Flexible Docking of Ribosomal Elongation Factor G

In collaboration with Prof. Joachim Frank (Wadsworth Center/HHMI), we recently developed multi-resolution flexible fitting methods and applied them to characterize the conformational changes in elongation factor G (EF-G) which binds to the E. coli ribosome to facilitate protein synthesis. EF-G acts as a GTP-dependent catalysts of protein synthesis through transient interactions with the ribosome. During the peptide elongation cycle, in which mRNA advances by one codon, EF-G facilitates the translocation of the A- and P- site tRNAs to the P and E sites, respectively. The crystal structures of EF-G in its nucleotide free form and in complex with GDP are very similar. It has been disputed for a long time whether the energy liberated by GTP hydrolysis is used for translocation or for release of the factor. The absence of any discernible nucleotide-dependent conformational change suggests that GTP hydrolysis induces an observed change in the ribosome mainly by modulation of EF-G's binding affinity to the ribosome, similar to other G proteins that perform a nucleotide-dependent activation of target protein binding sites.


Fig. 1 (click to enlarge): EF-G bound to the ribosome, visualized by difference mapping of 3D cryo-EM image reconstructions at 17 A resolution. The Figure was created by Rajendra K. Agrawal in Joachim Frank's lab. The colors code for the individual domains of EF-G (see Fig. 2a below). Individual sites on the 30S and 50S ribosomal subunit are also marked.

Frank's work has recently produced visually most intriguing evidence for induced-fit conformational differences in the form of EM image reconstructions of bound EF-G. These images, which were obtained from difference maps of free ribosomes and of ribosome-(fusidic acid)-EF-G complexes, are clearly incompatible with the shape of the crystal structure (see Fig. 2a). As a step towards a detailed understanding of the interactions of EF-G with the ribosome, and to identify the changes induced by binding, we refined an atomic resolution model of the bound protein against cryo EM data at 17 A resolution on the basis of information from the free structure of EF-G, secondary structure prediction methods, and a comparison with the structure of EF-Tu (Fig. 2).


Fig. 2 (click to enlarge): Rigid-body and flexible docking of EF-G. (a) The completed structure of EF-G (tube representation), fitted into the EM density map with the Situs package. The colors code for the original crystal coordinates (brown) of EF-G in complex with GDP, coordinates of domain III (green) extracted from the nucleotide-free structure, and coordinates added by secondary structure prediction (red). The position of EF-G's structural domains is indicated. The arrow points to the gap between Thr64 in the switch 1 loop (red) and the nearby beta phosphate of GDP. The EM density (blue) in Fig. 2 is contoured at 14% of the maximum value. The asterisk denotes a region of positive density that can be attributed to a conformational change in the ribosome. EF-G is rotated to visualize the GDP nucleotide (purple). (b) The flexibly fitted model of EF-G using five pairs of point landmarks (grey spheres). The arrows point to notable structural differences. (c) The final optimized protein structure (brown tube representation). Also represented by spheres are the positions of ten point landmarks (color and size-coded to distinguish their resolution and weight, see the literature below).

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