Conformational Changes of Three Periplasmic Receptors for Bacterial Chemotaxis and Transport: The Maltose-, Glucose/Galactose- and Ribose-binding Proteins.

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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.

Section: Fhud2 Function In S Aureusmentioning

confidence: 99%

Section: Fhud2 Function In S Aureusmentioning

confidence: 99%

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

Sebulsky¹,

Shilton

Speziali³

et al. 2003

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“…This decrease in R g of 0.04 nm is a relatively minor change, and is discernable only because of the very high quality of the SAXS data; nevertheless, it indicates that the protein undergoes a contraction upon binding ferrichrome. For comparison, maltose-binding protein (MBP) undergoes a large conformational change that has been well characterized by x-ray crystallography (28,29), and the R g of MBP shows a decrease of ϳ0.1 nm when it binds maltose in solution (30).…”

Section: Fhud1 In S Aureusmentioning

confidence: 99%

FhuD1, a Ferric Hydroxamate-binding Lipoprotein in Staphylococcus aureus

Sebulsky

Speziali

Shilton

et al. 2004

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Staphylococcus aureus can utilize ferric hydroxamates as a source of iron under iron-restricted growth conditions. Proteins involved in this transport process are: FhuCBG, which encodes a traffic ATPase; FhuD2, a post-translationally modified lipoprotein that acts as a high affinity receptor at the cytoplasmic membrane for the efficient capture of ferric hydroxamates; and FhuD1, a protein with similarity to FhuD2. Gene duplication likely gave rise to fhuD1 and fhuD2. While the genomic locations of fhuCBG and fhuD2 in S. aureus strains are conserved, both the presence and the location of fhuD1 are variable. The apparent redundancy of FhuD1 led us to examine the role of this protein. We demonstrate that FhuD1 is expressed only under conditions of iron limitation through the regulatory activity of Fur. FhuD1 fractions with the cell membrane and binds hydroxamate siderophores but with lower affinity than FhuD2. Using small angle x-ray scattering, the solution structure of FhuD1 resembles that of FhuD2, and only a small conformational change is associated with ferrichrome binding. FhuD1, therefore, appears to be a receptor for ferric hydroxamates, like FhuD2. Our data to date suggest, however, that FhuD1 is redundant to FhuD2 and plays a minor role in hydroxamate transport. However, given the very real possibility that we have not yet identified the proper conditions where FhuD1 does provide an advantage over FhuD2, we anticipate that FhuD1 serves an enhanced role in the transport of untested hydroxamate siderophores and that it may play a prominent role during the growth of S. aureus in its natural environments.

“…The proteins by x-ray scattering in solution (22). Until 1998, all of the known PBP structures could be classified into two groups depending on the topology of the connection between the two lobes (23).…”

mentioning

confidence: 99%

Crystal Structures of the Liganded and Unliganded Nickel-binding Protein NikA from Escherichia coli

Heddle

Scott

Unzai

et al. 2003

114

Bacteria have evolved a number of tightly controlled import and export systems to maintain intracellular levels of the essential but potentially toxic metal nickel. Nickel homeostasis systems include the dedicated nickel uptake system nik found in Escherichia coli, a member of the ABC family of transporters, that involves a periplasmic nickel-binding protein, NikA. This is the initial nickel receptor and mediator of the chemotactic response away from nickel. We have solved the crystal structure of NikA protein in the presence and absence of nickel, showing that it behaves as a "classical" periplasmic binding protein. In contrast to other binding proteins, however, the ligand remains accessible to the solvent and is not completely enclosed. No direct bonds are formed between the metal cation and the protein. The nickel binding site is apolar, quite unlike any previously characterized protein nickel binding site. Despite relatively weak binding, NikA is specific for nickel. Using isothermal titration calorimetry, the dissociation constant for nickel was found to be ϳ10 M and that for cobalt was approximately 20 times higher.