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Abstract
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 Mg2+ ion in the process.
Highlights
Elongation factor G (EF-G) belongs to the subfamily of translational G-proteins in the GTPase superfamily[1]
Our results show that the degree of defect in GTP hydrolysis varies in the H91 mutants with the exception of H91Q, which is not impaired in GTP hydrolysis
Our results show that GTP hydrolysis by EF-G gets strongly stimulated by the ribosome (Table 1)[26], suggesting that the ribosomal components act as the GTPase activating factor for EF-G30,36
Summary
Elongation factor G (EF-G) belongs to the subfamily of translational G-proteins in the GTPase superfamily[1]. The corresponding histidine on EF-Tu is H84; high resolution structures of EF-G and EF-Tu on the ribosome with GTP analogues place H91 and H84 in similar conformations, pointing towards the γ -phosphate of GTP through the hydrophobic gate[4,5,6] The role of this His in GTP hydrolysis by EF-Tu and EF-G has been extensively studied. Compared to the structures with the GTP analogue GDPCP or GDPNP, where the side chain of H91 points towards the γ -phosphate and remains tightly coupled via a salt bridge[4,5], in structures with GDP and fusidic acid, it is rotated by 140° in the opposite direction This striking observation prompted us to think that the ‘flip’ of the H91 following GTP hydrolysis might provide an efficient mechanism for Pi release. F94 is known to be important for conformational changes in EF-G on and off the ribosome[25,26] and mutation of F94 confers high level FA resistance to the bacteria, and leads to loss of fitness[25,27]
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