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The acquisition of ferrous iron in prokaryotes is achieved by the G-protein-coupled membrane protein FeoB. This protein possesses a large C-terminal membrane-spanning domain preceded by two soluble cytoplasmic domains that are together termed `NFeoB'. The first of these soluble domains is a GTPase domain (G-domain), which is then followed by an entirely α-helical domain. GTP hydrolysis by the G-domain is essential for iron uptake by FeoB, and various NFeoB mutant proteins from Streptococcus thermophilus have been con­structed. These mutations investigate the role of conserved amino acids from the protein's critical Switch regions. Five crystal structures of these mutant proteins have been determined. The structures of E66A and E67A mutant proteins were solved in complex with nonhydrolyzable GTP analogues, the structures of T35A and E67A mutant proteins were solved in complex with GDP and finally the structure of the T35S mutant was crystallized without bound nucleotide. As an ensemble, the structures illustrate how small nucleotide-dependent rearrangements at the active site are converted into large rigid-body reorientations of the helical domain in response to GTP binding and hydrolysis. This provides the first evidence of nucleotide-dependent helical domain movement in NFeoB proteins, suggesting a mechanism by which the G-protein domain could structurally communicate with the membrane domain and mediate iron uptake.

Supporting information

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Portable Document Format (PDF) file https://doi.org/10.1107/S0907444911039461/hv5199sup1.pdf
Supplementary material

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Quicktime video file https://doi.org/10.1107/S0907444911039461/hv5199sup2.mov
Supplementary Movie 1. Structural transitions throughout the GTPase cycle of NFeoB<inf><it>St</it></inf>. The movie was created using the following NFeoB<inf><it>St</it></inf> structures: apo-T35S (PDB entry 3b1z), mGMPPNP–WT (PDB code 3lx5), GDP–AlF<inf>4</inf><sup>−</sup>–WT (PDB entry 3sS8) and GDP–WT (PDB entry 3lx8). Throughout the movie, frames representing experimentally determined protein coordinates are labelled. The G-domain is coloured light blue with the Switch regions in yellow and the helical domain is coloured dark pink. Residues 118–123 are disordered in the structure of apo-T35S and are represented by a dashed line in the first scene.

PDB references: NFeoB, T35A mutant, GDPN complex, 3b1y; T35S mutant, apo, 3b1z; E67A mutant, mGMPPNP complex, 3b1v; E67A mutant, GDP complex, 3b1w; E66A mutant, GMPPNP complex, 3b1x


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