Table 1.
Crystallographic data processing and refinement statistics.
Figure 1.
Overall structure of NFeoBSt and the transitions associated with nucleotide hydrolysis.
Superposition between NFeoBSt structures bound to: a non-hydrolyzable GTP analogue mGMPPNP (red; 3LX5, [8]), GDP⋅AlF4- (orange; this work), and GDP (yellow; 3LX8, [8]). The regions of the protein that undergo the largest structural change in response to nucleotide hydrolysis are shown in the colors listed above. The transition state analogue is illustrated in spheres.
Figure 2.
Omit and anomalous-difference Fourier maps at the nucleotide binding site.
Shown in mesh are an FO-FC omit map about the GDP·AlF4- transition state analogue and the attacking water molecule, and likelihood-based anomalous difference Fourier map illustrating the position of the potassium ion. The omit map is colored green, and the anomalous difference Fourier map is colored orange. Both maps are contoured to 3 σ, and Switch I is colored light blue. Inset: FO-FC omit map around the aluminofluoride, contoured at 3 σ.
Figure 3.
Stereo view of metal coordination spheres at the nucleotide binding site.
The potassium ion is shown as a yellow sphere, the magnesium as a green sphere, and aluminum atom as a grey sphere. The attacking water is labeled ‘AW’. Switch I is colored light blue. The interactions with the potassium ion, primarily electrostatic in nature, are shown as long dashes.
Figure 4.
Primary sequence alignment and positions of conserved active site residues in the transition-state structure.
(A) Stereo view illustrating the orientation of active site residues in chain A when bound to GDP·AlF4−. Switches I and II are colored in light and dark blue, respectively. Switch I residues 24–34 have been omitted for clarity. The structure of MnmE bound to GDP·AlF4− (2GJ8, [9]) is overlayed, and is shown in transparent green cartoon. Its catalytic residue, Glu282, is shown as transparent sticks, and its active site waters as transparent green spheres. (B) Sequence alignment of FeoB from various organisms. Thr35 is highlighted in blue. Conserved residues in the vicinity of the GTP terminal phosphate that were mutated in the current study are highlighted in pink. Asn11 and Gly56 are shown in bold. Numbering is for the sequence from S. thermophilus. Abbreviations are as follows: St, S. thermophilus (Q5M586); Ec, E. coli (P33650); Mj, Methanococcus jannaschii (Q57986); Pf, Pyrococcus furiosus (Q8U2H8); Tm, Thermotoga maritima (Q9WXQ8); Kp, Klebsiella pneumoniae (C4X1R9); Lp, Legionella pneumophilia (Q8GNS3). Sequence alignment was performed using CLUSTALW2 [49].
Figure 5.
GTPase activities of NFeoBSt mutants.
Grey bars indicate the GTPase activity in KCl, and white bars (including inset) are rates in NaCl. Values are the mean from at least three independent measurements with errors bars indicating standard deviations. The wild-type rates are from a previous study [8].
Table 2.
Nucleotide binding and hydrolysis properties of NFeoBSt mutants.