GTP Conformation Research Articles

Pulvomycin-resistant mutants of E.coli elongation factor Tu.

This paper reports the generation of Escherichia coli mutants resistant to pulvomycin. Together with targeted mutagenesis of the tufA gene, conditions were found to overcome membrane impermeability, thereby allowing the selection of three mutants harbouring elongation factor (EF)-Tu Arg230-->Cys, Arg333-->Cys or Thr334-->Ala which confer pulvomycin resistance. These mutations are clustered in the three-domain junction interface of the crystal structure of the GTP form of Thermus thermophilus EF-Tu. This result shares similarities with kirromycin resistance; kirromycin-resistant mutations cluster in the domain 1-3 interface. Since both interface regions are involved in the EF-Tu switch mechanism, we propose that pulvomycin and kirromycin both act by specifically disturbing the allosteric changes required for the switch from EF-Tu-GTP to EF-Tu-GDP. The three-domain junction changes dramatically in the switch to EF-Tu.GDP; in EF-Tu.GDP this region forms an open hole. Structural analysis of the mutation positions in EF-Tu.GTP indicated that the two most highly resistant mutants, R230C and R333C, are part of an electrostatic network involving numerous residues. All three mutations appear to destabilize the EF-Tu.GTP conformation. Genetic and protein characterizations show that sensitivity to pulvomycin is dominant over resistance. This appears to contradict the currently accepted model of protein synthesis inhibition by pulvomycin.

Kinetic and structural analysis of the Mg(2+)-binding site of the guanine nucleotide-binding protein p21H-ras.

The coordination and binding of the Mg2+ ion in the nucleotide-binding site of p21 have been investigated using site-directed mutagenesis, kinetic methods, and phosphorous NMR. Mg2+ in the p21.nucleotide.Mg2+ complex appears to be in fast equilibrium with the solvent. The dissociation constant between Mg2+ and the p21.GDP complex was determined to be 2.8 microM. It decreases 30- or 16-fold on substituting Ser-17 or Asp-57 with alanine, respectively, whereas the T35A mutation has no effect. All three mutations influence the dissociation constants and the association and dissociation rate constants of the interaction between guanine nucleotides and p21, but to a different degree. We conclude that Thr-35 is only complexed to Mg2+ in the GTP conformation and both Asp-57 and Ser-17 appear to be critical for both GDP and GTP binding. 31P NMR spectra of the GDP and Gpp(NH)p (guanosine-5'-(beta,gamma-imido)triphosphate) complexes of mutated p21 show a remarkable perturbation of the guanine nucleotide-binding site compared to wild-type protein. The mutant proteins show reduced GTPase rates, which are not stimulated by the GTPase-activating protein GAP. p21(S17A) has been reported to function just as p21(S17N) as a dominant negative inhibitor of normal p21. We find that it inhibits oncogenic p21-induced survival of primary neurons.