron atom as a brown sphere, only one monomer is shown). Blue : Fe-Ent complex (2M6L), one NMR structure, yellow : apo, one NMR structure (2M6K). The orange and pale blue regions correspond to the ones where CSP is observed upon GaEnt binding. Figure S2 : Structures of C. jejuni CeuE. Left : ribbon representation of apo-CeuE (orange, 3ZKW) and holo-CeuE (Fe-DHBS2-complex, 5ADV, magenta); some of the green residues are completing iron coordination sphere. Right : CeuE surface potential. The iron atom is reresented as a blue-violet sphere. Bottom : structure of the Fe-MECAM-CeuE complex (2CHU), showing the two molecules of CeuE (blue ribbon) with their binding sites facing each other, and the two Fe-MECAM molecules (green and magenta, with the iron atoms as orange spheres).

Yersinia pestis

YiuA 6B2X 6B2Y