A conserved histidine in switch-II of EF-G moderates release of inorganic phosphate.
Elongation factor G (EF-G), a translational GTPase responsible for tRNA-mRNA translocation possesses a conserved histidine (H91 in Escherichia coli) at the apex of switch-II, which has been implicated in GTPase activation and GTP hydrolysis. While H91A, H91R and H91E mutants showed different degrees of defect in ribosome associated GTP hydrolysis, H91Q behaved like the WT. However, all these mutants, including H91Q, are much more defective in inorganic phosphate (Pi) release, thereby suggesting that H91 facilitates Pi release. In crystal structures of the ribosome bound EF-G•GTP a tight coupling between H91 and the γ-phosphate of GTP can be seen. Following GTP hydrolysis, H91 flips ~140° in the opposite direction, probably with Pi still coupled to it. This, we suggest, promotes Pi to detach from GDP and reach the inter-domain space of EF-G, which constitutes an exit path for the Pi. Molecular dynamics simulations are consistent with this hypothesis and demonstrate a vital role of an Mg(2+) ion in the process.
Department of Cell and Molecular Biology, Uppsala University, Box-596, BMC, 75124, Uppsala, Sweden.
Ravi Kiran
RK
Koripella
Department of Cell and Molecular Biology, Uppsala University, Box-596, BMC, 75124, Uppsala, Sweden.
Mikael
M
Holm
Department of Cell and Molecular Biology, Uppsala University, Box-596, BMC, 75124, Uppsala, Sweden.
Daniel
D
Dourado
Department of Cell and Molecular Biology, Uppsala University, Box-596, BMC, 75124, Uppsala, Sweden.
Chandra Sekhar
CS
Mandava
Department of Cell and Molecular Biology, Uppsala University, Box-596, BMC, 75124, Uppsala, Sweden.
Samuel
S
Flores
Department of Cell and Molecular Biology, Uppsala University, Box-596, BMC, 75124, Uppsala, Sweden.
Suparna
S
Sanyal
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D006160:Guanosine Triphosphate / Q000737:chemistry; D006639:Histidine / Q000737:chemistry*; D006868:Hydrolysis; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D020653:Peptide Elongation Factor G / Q000737:chemistry*; D010710:Phosphates / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid
D020653:Peptide Elongation Factor G; D010710:Phosphates; D006639:Histidine; D006160:Guanosine Triphosphate