The Role of FhuD2 in Iron(III)-Hydroxamate Transport in Staphylococcus aureus

Abstract

The fhuD2 gene encodes a lipoprotein that has previously been shown to be important for the utilization of iron(III)-hydroxamates by Staphylococcus aureus. We have studied the function of the FhuD2 protein in greater detail, and demonstrate here that the protein binds several iron(III)-hydroxamates. Mutagenesis of FhuD2 identified several residues that were important for the ability of the protein to function in iron(III)-hydroxamate transport. Several residues, notably Tyr-191, Trp-197, and Glu-202, were found to be critical for ligand binding. Moreover, mutation of two highly conserved glutamate residues, Glu-97 and Glu-231, had no affect on ligand binding, but did impair iron(III)-hydroxamate transport. Interestingly, the transport defect was not equivalent for all iron(III)-hydroxamates. We modeled FhuD2 against the high resolution structures of Escherichia coli FhuD and BtuF, two structurally related proteins, and showed that the three proteins share a similar overall structure. FhuD2 Glu-97 and Glu-231 were positioned on the surface of the N and C domains, respectively. Characterization of E97A, E231A, or E97A/ E231A mutants suggests that these residues, along with the ligand itself, play a cumulative role in recognition by the ABC transporter FhuBGC 2 . In addition, small angle x-ray scattering was used to demonstrate that, in solution, FhuD2 does not undergo a detectable change in conformation upon binding iron(III)-hydroxamates. Therefore, the mechanism of binding and transport of ligands for binding proteins within this family is significantly different from that of other well studied binding protein families, such as that represented by maltosebinding protein.

FhuD1, a Ferric Hydroxamate-binding Lipoprotein in Staphylococcus aureus

“…The cell wall was digested using lysostaphin (50 g), followed by sonication to lyse cells. Triton X-114 phase partitioning was performed as previously described (14). Before electrophoresis on SDS-polyacrylamide gels, the detergent pellets were diluted 1:1 with water followed by the addition of sample loading buffer.…”

“…Protein Overexpression and Purification-The fhuD1 gene, without the predicted signal sequence codons, was PCR-amplified as a 1.0-kb DNA fragment, digested with BamHI and EcoRI (sites were incorporated into the oligonucleotides) and then cloned into BamHI-and EcoRI-digested pGEX-2T-TEV (14). The resulting plasmid, pMTS78, from which a translational fusion between glutathione S-transferase (GST) and FhuD1 is encoded, was introduced into E. coli strain DH5␣ to generate strain H659.…”

“…The excitation and emission slits were set at 1 and 10 nm, respectively, with excitation and emission wavelengths set at 283 and 348 nm, respectively. Dissociation constants (K D ) were calculated as previously described (14).…”

“…One group of xenosiderophores that S. aureus can utilize is the hydroxamateclass of siderophore, including aerobactin, coprogen, ferrioxamine B (Desferal™), ferrichrome, and rhodotorulic acid (12). In our previous studies, we showed that S. aureus strain RN6390 possessed at least five different iron-regulated genes whose products were involved in the ferric hydroxamate uptake process (12)(13)(14). A three gene operon, fhuCBG, encodes a classical traffic ATPase (for a review of traffic ATPases, see Ref.…”

“…A three gene operon, fhuCBG, encodes a classical traffic ATPase (for a review of traffic ATPases, see Ref. 15) while a fourth gene, fhuD2, codes for a lipoprotein (12) that functions as a high affinity receptor for aerobactin, coprogen, Desferal™, ferrichrome, and rhodotorulic acid (14). The fifth gene involved in this transport system, fhuD1, is predicted to encode a protein with 50% total similarity to FhuD2.…”