The Crystal Structure of Klebsiella pneumoniae FeoA Reveals A Site For Protein-Protein Interactions

Abstract

In order to establish infection, pathogenic bacteria must obtain essential nutrients such as iron. Under acidic and/or anaerobic conditions, most bacteria utilize the Feo system in order to acquire ferrous iron (Fe²⁺) from their host environment. The mechanism of this process, including its regulation, remains poorly understood. In this work, we have determined the crystal structure of FeoA from the nosocomial agent Klebsiella pneumoniae (KpFeoA). Our structure reveals an SH3-like domain that mediates interactions between neighboring polypeptides via hydrophobic intercalations into a Leu-rich surface ridge. Using docking of a small peptide corresponding to a postulated FeoB partner binding site, we demonstrate the KpFeoA can assume both “open” and “closed” conformations, controlled by binding at this Leu-rich ridge. We propose a model in which a “C-shaped” clamp along the FeoA surface mediates interactions with its partner protein, FeoB. These findings are the first to demonstrate atomic-level details of FeoA-based protein-protein interactions and provide a future framework to test for FeoA-FeoB interactions, which could be exploited for future antibiotic developments.

INTRODUCTION

The acquisition of iron is an essential virulence factor for the establishment of infection by a wide array of bacterial pathogens,^1–2 including one of the major causative agents of nosocomial (hospital-acquired) infections, Klebsiella pneumoniae.^3–4 The environmental source of iron is typically the host, where it may be found in multiple oxidation and coordination states, necessitating pathogens such as K. pneumoniae to adapt to acquire iron in ferric (Fe³⁺), ferrous (Fe²⁺), and even chelated forms. Under oxidizing conditions, siderophore- and heme-based acquisition systems are essential to stabilize, to solubilize, and to transport ferric iron.¹ However, under acidic, micro-aerobic, and/or anaerobic conditions, such as those found in the gut or within biofilms, iron may be prevalent in the reduced, ferrous form.^5–6 Because ferrous iron has differences in solubility, lability, and even coordination properties compared to ferric iron, bacteria such as K. pneumoniae must employ orthogonal transport systems to acquire and to handle Fe²⁺.^5–6

The most prevalent prokaryotic transport system dedicated to the transport of Fe²⁺ is the ferrous iron transport system, also known as Feo (Fig. 1).⁶ In K. pneumoniae, this system is found along the feo operon (Fig. 1A), which encodes for three proteins: FeoA, FeoB, and FeoC.⁷ FeoA and FeoC are predicted to be small (~8 kDa), cytosolic proteins, whereas FeoB is a large (~90 kDa), polytopic membrane protein bearing a N-terminal GTP-binding domain (NFeoB; Fig. 1B). These three proteins are postulated to function in concert to regulate the movement of ferrous iron from the periplasm into the cytosol (Fig. 1B), where it is presumably handed off to an unknown ferrous iron chaperone for assembly into iron-containing proteins and/or intracellular storage.

Figure 1.

The Feo system in K. pneumoniae. A. The feo operon within K. pneumoniae encodes for three proteins: FeoA, FeoB, and FeoC. B. In K. pneumoniae, FeoA (red) and FeoC (green) are small, cytosolic proteins that are predicted to function as accessories to ferrous (Fe²⁺) transport across the intracellular membrane, mediated by the polytopic membrane protein FeoB (purple). At the N-terminus of FeoB is the soluble GTP-binding domain known as NFeoB (teal), which is capable of hydrolyzing GTP and may drive ferrous iron transport in an active manner.

We recently described the preparation and biophysical properties of detergent-solubilized K. pneumoniae FeoB (KpFeoB).⁷ Of note was the ability of KpFeoB to hydrolyze GTP at a rate comparable to the hydrolysis of ATP by some ABC^8–9 and P_1B-ATPase^10–11 metallotransporters, suggesting that ferrous iron transport could be driven in an active manner by GTP hydrolysis. However, this hydrolysis rate (~0.1 s⁻¹) is still considered sluggish by means of most active transporters.⁷ The slow rate of GTP hydrolysis by FeoB has lead us and others to consider that an additional stimulatory factor may exist to upregulate hydrolysis under changing intracellular conditions in order to drive ferrous iron uptake.^{6–7, 12–14} It is postulated that this stimulatory factor is FeoA.

At least a few three-dimensional structures of FeoA exist,^{13, 15} and these structures reveal the presence of an Src-homology 3 (SH3)-like fold. SH3 folds, which are common to eukaryotes and are characterized by small (<100 amino acids) β-barrels, are often involved in mediating protein-protein interactions and are even utilized to activate eukaryotic GTPases.^16–19 SH3 folds typically interact with binding sites on partner proteins bearing a consensus motif of PxxP, with “x” frequently being a hydrophobic amino acid.²⁰ To our knowledge, every FeoB sequence that we have examined contains a candidate PxxP binding site, leading to the consideration that this site is the location for FeoA-FeoB interactions. However, how FeoA may facilitate this interaction remained unknown until now.

MATERIALS AND METHODS

Materials.

The pET-21a(+) expression plasmid was purchased from EMD-Millipore (MilliporeSigma). A modified BL21(DE3) E. coli expression cell line in which the gene for the multidrug exporter AcrB (a common contaminant from E. coli membranes) had been deleted (BL21(DE3) ΔacrB) was a generous gift of Prof. Edward Yu (Iowa State University). All materials used for buffer preparation, protein expression, and protein purification were purchased from RPI, MilliporeSigma, and/or VWR and were used as received.

Cloning, Expression, and Purification of KpFeoA.

DNA encoding for the gene corresponding to FeoA from Klebsiella pneumoniae (subsp. pneumoniae) (Uniprot identifier A0A0M1TF23) (KpFeoA) was commercially synthesized by GenScript (Piscataway, NJ), with an additionally engineered DNA sequence encoding for a C-terminal TEV-protease cleavage site (ENLYFQS). This gene was subcloned into the pET-21a(+) expression plasmid using the NdeI and XhoI restriction sites, encoding for a C-terminal (His)₆ affinity tag when read in-frame. The complete expression plasmid was transformed into chemically competent BL21(DE3) ΔacrB cells, spread onto Luria-Bertani (LB) agar plates supplemented with 100 μg/ml ampicillin, and grown overnight at 37°C. Colonies from these plates served as the source of E. coli for small-scale starter cultures (generally 100 mL LB supplemented with 100 μg/ml ampicillin). Large-scale expression of KpFeoA was accomplished in 12 baffled flasks each containing 1 L sterile LB supplemented with 100 μg/ml (final) ampicillin and inoculated with a pre-culture. Cells were grown by incubating these flasks at 37°C with shaking of 200 r.p.m. until OD₆₀₀ reached ~0.6−0.8. The flasks containing cells and media were then chilled to 4°C for 2 h, after which protein expression was induced by the addition of isopropyl β-D-l-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. The temperature of the incubator shaker was lowered to 18°C with continued shaking of 200 r.p.m. After ~18–20 h, cells were harvested by centrifugation at 4800×g, 10 min, 4°C. Cell pellets were subsequently resuspended in resuspension buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 5% (v/v) glycerol), flash-frozen on N_2(l), and stored at −80°C until further use.

For SeMet-substituted KpFeoA, a single colony from LB agar plates supplemented with 100 μg/ml ampicillin (vide supra) was used to inoculate 5 mL of sterile LB supplemented with 100 μg/ml ampicillin (final). After ~8 h, ~1 mL of this culture was added to 100 mL of 1× minimal media composed of 1× (final) M9 salts, 0.4% (w/v; final) D-glucose, 2 mM (final) MgSO₄, 100 μM (final) CaCl₂ supplemented with 100 μg/ml ampicillin. This culture was then grown by incubating these flasks at 37°C with shaking of 200 r.p.m overnight. Large-scale expression of SeMet KpFeoA was accomplished in 12 baffled flasks each containing 1 L sterile 1× supplemented minimal media with 100 μg/ml (final) ampicillin and inoculated with the minimal media pre-culture. Cells were grown by incubating these flasks at 37°C with shaking of 200 r.p.m. until OD₆₀₀ reached ~0.6–0.8. After 2 hr cold shock at 4°C, each 1 L flask of 1× minimal media was supplemented with a solution delivering 100 mg L-Phe, 100 mg L-Lys, 100 mg L-Thr, 50 mg L-Ile, 50 mg L-Leu, 50 mg L-Val, and 60 mg L-SeMet. These flasks were then incubated at 18°C with shaking of 200 r.p.m. for 15 min. After this incubation period, protein expression was induced by the addition of IPTG to a final concentration of 1 mM. Protein expression and cell harvest were accomplished as with the native protein (vide supra).

All steps for the purification of KpFeoA were performed at 4°C unless otherwise noted. Frozen cells were thawed and stirred until the solution was homogeneous. Solid phenylmethylsulfonyl fluoride (PMSF; ~50−100 mg) was added immediately prior to cellular disruption using a Q700 ultrasonic cell disruptor (QSonica) set to 70% maximal amplitude, 30 s pulse on, 30 s pulse off, for a total pulse on time of 12 min. Cellular debris was cleared by ultracentrifugation at 163000×g for 1 h. The supernatant was then applied to a 5 mL HiTrap IMAC FF column (GE Healthcare) that had been charged with Ni²⁺ and equilibrated with 8 column volumes of wash buffer (50 mM Tris, pH 8.0, 200 mM NaCl, 10% (v/v) glycerol) with 21 mM imidazole. The column was then washed with 12 column volumes of wash buffer with 30 mM imidazole. Protein was then eluted by wash buffer containing 300 mM imidazole. Fractions were concentrated using a 15 mL Amicon 3 kDa molecular-weight cutoff (MWCO) spin concentrator (MilliporeSigma). Protein was then applied to a 120 mL Superdex 75 (GE Healthcare) gel filtration column that had been pre-equilibrated with 25 mM Tris, pH 7.5, 200 mM NaCl, and 5% (v/v) glycerol. The eluted fractions of the colorless KpFeoA, which corresponded to either dimeric protein (~20 kDa) or monomeric protein (~10 kDa), were pooled and concentrated with a 4 mL Amicon 3 kDa MWCO spin concentrator. All additional size-exclusion experiments were performed in a similar manner. Protein concentration was determined using the Lowry assay, and purity was assessed via 15% SDS-PAGE analysis.

Crystallization, Data Analysis, and Structure Determination.

Crystals of native and SeMet-substituted KpFeoA were obtained by sitting-drop vapor-diffusion using MiTeGen-XtalQuest Plates with a 1:1 (v:v) KpFeoA (~7.5 mg/mL) and reservoir solution mixture at room temperature. The precipitant solution for both native and SeMet KpFeoA consisted of 2.0 M ammonium sulfate, 0.1 M ammonium fluoride, and 3% (v/v) glycerol. Small three-dimensional, colorless octagons appeared within ~24 hr and reached their maximal size within ~2–3 days. Crystals were transferred into cryoprotectant consisting of 1.5 M ammonium sulfate and 50% (v/v) glycerol. After soaking for ~1 min, crystals were then looped, flash-frozen, and stored at 77 K.

Data sets were collected on beamline LS-CAT 21-ID-D at the Advanced Photon Source, Argonne National Laboratory, using a Dectris Eiger 9M detector. Data were processed automatically with Xia2.²¹ Phases and initial models of SeMet KpFeoA were generated using the AutoSol and AutoBuild programs in Phenix.²² This model served as the input for solving native KpFeoA datasets via molecular replacement using Phaser in Phenix.²² Extended model building and refinement cycles were performed in Coot²³ and REFMAC5²⁴, respectively. Final validations were performed using Phenix Validate²², and the final model consists of residues 1–80; the final 10 residues (81–90) comprising chiefly the purification tag are not visible in the electron density map. Data collection and refinement statistics are presented in Table S1. Structural overlays, Cα rmsd values, and surface representations were generated and calculated by utilizing UCSF Chimera (v. 1.11)²⁵, whereas electron density map overlays were generated using Mac PyMOL (v. 1.7.4.5). Topology images were generated in part by the Pro-Origami server.²⁶ The atomic coordinates for native KpFeoA have been deposited in the Protein Data Bank (deposition ID: 6E55).

Docking and Bioinformatics.

Docking models were created using the ClusPro online server^27–28 with an input of either the KpFeoA crystal structure (chain A; PDB ID 6E55) or the KpFeoA NMR structure (chain A; PDB ID 2GCX) and the 10 amino acid peptide corresponding to LGCPVIPLVS excised from the crystal structure of KpNFeoB bound to GMP-PNP (PDB ID 2WIC)²⁹. Docking was performed without modification of the default restraints. The models shown represent those with the lowest balanced, weighted score, but these models are generally representative of observed behavior among all output models.

For bioinformatics analyses, all FeoA sequences were obtained from the Universal Protein Resource (UniProt) Knowledgebase and Reference Clusters (http://www.uniprot.org) or the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Multiple sequence alignments were performed using JalView v. 2.7³⁰ implementing the ClustalW algorithm³¹ and the Blosum62 matrix³².

RESULTS

In order to investigate the function of KpFeoA, we cloned, expressed, and purified KpFeoA to good yield and high purity (Fig. S1). Circular dichroism studies indicated our overexpressed, purified protein was folded (data not shown), and gel filtration studies demonstrated the presence of two oligomeric species whose molecular weights were consistent with monomeric (~10 kDa) and dimeric (~20 kDa) (His)₆-tagged KpFeoA (Fig. S2). To our knowledge, no other reports indicating the presence of both monomeric and dimeric FeoA in solution exist. We observed no trimeric or higher-order oligomerization of KpFeoA under these conditions. Moreover, our oligomeric states appeared to be static under our gel filtration conditions, as dilution of dimeric KpFeoA and reinjection preserved oligomeric homogeneity (Fig. S2). To characterize these states further, we attempted to crystalize both oligomeric species independently; however, we were only successful in generating diffraction-quality crystals with monomeric KpFeoA as our protein input into the crystallization drop.

Crystals of unmodified, tagged KpFeoA diffracted to <2 Å, and our best native dataset was processed to a resolution of 1.57 Å (Table S1). Despite exhaustive efforts to utilize molecular replacement (MR) to determine phases, including the use of an unpublished NMR structure of KpFeoA, we were unable to find a suitable MR solution, suggesting our structure adopted a conformation distinct from previously determined structures. Subsequently, we expressed, purified, and crystallized SeMet-derived KpFeoA, and were able to establish phases utilizing single-wavelength anomalous dispersion (SAD). This model then was used to establish phases definitively for our native data via MR. Our final refined model converged to R_work/R_free of 0.174/0.193 (Table S1), and all residues comprising the full length of the KpFeoA polypeptide (1–75) and part of the tag cleavage site (76–80) were unambiguously present in the electron density (Fig. S3).

Initial inspection of a single polypeptide comprising the X-ray crystal structure of KpFeoA reveals the expected SH3-like fold present in other FeoA structures (Fig. 2A).^{13, 15} In particular, we observe five β strands (β1–β5) that comprise the β barrel, complemented by two additional α helices (α1,α3) and a helical turn (α2) that appear to be unique to FeoAs (Fig. 2B).^{13, 15} A fourth, short helix (α4) appears at the C-terminal tail in the visible electron density, but this helix is composed of residues that are part of the tag cleavage site and are not part of the native sequence. Importantly, we observe no electron density for the presence of the intact His tag, and α4 (part of the TEV site) does not contribute to protein-protein interactions (vide infra). However, there are major differences in our structure that have never been observed in other FeoA structures.

Figure 2.

The crystal structure of a single KpFeoA polypeptide (PDB ID 6E55). A. The ribbon structure of KpFeoA reveals a small β barrel comprising an SH3-like fold. The right panel represents a 90° rotation of the left panel. B. Secondary-structure topology diagram of a single KpFeoA polypeptide. α helices and β sheets are numbered sequentially. “N” and “C” represent the location of the N- and C-termini, respectively.

Intriguingly, analysis of the KpFeoA asymmetric unit (ASU) composed of 6 polypeptides reveals relevant hydrophobic interactions among neighboring KpFeoA molecules (Fig. 3A). Within the ASU there are 2 KpFeoA dimers and 2 “independent” KpFeoA polypeptides. Comprising the directly-interacting dimers, two KpFeoA chains participate in hydrophobic intercalations into a Leu-rich region present on their dimeric partner KpFeoA chains, allowing for the exclusion of a modest amount (~29 Ų) of hydrophobic surface area (Fig. 3B,C). This dimerization is distinct from the Zn²⁺-cross-linked dimer seen in S. maltophilia FeoA¹⁵, which is facilitated by ionic interactions. The Leu-rich ridge on a single KpFeoA polypeptide is composed of four residues along a surface ridge that forms a “C-shaped” clamp (Fig. 3D): Leu²⁶ and Leu²⁹ (present along α1) and Leu⁵⁸ and Leu⁶⁰ (present along β4). Intercalated into this hydrophobic patch are two residues of the neighboring KpFeoA chain along β5: Ala⁷² and Ala⁷⁴ (Fig. 3D). This intercalation appears to displace Leu²⁹ from the central portion of this hydrophobic ridge towards the Ala residues along β5 (Fig 3D). There is a slight asymmetry in this dimerization that we observe in the crystal, as superposition of the two dimeric pairs show Cα rmsd of ~0.47 Å over 160 residues. In one pair of dimers, the protein-protein interactions appear to be tighter, as α1 appears to be placed ~2 Å in closer proximity to its interacting partner’s β5 (Fig. S4), leading to a slight difference in the dimeric interfaces with respect to one another. To our knowledge, these dimers represent the first FeoA-FeoA interactions directly observed at the atomic level.

Figure 3.

Features of KpFeoA interactions. A. Ribbon representation of the asymmetric unit (ASU) of the crystal structure of KpFeoA, which contains 6 molecules. B. Space-filling model of two adjacent, interacting KpFeoA polypeptides. The individual polypeptides are colored in red and blue. C. The same two interacting KpFeoA polypeptides in B with one polypeptide displayed as space-filling and the other displayed as ribbon, emphasizing the intercalation of one molecule into the other. D. Ribbon representation of the KpFeoA-KpFeoA interaction, with key hydrophobic residues represented as gray balls and sticks. Inset: two Ala residues along β5 (Ala⁷² and Ala⁷⁴) intercalate into hydrophobic surface ridge comprising 4 Leu residues: Leu²⁶ and Leu²⁹ (along α1) and Leu⁵⁸ and Leu⁶⁰ (along β4). Leu²⁹ becomes displaced from hydrophobic ridge as it interacts with the Ala residues along β5 of the neighboring polypeptide.

In addition to these dimeric partners, we observe two additional polypeptides in the ASU that ostensibly appeared to lack interacting partners (Fig. 3A; Fig. S5A). However, when one of these independent chains is superposed onto one of the dimeric KpFeoA chains, there is good agreement in the two conformations (Cα rmsd of ~0.39 Å over 80 residues; Fig. S5B) including the α1 displacement, which is driven by hydrophobic interactions with the dimeric partners. This similarity can be rationalized when considering the neighboring ASU, in which we see interactions among ASUs (Fig. S6). In fact, this interaction is repeated throughout the crystal (Fig. S7), and thus the two “orphaned” KpFeoA polypeptides actually interact with the β5 of the adjacent ASU through their “C-shaped” clamps comprising α1 and β4, thus repeating direct interactions throughout the entire crystalline lattice and recapitulating the observed oligomerization.

This direct interaction results in a significant “closing” or “clamping” of the KpFeoA SH3-like fold onto its neighboring polypeptide. We compared our crystal structure to the unpublished NMR structure of KpFeoA (PDB ID 2GCX). Superposition of our structure onto 2GCX chain A shows conservation of the global fold, but there are dramatic structural changes resulting in a rmsd of ~2.3 Å over 74 Cα atoms (Fig. 4A). This structural deviation likely explains the failure of the NMR model for MR. The most striking structural differences observed are the closing of the space between the β3-β4 turn and that of the C-terminal side of α1 (Fig. 4A). The two residues anchoring the ends of this region are Leu²⁹ and Arg⁵⁵. In the NMR structure, this area is quite open: the distance of Cα Leu²⁹ to Cα Arg⁵⁵ is ~10.5 Å. In stark contrast, our crystal structure reveals that this region has closed along β5 of the neighboring molecule: the distance of Cα Leu²⁹ to Cα Arg⁵⁵ has decreased to ~6.6 Å, representing a nearly 4 Å narrowing. Previous analyses of the NMR structure of EcFeoA have indicated dynamicism to be present with this same region and, in particular, along β4,¹³ suggesting this region may be flexible. Based on these prior observations and our current data, we propose that 2GCX represents the “open” form of KpFeoA, and that every polypeptide in our structure represents the “closed” form of KpFeoA bound to a protein partner, which is a significant revelation.

Figure 4.

Structural analysis of KpFeoA. A. Superposition of our crystal structure of KpFeoA (red) with the NMR structure of KpFeoA (goldenrod; PDB ID 2GCX). While the global fold is conserved, there is a significant closure (~4 Å) of the C-terminal side of α1 and the β3-β4 turn in our structure compared to the NMR structure. B. Docking studies of the 10 amino acid sequence (cornflower) postulated to be the FeoA recognition site along NFeoB. In the “open” NMR structure (goldenrod), the 10-mer peptide docks precisely in the same location we observe interactions between KpFeoA dimers in our crystal structure. In the “closed” crystal structure (red), the 10-mer peptide fails to dock in the “C-shaped” clamp due to the closing of this binding region. C. We hypothesize that the open form of KpFeoA (goldenrod) binds to the PxxP recognition site on NFeoB (orange; Leu and Val residues shown as ball and stick of KpNFeoB bound to GMP-PNP, PDB ID: 2WIC). We postulate this binding can be directly communicated to the GTP-binding site through the short helical loop containing hydrogen bonds to the guanine nucleobase. The right panel of C represents a 30° rotation of the left panel of C.

We believe our structure points to the location along FeoA that may mediate interactions with its corresponding binding site along NFeoB. As an initial test of our “open-to-closed” hypothesis, we performed in silico docking experiments^27–28 of both KpFeoA models with a hydrophobic 10 AA sequence (LGCPVIPLVS) representing the postulated partner binding site present on NFeoB. We excised the structure of this 10-mer directly from the crystal structure of KpNFeoB bound to GMP-PNP (PDB ID 2WIC).²⁹ Wholly consistent with our hypothesis, the lowest-energy docking model of this peptide with the NMR structure of KpFeoA predicts binding directly within the “C-shaped” clamp of the “open” conformer (Fig. 4B). In contrast and as predicted, the lowest-energy docking model of this peptide with our crystal structure of KpFeoA fails to dock into the now “closed” binding site (Fig. 4B).

DISCUSSION

In light of our new structure, by comparison to the monomeric NMR structure, and based on our docking models, we propose a mechanism of FeoA-NFeoB interactions that may link to the status, or alter the state, of bound nucleotide (Fig. 4C). We posit that the “open” conformer of KpFeoA uses its “C-shaped” clamp region (defined by α1 and β4) to interact with the PxxP recognition site on NFeoB. This location is rich in hydrophobic residues (Val and Leu) that would likely intercalate into the Leu-rich ridge along KpFeoA (Fig. 4C), similar to what we observe in our crystal structure (Fig. 3D). Moreover, hydrophobic residues are strongly conserved within this hydrophobic ridge despite low overall conservation of FeoA sequence (Fig. S8), emphasizing the functional importance of hydrophobicity in this vicinity. We envision a scenario in which the FeoA-NFeoB binding event is communicated to, or even linked to the state of, GTP/GDP bound on the surface of NFeoB. In the both the GMP-PNP- and GDP-bound KpNFeoB structures, the guanine nucleobase is connected via hydrogen bonding directly to the PxxP binding site by a short helical turn.²⁹ Thus, FeoA binding at this site could either increase the rate of GTP hydrolysis, facilitate nucleotide release, or both. Research to probe this mechanism is currently underway.

Our discovery of a site for FeoA-mediated protein-protein interactions provides insight into the function of prokaryotic SH3-like domains and opens up several exciting avenues for future research on the Feo system. Numerous studies have demonstrated that FeoA is a virtually indispensable component of the bacterial Feo system,^33–36 likely due to FeoA’s regulatory role in modulating ferrous iron import through its interaction with FeoB. Furthermore, the disruption of protein-protein interactions mediated by complex surface recognition sites along SH3-like domains like FeoA is an active area of pharmacological development in eukaryotes.^{20, 37} This targeted approach could extend to antibiotic development aimed at nutrient uptake pathways such as Feo. We imagine a future scenario in which small, hydrophobic molecules could be developed to disrupt FeoA-FeoB interactions as a novel means of attenuating bacterial virulence through the limitation of iron uptake.

CONCLUSION

We have determined the crystal structure of K. pneumoniae FeoA, which reveals an SH3-like domain that mediates interactions between neighboring polypeptides via hydrophobic intercalations into a Leu-rich surface ridge. By comparison to a previously unpublished, monomeric NMR structure, we demonstrate the KpFeoA can assume both “open” and “closed” conformations, controlled by binding at this Leu-rich ridge. We have used peptide docking to construct a model of interaction between FeoA and FeoB, which we are actively testing. We propose that these FeoA-FeoB protein-protein interactions are likely utilized to control ferrous iron uptake, a key nutrient acquisition pathway used by pathogenic bacteria to establish infection in acidic and/or anaerobic niches within human hosts.

ABREVIATIONS

ASU

asymmetric unit

Feo

ferrous iron transport system

GDP

guanosine-5’-diphosphate

GMP-PNP

5’-guanylyl imidodiphosphate

GTP

guanosine-5’-triphosphate

MR

molecular replacement

NMR

nuclear magnetic resonance

rmsd

root-mean-square deviation

SAD

single-wavelength anomalous dispersion

SH3

Src-homology 3-like fold