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Originally published In Press as doi:10.1074/jbc.M411238200 on December 2, 2004

J. Biol. Chem., Vol. 280, Issue 7, 5820-5827, February 18, 2005
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Novel Anion-independent Iron Coordination by Members of a Third Class of Bacterial Periplasmic Ferric Ion-binding Proteins*

Stephen R. Shouldice{ddagger}§, Duncan E. McRee¶, Douglas R. Dougan||, Leslie W. Tari¶, and Anthony B. Schryvers{ddagger}**

From the {ddagger}Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta T2N 4N1, Canada and ActiveSight and ||Syrrx Inc., San Diego, California 92121

Received for publication, October 1, 2004 , and in revised form, November 30, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The uptake of the element iron is vital for the survival of most organisms. Numerous pathogenic Gram-negative bacteria utilize a periplasm-to-cytosol ATP-binding cassette transport pathway to transport this essential atom in to the cell. In this study, we investigated the Yersinia enterocolitica (YfuA) and Serratia marcescens (SfuA) iron-binding periplasmic proteins. We have determined the 1.8-Å structures of iron-loaded (YfuA) and iron-free (SfuA) forms of this class of proteins. Although the sequence of these proteins varies considerably from the other members of the transferrin structural superfamily, they adopt the same three-dimensional fold. The iron-loaded YfuA structure illustrates the unique nature of this new class of proteins in that they are able to octahedrally coordinate the ferric ion in the absence of a bound anion. The iron-free SfuA structure contains a bound citrate anion in the iron-binding cleft that tethers the N- and C-terminal domains of the apo protein and stabilizes the partially open structure.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Metal ions are indispensable components of biological systems. Iron is required for the growth and survival of nearly all living organisms (1). Although iron is the most abundant transition element and the fourth most plentiful element in the Earth's crust, this important atom remains a limiting factor for growth for the vast majority of organisms. Because of the extremely low solubility of Fe3+ (~10-18 M) at neutral pH in aerobic environments most organisms are faced with the problem of obtaining enough iron from their environment (2). Although iron is not readily available to most organisms, in vivo iron is a key component of a number of essential metabolic enzymes, including ribonucleotide reductase and cytochromes. Its biological function is almost entirely dependent on its incorporation into protein molecules. Through the variation of the coordinating ligands surrounding iron, its redox potential can be altered between -300 and +700 mV, making it ideally suited to participate in a wide range of electron transfer reactions involved in intermediary metabolism (3). Although the reactivity of iron makes it uniquely suited for numerous biological applications, it also contributes to a variety of undesirable effects. Free iron has the ability to generate toxic derivatives within the body (4). The highly reactive hydroxyl radical, a product of Fenton chemistry, can damage lipids by inducing the formation of unsaturated bonds, decrease membrane fluidity, and cause cell lysis. Thus, it is vital that biological systems maintain control of the chemical environment of iron.

Most organisms have developed specialized systems for retrieval, transport, and storage of this vital element to maintain it in a non-toxic state. In higher organisms such as vertebrates, there is little free iron present in the extracellular compartment because of the presence of the monomeric glycoproteins transferrin (in sera) and lactoferrin (on mucosal surfaces), which are high affinity bilobed iron-binding proteins. Lactoferrin and transferrin are each composed of a single polypeptide chain of ~650 amino acid residues that are arranged in two homologous globular lobes, the N- and C-lobes, representing the N- and C-terminal halves of the molecules (5). Each lobe is further subdivided into two domains with the specific iron binding sites in the intradomain cleft of each lobe. The iron atom is bound by identical iron-binding ligands in each lobe, two tyrosines, a histidine, and an aspartic acid residue (6, 7). The ability of transferrin and lactoferrin to bind iron also depends on the presence of a carbonate anion. The presence of these glycoproteins throughout the body of the host organism reduces the levels of free ferric iron below that required to support bacterial growth.

In response to the problem of iron scarcity, extracellular Gram-negative pathogenic bacteria have developed a variety of different high affinity iron acquisition systems to survive. One strategy that is effective in a variety of environments involves the synthesis and secretion of small iron chelating molecules, termed siderophores (8). Siderophores function by complexing and removing iron from the host proteins or by scavenging it from precipitates of ferric hydroxide, as they possess affinity constants in excess of host proteins. An alternate iron uptake system utilized by Gram-negative bacteria has also been discovered in a number of human and veterinary pathogens (9, 10). These bacteria possess outer membrane surface receptors that are used for acquiring iron directly from transferrin and/or lactoferrin. The bacteria use this alternative mechanism for iron acquisition as they are unable to produce siderophores. Even though the specific lactoferrin and transferrin outer membrane receptors are distinct complexes, the mechanism of iron removal is thought to occur through similar mechanisms (9).

Following transport across the outer membrane, the ensuing transport of iron into the cell is mediated by an ATP-binding cassette transport system consisting of a periplasmic-binding protein and an inner membrane transport complex (11). This transport system belongs to the superfamily of ATP-binding cassette transporters that includes a broad and diverse group of import and export systems found in prokaryotes and eukaryotes (12). The periplasmic-binding protein is required to transport the complex across the periplasm and release it at the inner membrane permease complex. This inner membrane complex consists of at least two proteins, one to span the membrane and transport the substrate across the inner membrane and another that contains an ATP-binding cassette and that can hydrolyze ATP to provide the energy required for transport (13).

The iron uptake pathways from host transferrin in pathogenic Neisseria species and Haemophilus influenzae have received considerable attention because of their predicted importance in vivo, which has been established experimentally for gonococcal infection in humans (14). This pathway is dependant upon the periplasmic FbpA (ferric-binding protein A) (15, 16). FbpA was shown to possess similar properties to the transferrins (17, 18) to the extent that they have been called bacterial transferrins (19). Structural studies revealed a nearly identical set of amino acid residues involved in iron coordination (20) that led to the suggestion that the forces of convergent evolution had selected for an optimal coordination mode. However, sequence and spectral differences observed with a functional homologue from the bovine pathogen Mannheimia (Pasteurella) haemolytica (21) suggested that the similarity with transferrin may not be a general property of this class of proteins. Indeed, structural studies with the M. haemolytica protein revealed a novel mode of iron coordination involving three tyrosyl residues and a carbonate anion (22).

SfuA (Serratia ferric-iron uptake) of Serratia marcescens (23) and YfuA (Yersinia ferric-iron uptake) of Yersinia enterocolitica (24) are also functional homologues of FbpA, because they are capable of binding and transporting the Fe3+ ion (25, 26). The sequence alignments of YfuA and SfuA with the FbpAs from Haemophilus and Neisseria reveal a considerable level of sequence identity and conservation of key iron liganding amino acids. However, the spectral properties of the iron-loaded forms of these proteins differ from transferrins and from the Haemophilus and Neisseria FbpAs, suggesting that they may utilize a novel mode of iron coordination. In this study, we present the 1.8 Å resolution structure of iron-loaded YfuA and the 1.8 Å iron-free SfuA structure. A comparison between the iron-loaded and iron-free structures has allowed us to characterize the precise atomic details of iron binding, and the role of the ferric ion in mediating protein conformation. Our results reveal that YfuA and SfuA coordinate iron in a unique fashion. Surprisingly, YfuA and SfuA do not require a synergistic anion to bind a ferric ion. This new class of bacterial ferric ion-binding proteins is the only characterized member of the transferrin structural superfamily able to bind and transport iron in the absence of a synergistic anion.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning and Purification—The intact sfuA gene was amplified from a clinical isolate of S. marcescens obtained from the Foothills Hospital (Calgary, Alberta) with a forward primer introducing an NdeI site at the start codon preceded immediately by a BclI site and with a reverse primer introducing a HindIII site immediately after the stop codon. The resulting PCR product was digested with restriction enzymes BclI and HindIII and ligated into a derivative of the pT7-7 vector digested with BamHI and HindIII. The modified pT7-7 vector has a BamHI site introduced between the ribosomal binding site and start codon. A similar strategy was utilized to clone the yfuA gene from Y. enterocolitica strain 0.9 obtained from Dr. Jessica Boyd, Halifax, Nova Scotia, except that the forward primer included a BamHI site in lieu of a BclI site. The cloned genes were sequenced, and the sequences were compared with the published sequences for YfuA (GenBankTM 619573) and SfuA (GenBankTM 152860). The resulting plasmids were transformed into Escherichia coli BL21(DE3)/pLysS for expression of SfuA and YfuA. Both sfuA and yfuA were cloned with their native signal sequences so that when the plasmids were introduced into E. coli the functional proteins would be expressed under isopropyl-1-thio-{beta}-D-galactopyranoside induction and then exported to the periplasm.

The proteins were expressed, and the periplasmic fraction isolated using a previously described modified osmotic shock procedure (27). The protein preparations were dialyzed extensively against 20 mM ethanolamine buffer, pH 9.0, at 4 °C and subjected to anion exchange chromatography as a final purification step. The resulting samples were dialyzed against 10 mM HEPES, pH 7.4, at 4 °C and concentrated using Millipore Amicon Ultra centrifugal filter devices with a 10,000 molecular weight cut-off (final concentrations of 19.6 mg/ml for SfuA and 17.8 mg/ml for YfuA). The purity of the preparations was confirmed by SDS-PAGE analysis. Samples were either utilized in crystallization experiments immediately or stored at 4 °C until required.

Absorption Spectra—UV-visible spectra were recorded on a Nano-Drop® ND-1000 spectrophotometer. A buffer solution consisting of 10 mM HEPES, pH 7.4, served as the reference for the full-range spectra from 220 to 750 nm. Each sample was done in triplicate.

Crystallization and X-ray Data Collection—Crystallization conditions for SfuA and YfuA were initially determined by high throughput screening. A single colorless crystal form was obtained for SfuA, and two colored (purple) crystal forms of YfuA were observed. Crystals used for diffraction analysis were grown at 4 °C for SfuA and at 20 °C for YfuA by the sitting drop vapor-diffusion technique, with 2-µl drops (1 µl of protein sample and 1 µl of reservoir solution) and an 85-µl reservoir. The reservoir for SfuA crystallization was 1.0 M lithium chloride, 30% (w/v) polyethylene glycol 6000, 0.1 M citric acid, pH 4.0, and for YfuA (superior, diffraction quality crystals) was 0.2 M zinc acetate dihydrate, 18% (w/v) polyethylene glycol 8000, and 0.1 M sodium cacodylate, pH 6.5. Large colorless crystals of SfuA belonging to the monoclinic space group P21 appeared after 1 week, and purple YfuA crystals belonging to the monoclinic space group C2 typically grew in 6 days.

The crystals were harvested by scooping them from the drop with a nylon loop. The SfuA crystal was dipped into a cryoprotectant solution containing 20% (v/v) ethylene glycol, 30% (w/v) polyethylene glycol 6000, 1.0 M lithium chloride, and 0.1 M citric acid, pH 4.0, for ~30 s before it was cooled in liquid nitrogen. The YfuA crystal was dipped into crystallization mother liquor supplemented with 25% (v/v) ethylene glycol. The crystals of both proteins were maintained at 100 K during data collection. All data were collected on a Rigaku HTC image plate detector mounted on a Rigaku FR-E rotating anode generator with a copper target. X-ray data were integrated and scaled using the d*TREK program suite (28). Individual data processing statistics for the proteins are listed in Table I.


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  		TABLE I
X-ray data collection and refinement statistics for the YfuA and SfuA crystals

 
Structure Solution and Refinement—The iron-loaded YfuA structure was solved first and used as a molecular replacement model to solve the SfuA structure. To solve the YfuA structure, single wavelength anomalous diffraction x-ray data were analyzed using the program SHELXD (29) to find the heavy atom substructure, locating the six sulfur atoms from methionine residues, the iron, and five bound zinc atoms. Single wavelength anomalous diffraction phases were calculated to 1.7 Å from these heavy atom positions by use of the SHELXE program (30). The phases were then improved by solvent flattening and histogram matching with the program DM (31). The resulting experimental phases were of sufficient quality to allow automated building of almost the entire protein structure in electron density with the program ARP/wARP (32). The resulting structure and electron density maps were interpreted using the program XtalView/Xfit (33). Iterative cycles of interactive manual building and refitting of the model using the program XtalView/Xfit (33) and refinement with Refmac5 (34) were carried out to complete and correct the model. Restrained refinement using a maximum likelihood target function and isotropic temperature factors for individual atoms was carried out followed by TLS refinement. During the later stages of refinement, difference maps (Fo - Fc maps) were used to place the bound iron, zinc, carbonate, ethylene glycol, and water molecules. The high-resolution apo SfuA crystal structure was determined by a molecular replacement procedure using the program Molrep from the CCP4 suite of programs (35). Because the structure of YfuA was in a closed conformation compared with the SfuA protein, two independent search models comprising the N- and C-terminal domains of the protein had to be positioned independently to solve the structure. Iterative cycles of interactive manual refitting of the model using the program XtalView/Xfit (33) made use of maps created with ARP/wARP (32), and refinement with Refmac5 (34) was carried out to complete and correct the model. Restrained refinement using a maximum likelihood target function and isotropic temperature factors for individual atoms was carried out. During the later stages of refinement, difference maps (Fo - Fc maps) were used to place the bound citrate and water molecules. The final crystallographic refinement statistics for each structure are reported in Table I.

Structure Analysis—The refined coordinates of SfuA and YfuA in complex with citrate and iron, respectively, have each been deposited in the Protein Data Bank (codes 1XVY [PDB] and 1XVX). Ramachandran plots of the two structures reveal the satisfactory location of all residues in allowed regions of conformational space. The DALI server was used to find structurally similar proteins in the Fold classification based on structure-structure alignment of the proteins data base (36).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Protein Production, Crystallization, and Structure Solution—The intact genes encoding SfuA and YfuA were amplified from clinical isolates and subcloned into a T7-based expression vector. The sequences of the cloned genes encoded proteins with several amino acid changes relative to YfuA and SfuA sequences in GenBankTM (Fig. 1). The presence of the native leader peptides resulted in efficient production and export of the proteins into the periplasm of E. coli providing yields of up to 100 mg protein/liter of culture. The recombinant SfuA and YfuA proteins were isolated from the periplasm of E. coli and purified by ion-exchange chromatography. Following purification and buffer exchange, the protein samples were concentrated to greater than 15 mg/ml prior to crystallization. The protein samples had a distinctive purple color in contrast to iron-loaded transferrins and FbpAs from Neisseria and Haemophilus that are deep red at these concentrations. Spectral analysis of the protein preparations demonstrated that visible absorption maximum at 513 and 510 nm wavelengths for YfuA and SfuA, respectively, contrasting the maximum at 480 nm for the H. influenzae FbpA (HiFbpA).1



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  		FIG. 1.
Alignment of the amino acid sequences of representative periplasmic ferric ion-binding proteins. The alignment was obtained by superimposing the structures of the ferric ion-binding proteins from Y. enterocolitica (YfuA, PDB code 1XVX [PDB] ), S. marcescens (SfuA, PDB code 1XVY [PDB] ), H. influenzae (HiFbpA, PDB code 1MRP [PDB] ), and M. (Pasteurella) haemolytica (MhFbpA, PDB code 1SI0 [PDB] ) in Swiss-PdbViewer, performing a structural alignment, and then exporting the amino acid alignment. Conserved amino acids are indicated by an asterisk, and residues common to three of the sequences are indicated by a period. The ligands directly involved in co-coordinating iron are indicated by single underline, the residues involved in coordinating anions are indicated by double underlines, and the residues that differ from the GenBankTM sequences are written in italics.

 

Following purification, an extensive high throughput screen was performed yielding a high quality crystal form of YfuA. The structure of the iron-loaded YfuA sample was solved initially by single wavelength anomalous diffraction phasing methods. An additional set of crystallization screens was performed for the SfuA protein but only yielded uncolored crystals. The structure of YfuA successfully served as a search model in molecular replacement to obtain the SfuA structure.

General Structural Features—Data collection and refinement statistics are reported in Table I. For almost all of the protein side chains, the electron density is clear and unambiguous. In the SfuA structure the first two residues of the N terminus were not visible in the electron density map and were omitted. Similarly, the first residue of the YfuA model was not well defined by electron density and thus was omitted from the final structure. The stereochemical parameters (Table I) and the Ramachandran plots for each structure, which reveal that all of the residues are in allowed regions as defined by the program PROCHECK (35), illustrate that these crystal structures are of high quality.

SfuA and YfuA share 86% sequence identity at the amino acid level (Fig. 1) and share identical polypeptide folds (Fig. 2). Crystal structures of all of the periplasmic ferric ion-binding proteins deposited to date in the PDB display an almost identical topology. With approximate dimensions of 60 x 30 x 40 Å, SfuA and YfuA have a similar overall structure to the wild type H. influenzae periplasmic FbpA. A search utilizing the DALI (36) server for proteins with three-dimensional folds similar to SfuA and YfuA in the PDB reveals that these proteins possess the highest structural similarity with HiFbpA (PDB code 1MRP [PDB] , see Ref. 20). SfuA and YfuA only share ~35% sequence identity with HiFbpA. However, the C{alpha} backbones of the HiFbpA and YfuA structures overlay with a positional root mean square deviation of 1.84 Å over 95% of the residues. Similar to most other periplasmic-binding protein structures and other members of the transferrin structural superfamily, the SfuA and YfuA molecules are composed of two domains termed the N- and C-domains. The polypeptide chain of SfuA and YfuA crosses between the N- and C-domains twice. Between these two domains lies the iron-binding cleft. Each domain is composed of a twisted mixed {beta}-sheet surrounded by {alpha}-helices. The two domains are connected by two anti-parallel {beta}-strands that serve as a hinge between the two domains.



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  		FIG. 2.
Stereo ribbon diagram of YfuA structure. YfuA possesses two {alpha}/{beta}-domains linked by a {beta}-strand hinge. {alpha}-Helices are shown in gold. {beta}-strands are shown in cyan. The relative positions of the iron (orange sphere), water molecule (red sphere), and iron ligands in the binding cleft are shown. For reference, the orientation of the protein shown here is slightly rotated from the orientation used to generate Fig. 3.

 
YfuA Structure—YfuA is able to coordinate a single Fe3+ ion in an octahedral fashion, utilizing five amino acid residues and a water molecule (Fig. 3, A and B). The coordination scheme observed in this structure is novel because of the presence of an additional amino-acid-coordinating residue and the absence of a coordinating anion. The square-planar base of the octahedron is composed of a water molecule, Glu-62, Tyr-198, and Tyr-199. To complete the coordination sphere, His-14 and Asp-144 are located at the axial positions. The iron coordination is distorted from ideal octahedral geometry with ligation distances ranging from 2.04 to 2.21 Å (Table II). The YfuA crystal structure represents a novel class of ferric ion-binding periplasmic proteins that do not require a synergistic anion to complete the iron coordination. Overlays involving the iron binding residues of YfuA and HiFbpA (PDB code 1MRP [PDB] ) reveal that Asp-144 in YfuA is able to functionally replace the phosphate anion in iron coordination.



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  		FIG. 3.
Stereo views of the iron binding site of YfuA and SfuA. A, electron density maps of the iron-loaded YfuA structure (green for the 2Fo - Fc map contoured at 1{sigma}, and red for the Fo - Fc map contoured at 5{sigma} obtained using the reflection data after refinement of the model in which the iron atom (orange sphere) was omitted). B, the iron-liganding residues are shown (water is a cyan sphere) with bonds to the iron atom. C, electron density maps of the iron-free SfuA structure (green for the 2Fo - Fc map contoured at 1{sigma}, and red for the Fo - Fc map contoured at 3{sigma} obtained using the reflection data after refinement of the model in which the citrate molecule was omitted). D, potential hydrogen bonding interactions in SfuA are shown as green dotted lines. This figure was generated using Raster3D

 


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  		TABLE II
Geometry of the iron binding site in YfuA

 
As a result of the high zinc concentrations in the crystallization solution used for YfuA, there are five zinc atoms bound to the final structure. Although these ions apparently have no role in the function of the protein, they appear to stabilize some of the mobile portions of the molecule for crystallization. Zincs 314, 317, and 318 utilize amino acid residues from two symmetry-related molecules, which likely aided in the crystallization process. It was previously shown that heavy metals were important for the successful crystallization of the substrate-binding protein HisJ (37). In addition to three water molecules and an aspartic acid residue, zinc 315 is also coordinated by a bicarbonate anion in the current YfuA structure. The coordination states range from 4 to 6 coordinating ligands surrounding each zinc ion. Similar ligation distances and coordination geometries for zinc ions in proteins have been reported in other protein structures (38-41).

SfuA Structure—The wild type ferric ion-binding periplasmic protein structures determined to date undergo large (~20°) rotation in the hinge region when they convert from the iron-loaded (closed) form to the iron-free (open) form. In contrast, a structural comparison of the apo (SfuA) and holo (YfuA) forms reveals only a 5° rotation about the hinge region. The apo- and holo-structures are very similar with a root mean square deviation in the C{alpha} backbone of 1.42 Å. This reduced movement of the two domains compared with other FbpA structures is likely because of the presence of the citrate molecule observed in the iron-binding cleft of the SfuA structure that tethers the domains together. The high resolution electron density maps (2Fo - Fc and Fo - Fc) permitted the unambiguous placement of the citrate in the binding cleft (Fig. 3, C and D). Other anions were considered based on buffers used during the purification and crystallization protocols, but they were later rejected because of their lack of agreement with the electron density maps. The citrate anion is able to tether the two domains together by forming hydrogen bonds with two residues of each domain located in the ligand-binding cleft (Fig. 3). From the N-terminal domain, residue His-11 forms a hydrogen bond (3.29 Å) with O-13 of citrate, and Glu-59 forms two bonds with O-9 (2.46 Å) and O-10 through a water molecule. Ser-176 from the C-terminal domain forms a hydrogen bond with the O-7 (3.44 Å) of citrate. Also, Tyr-196 from the C-domain is able to form two hydrogen bonds with O-11 (2.70 Å) and O-12 (3.24 Å) of the citrate anion. The citrate anion is not bound in a basic pocket at the base of the binding cleft in an analogous fashion to the synergistic anions observed in other members of the transferrin structural superfamily. In contrast, the citrate anion is bound at the solvent exposed surface of the iron-binding cleft and plays no apparent role in iron coordination. In fact, the citrate molecule may have been responsible for displacing a bound ferric ion. As already stated, no anion was observed in the iron-loaded YfuA protein.

A further comparison of the SfuA and YfuA structures reveals that the citrate anion only effects the orientation of the N- and C-terminal domains as mentioned above and does not alter the relative conformations of key residues in the solvent-exposed regions of the iron-binding pocket that sequester the ferric ion from solvent. The N-C-domain interfaces in YfuA and SfuA are populated primarily by polar residues and are compositionally similar to those observed in HiFbpA and NFbpA. Thus, it is reasonable to assume that YfuA and SfuA are capable of large interdomain movements similar in magnitude to those observed in the HiFbpA and NFbpA structures (42). Based on our structural analysis it seems likely that interdomain movement is restricted in the SfuA structure by the bound citrate molecule, and the SfuA structure does not correspond to a fully open non-liganded form.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The SfuA and YfuA crystal structures adopt the periplasmic ligand-binding protein fold common to most periplasmic ligand-binding proteins (43). These proteins possess bilobate structures in which the two domains are connected by two flexible, anti-parallel {beta}-strands (Fig. 2). Along with previously characterized periplasmic ferric ion-binding proteins (20, 22), they belong to the periplasmic-binding protein-like II superfamily (44). Although the C{alpha} backbones of these proteins are virtually superimposable, our findings suggest that YfuA and SfuA represent a new class in this iron-binding family of proteins based on metal coordination (Fig. 4).



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  		FIG. 4.
Coordination of ferric ion. The geometry for coordination of ferric ion in the bacterial periplasmic ferric ion-binding proteins HiFbpA (class 1) (A), YfuA (class 2) (B), MhFbpA (class 3) (C) is illustrated.

 
Phylogenetic clustering, in conjunction with analysis of the iron- and anion-binding ligands identified by structural studies (Fig. 1), suggest that homologues of known periplasmic iron-binding proteins can roughly be grouped into at least three classes (Fig. 5). The first class, represented by H. influenzae FbpA (20), possesses a set of four iron-liganding amino acids similar to transferrin and amino acids required for coordinating the bound phosphate anion that completes the coordination complex along with a water molecule (Fig. 4). Most of these proteins are from host-restricted, human respiratory pathogens possessing a receptor-mediated pathway for acquiring iron directly from transferrin and/or lactoferrin and lacking the capacity to produce siderophores. Because transferrin and lactoferrin are the primary source of iron for the FbpA-mediated pathway in these pathogens, these FbpAs may be optimally designed for this role. The homologues from the Pseudomonas species do not contain an amino acid equivalent to the glutamine 58 of HiFbpA that is involved in coordinating the phosphate anion and thus have been placed in the subclass 1b.



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  		FIG. 5.
Phylogram of organisms containing a predicted ferric ion-binding protein. The sequences were obtained by performing a BLAST search using the S. marcescens SfuA sequence as the reference. Only those sequences containing the dityrosine motif and possessing a greater than 25% identity to SfuA were used in the final alignment. The alignment was then created using ClustalW to create the phylogram.

 
The results in this study demonstrated that although YfuA and SfuA possess all of the liganding amino acids found in the HiFbpA and transferrin (Fig. 1), the mode of iron coordination is novel and represents a separate class of periplasmic ferric binding proteins (Fig. 4). Like HiFbpA, the title proteins possess two tyrosine residues in the C-domain, along with a histidine and glutamic acid residue from the N-terminal domain that coordinate the ferric ion. However, instead of using a synergistic phosphate anion in the coordination of the iron atom, YfuA utilizes an Asp residue (Asp-144). When these two structures are overlaid, this side chain occupies the same position as the phosphate anion and is able to coordinate the ferric ion in a similar manner. With the anion position occupied by a protein side chain, this class of proteins cannot undergo the proton-assisted dissociation of the synergistic anion that is believed to be a trigger for Fe3+ release for both Neisseria FbpA, and human transferrin (45).

Currently, the physiological source of iron for YfuA and its homologues is unknown and thus the potential advantage of an iron chelation strategy that does not require a synergistic anion is uncertain. In Yersinia species, the ybt locus that encodes the enzymes required for production of the siderophore, yersiniabactin, and the yfe locus that encodes another metal/iron uptake pathway, were shown to be essential for infection in mice (46), presumably because of an acquisition of iron at different stages of infection. The YfeA protein is not related to the periplasmic iron-binding proteins described in this study but belongs to a family of predominantly zinc- and manganese-binding proteins represented by the zinc-binding TroA protein from Treponema pallidum (47), that has a helical backbone distinguishing it from most periplasmic ligand-binding proteins. The Yfu ATP-binding cassette pathway is not required for infection in mice (24, 25) nor for growth under any in vitro conditions that have been tested. A potential role for survival in the flea gut has been suggested but not confirmed experimentally.

The first two classes of ferric ion-binding proteins may have been derived from a common progenitor, because they share four conserved iron-coordinating residues (Fig. 4) and cluster into the same grouping based on overall sequence similarity (Fig. 5). Although the rest of the proteins that cluster nearest to the class 1 and 2 FbpAs contain the dityrosine motif shown to be vital for iron coordination, there is no experimental evidence to confirm metal binding. It is possible that these proteins will represent additional classes of ferric ion-binding proteins based on alternate coordination chemistries or perhaps will even represent periplasmic proteins responsible for transport of other substrates, such as anions.

The FbpA from M. haemolytica (MhFbpA) (22) is a representative member of a third class that is characterized by a unique iron coordination scheme involving three tyrosines and a bound carbonate anion that provides five rather than six coordinating moieties (Fig. 4). All members of this class have an arginine residue at position 10 that is utilized to coordinate the carbonate anion in MhFbpA. The residue located at position 11 involved in coordinating the carbonate anion in MhFbpA varies among members of this class, which may reflect differences in anion coordination. Although the closest homologues of MhFbpA are found in other ruminant pathogens that possess receptor-mediated uptake systems capable of acquiring iron directly from transferrin, there is considerable diversity in the types of bacteria that possess unambiguous homologues to this protein (48). Thus, it is likely that this class of FbpA is involved in acquiring iron from a diverse range of iron sources.

It is evident from the analysis in Fig. 5 that the relationship between the ferric ion-binding proteins does not reflect the phylogenetic relationship among the species expressing these proteins. In this respect it is also interesting to note that the host adapted human pathogens that utilize receptor-mediated uptake from transferrin (H. influenzae, Neisseria sp., Actinobacillus actinomycetemcomitans) have class 1 FbpAs whereas the comparable bovine pathogens (M. haemolytica, Haemophilus somnus, Pasteurella multocida) possess class 3 FbpAs. The presence of two distinct classes of FbpAs within members of the pasteurellae suggest that the genes may have been acquired by separate horizontal exchange events or that both classes of proteins were originally present in an early predecessor of the current species.

Although the different modes of iron coordination in FbpA homologues could confer specific advantages for the physiological role they serve, there currently is no experimental evidence to support this concept. The intact pathways are all capable of mediating the uptake of iron in reconstituted systems in a strain of E. coli defective in siderophore production (18, 21, 24, 26). However, this system relies on a non-physiological source of iron provided by the addition of the exogenous iron chelator, dipyridyl, that is capable of facilitating movement of iron across the outer membrane. Thus, whether the different classes of FbpAs would be equally capable of acquiring ferric ions from different physiological sources remains an open question. Comparisons of the different modes of iron coordination could also potentially provide insights into the mechanism of iron removal and transport across the inner membrane, particularly if the mechanism was conserved among the various homologues. However, at this juncture little is known about the mechanism, and there is no experimental evidence supporting the concept that the mechanism is conserved among the various homologues.

In summary, we have determined the first two crystal structures of a new class of bacterial iron-binding proteins. Five protein side chains are utilized along with a water molecule for octahedral iron coordination. It is also apparent that this class of proteins has the ability to bind large anions such as the citrate present in the SfuA model. To better understand the iron release process by this class of proteins, our structural analysis will have to be coupled with additional biochemical and biophysical work.


   FOOTNOTES
 
The atomic coordinates and structure factors (codes 1XVY and 1XVX) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by Grant 49603 from the Canadian Institutes for Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported by a Studentship from University Technologies International. Back

** To whom correspondence should be addressed. Tel.: 403-220-3703; Fax: 403-270-2772; E-mail: schryver{at}ucalgary.ca.

1 The abbreviations used are: HiFbpA, H. influenzae FbpA; MhFbpA, M. haemolytica FbpA. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Guerinot, M. L. (1994) Annu. Rev. Microbiol. 48, 743-772[CrossRef][Medline] [Order article via Infotrieve]
  2. Braun, V., and Killmann, H. (1999) Trends Biochem. Sci. 24, 104-109[CrossRef][Medline] [Order article via Infotrieve]
  3. Andrews, S. C., Robinson, A. K., and Rodriguez-Quinones, F. (2003) FEMS Microbiol. Rev. 27, 215-237[CrossRef][Medline] [Order article via Infotrieve]
  4. Wessling-Resnick, M. (1999) Crit. Rev. Biochem. Mol. Biol. 34, 285-314[CrossRef][Medline] [Order article via Infotrieve]
  5. Baker, E. N., Anderson, B. F., Baker, H. M., MacGillivray, R. T. A., Moore, S. A., Peterson, N. A., Shewry, S. C., and Tweedie, J. W. (1998) Adv. Exp. Med. Biol. 443, 1-14[Medline] [Order article via Infotrieve]
  6. Anderson, B. F., Baker, H. M., Norris, G. E., Rice, D. W., and Baker, E. N. (1989) J. Mol. Biol. 209, 711-734[CrossRef][Medline] [Order article via Infotrieve]
  7. Bailey, S., Evans, R. W., Garrat, R. C., Gorinsky, B., Hasnain, S., Horsburgh, C., Jhoti, H., Lindley, P. F., Mydin, A., Sarra, R., and Watson, J. L. (1988) Biochemistry 27, 5804-5812[CrossRef][Medline] [Order article via Infotrieve]
  8. Neilands, J. B. (1995) J. Biol. Chem. 270, 26723-26726[Free Full Text]
  9. Gray-Owen, S. D., and Schryvers, A. B. (1996) Trends Microbiol. 4, 185-191[CrossRef][Medline] [Order article via Infotrieve]
  10. Schryvers, A. B., and Stojiljkovic, I. (1999) Mol. Microbiol. 32, 1117-1123[CrossRef][Medline] [Order article via Infotrieve]
  11. Koster, W. (2001) Res. Microbiol. 152, 291-301[Medline] [Order article via Infotrieve]
  12. Schmitt, L., and Tampe, R. (2002) Curr. Opin. Struct. Biol. 12, 754-760[CrossRef][Medline] [Order article via Infotrieve]
  13. Higgins, C. F. (2001) Res. Microbiol. 152, 205-210[Medline] [Order article via Infotrieve]
  14. Cornelissen, C. N., Kelley, M., Hobbs, M. M., Anderson, J. E., Cannon, J. G., Cohen, M. S., and Sparling, P. F. (1998) Mol. Microbiol. 27, 611-616[CrossRef][Medline] [Order article via Infotrieve]
  15. Khun, H. H., Kirby, S. D., and Lee, B. C. (1998) Infect. Immun. 66, 2330-2336[Abstract/Free Full Text]
  16. Kirby, S. D., Gray-Owen, S. D., and Schryvers, A. B. (1997) Mol. Microbiol. 25, 979-987[CrossRef][Medline] [Order article via Infotrieve]
  17. Nowalk, A. J., Tencza, S. B., and Mietzner, T. A. (1994) Biochemistry 33, 12769-12775[CrossRef][Medline] [Order article via Infotrieve]
  18. Adhikari, P., Kirby, S. D., Nowalk, A. J., Veraldi, K. L., Schryvers, A. B., and Mietzner, T. A. (1995) J. Biol. Chem. 42, 25142-25149
  19. Taboy, C. H., Vaughan, K. G., Mietzner, T. A., Aisen, P., and Crumbliss, A. L. (2001) J. Biol. Chem. 276, 2719-2724[Abstract/Free Full Text]
  20. Bruns, C. M., Norwalk, A. J., Avrai, A. S., McTigue, M. A., Vaughan, K. A., Mietzner, T. A., and McRee, D. E. (1997) Nat. Struct. Biol. 4, 919-924[CrossRef][Medline] [Order article via Infotrieve]
  21. Kirby, S. D., Lainson, F. A., Donachie, W., Okabe, A., Tokuda, M., Hatase, O., and Schryvers, A. B. (1998) Microbiology 144, 3425-3436[Abstract/Free Full Text]
  22. Shouldice, S. R., Skene, R. J., Dougan, D. R., Snell, G., McRee, D. E., Schryvers, A. B., and Tari, L. W. (2004) J. Bacteriol. 186, 3903-3910[Abstract/Free Full Text]
  23. Zimmermann, L., Angerer, A., and Braun, V. (1989) J. Bacteriol. 171, 238-243[Abstract/Free Full Text]
  24. Saken, E., Rakin, A., and Heesemann, J. (2000) Int. J. Med. Microbiol. 290, 51-60[Medline] [Order article via Infotrieve]
  25. Gong, S., Bearden, S. W., Geoffroy, V. A., Fetherston, J. D., and Perry, R. D. (2001) Infect. Immun. 69, 2829-2837[Abstract/Free Full Text]
  26. Angerer, A., Klupp, B., and Braun, V. (1992) J. Bacteriol. 174, 1378-1387[Abstract/Free Full Text]
  27. Shouldice, S. R., Dougan, D. R., Skene, R. J., Tari, L. W., McRee, D. E., Yu, R.-H., and Schryvers, A. B. (2003) J. Biol. Chem. 278, 11513-11519[Abstract/Free Full Text]
  28. Pflugrath, J. W. (1999) Acta Crystallogr. Sect. D Biol. Crystallogr. 55 (Pt 10), 1718-1725[CrossRef][Medline] [Order article via Infotrieve]
  29. Schneider, T. R., and Sheldrick, G. M. (2002) Acta Crystallogr. Sect. D Biol. Crystallogr. 58, 1772-1779[CrossRef][Medline] [Order article via Infotrieve]
  30. Sheldrick, G. M. (2002) Zeitschrift fur Kristallogrphie 217, 644-650[CrossRef]
  31. Cowtan, K., and Main, P. (1998) Acta Crystallogr. Sect. D Biol. Crystallogr. 54, 487-493[CrossRef][Medline] [Order article via Infotrieve]
  32. Morris, R. J., Perrakis, A., and Lamzin, V. S. (2002) Acta Crystallogr. Sect. D Biol. Crystallogr. 58, 968-975[CrossRef][Medline] [Order article via Infotrieve]
  33. McRee, D. E. (1999) J. Struct. Biol. 125, 156-165[CrossRef][Medline] [Order article via Infotrieve]
  34. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Acta Crystallogr. Sect. D 53, 240-255[CrossRef][Medline] [Order article via Infotrieve]
  35. Collaborative Computational Project and 4 (1994) Acta Crystallogr. Sect. D 50, 760-763[CrossRef][Medline] [Order article via Infotrieve]
  36. Holm, L., and Sander, C. (1997) Nucleic Acids Res. 25, 231-234[Abstract/Free Full Text]
  37. Oh, B. H., Kang, C. H., De Bondt, H., Kim, S. H., Nikaido, K., Joshi, A. K., and Ames, G. F. (1994) J. Biol. Chem. 269, 4135-4143[Abstract/Free Full Text]
  38. Ma, L., and Kantrowitz, E. R. (1996) Biochemistry 35, 2394-2402[CrossRef][Medline] [Order article via Infotrieve]
  39. Johansson, K., Ramaswamy, S., Saarinen, M., Lemaire-Chamley, M., Issakidis-Bourguet, E., Miginiac-Maslow, M., and Eklund, H. (1999) Biochemistry 38, 4319-4326[CrossRef][Medline] [Order article via Infotrieve]
  40. Korndorfer, I. P., Fessner, W. D., and Matthews, B. W. (2000) J. Mol. Biol. 300, 917-933[CrossRef][Medline] [Order article via Infotrieve]
  41. Holland, D. R., Cousens, L. S., Meng, W., and Matthews, B. W. (1994) J. Mol. Biol. 239, 385-400[CrossRef][Medline] [Order article via Infotrieve]
  42. Bruns, C. M., Anderson, D. S., Vaughan, K. G., Williams, P. A., Nowalk, A. J., McRee, D. E., and Mietzner, T. A. (2001) Biochemistry 40, 15631-15637[CrossRef][Medline] [Order article via Infotrieve]
  43. Quiocho, F. A., and Ledvina, P. S. (1996) Mol. Microbiol. 20, 17-25[Medline] [Order article via Infotrieve]
  44. Andreeva, A., Howorth, D., Brenner, S. E., Hubbard, T. J., Chothia, C., and Murzin, A. G. (2004) Nucleic Acids Res. 32, D226-D229[Abstract/Free Full Text]
  45. Boukhalfa, H., Anderson, D. S., Mietzner, T. A., and Crumbliss, A. L. (2003) J Biol. Inorg. Chem. 8, 881-892[CrossRef][Medline] [Order article via Infotrieve]
  46. Bearden, S. W., and Perry, R. D. (1999) Mol. Microbiol. 32, 403-414[CrossRef][Medline] [Order article via Infotrieve]
  47. Lee, Y. H., Deka, R. K., Norgard, M. V., Radolf, J. D., and Hasemann, C. A. (1999) Nat. Struct. Biol. 6, 628-633[CrossRef][Medline] [Order article via Infotrieve]
  48. Shouldice, S. R., Dougan, D. A., Williams, P. W., Skene, R. J., Snell, G., Scheibe, D., Kirby, S. M., Hosfield, D. J., McRee, D. E., Schryvers, A. B., and Tari, L. W. (2003) J. Biol. Chem. 278, 41093-41098[Abstract/Free Full Text]

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