Molecular Dynamics Simulations of the Periplasmic Ferric-hydroxamate Binding Protein FhuD

Abstract

FhuD is a periplasmic binding protein (PBP) that, under iron-limiting conditions, transports various hydroxamate-type siderophores from the outer membrane receptor (FhuA) to the inner membrane ATP-binding cassette transporter (FhuBC). Unlike many other PBPs, FhuD possesses two independently folded domains that are connected by an alpha-helix rather than two or three central beta-strands. Crystal structures of FhuD with and without bound gallichrome have provided some insight into the mechanism of siderophore binding as well as suggested a potential mechanism for FhuD binding to FhuB. Since the alpha-helix connecting the two domains imposes greater rigidity on the structure relative to the beta-strands in other 'classical' PBPs, these structures reveal no large conformational change upon binding a hydroxamate-type siderophore. Therefore, it is difficult to explain how the inner membrane transporter FhuB can distinguish between ferrichrome-bound and ferrichrome-free FhuD. In the current study, we have employed a 30 ns molecular dynamics simulation of FhuD with its bound siderophore removed to explore the dynamic behavior of FhuD in the substrate-free state. The MD simulation suggests that FhuD is somewhat dynamic with a C-terminal domain closure of 6 degrees upon release of its siderophore. This relatively large motion suggests differences that would allow FhuB to distinguish between ferrichrome-bound and ferrichrome-free FhuD.

Albomycin Uptake via a Ferric Hydroxamate Transport System ofStreptococcus pneumoniaeR6

“…3B) according to the nomenclature introduced by Clancy et al (12), who studied siderophore-dependent iron acquisition of a group B streptococcus. We refrained from using the pia gene nomenclature introduced by Brown et al (9) because the fhu designation specifically refers to ferric hydroxamate uptake, which we introduced for E. coli (26) and has been used since then for gram-negative and gram-positive bacteria (5,8,30,31). spr0937 is an open reading frame on the cDNA strand and is probably not related to ferrichrome transport.…”

Interactions between TonB from Escherichia coli and the Periplasmic Protein FhuD

“…However, the TonB-binding surface on FhuD as identified by phage display both overlaps the siderophore-binding site and extends beyond it; competition would not be expected. Furthermore, there is no evidence to suggest large conformational changes in FhuD upon binding siderophore (19). In the siderophore-bound state, FhuD probably maintains a rigid backbone; this rigidity would not influence binding of TonB.…”

“…If there was a continuous landscape of all four regions, it would require a displacement of loop 3 from FhuD to accommodate TonB binding. Recent molecular dynamics simulations on the FhuD structure (19) indicated that the C-terminal domain of FhuD has more overall mobility than the N-terminal domain. However, within the relatively static N-terminal domain, loop 3 was observed to be the most mobile region.…”

“…The structure of FhuD and the hydrophobicity of the siderophore-binding site suggest that large scale opening and closing of the binding site does not occur upon siderophore binding and release (18). Molecular dynamics simulations also suggest that FhuD is conformationally rigid but that subtle conformational differences in the C-terminal domain between the apo-and holo-forms may be sufficient for discrimination by FhuB (19).…”

Siderophore-Based Iron Acquisition and Pathogen Control

“…Most importantly, while the two domains of these proteins are usually linked by flexible antiparallel ␤-strands, which are also found in ferric ion binding proteins such as FbpA (235), SfuA, and YfuA (301), belonging to the transferrin structural superfamily, the bidomains of FhuD and CeuE are connected via ␣-helical elements, making their backbones more rigid, and abolish broad conformational changes upon ligand binding. However, molecular dynamic simulations suggested a 6°clo-sure of the FhuD C-terminal domain upon ferrichrome release, and this motion might lead to FhuD conformers that could selectively interact with the FhuB transmembrane component of the transporter (177). The ligand binding site is commonly the most variable part in periplasmic binding protein structures and can be used for phylogenetic classification as in the case of periplasmic ferric ion binding proteins that are currently divided into three classes depending on their coordination modes for the Fe(III) ligand (301).…”