Dubiez Etienne, Aleksandrov Alexey, Lazennec-Schurdevin Christine, Mechulam Yves, Schmitt Emmanuelle
Laboratoire de Biochimie, Unité Mixte de Recherche 7654, Ecole Polytechnique, Centre National de la Recherche Scientifique, F-91128 Palaiseau cedex, France.
Laboratoire de Biochimie, Unité Mixte de Recherche 7654, Ecole Polytechnique, Centre National de la Recherche Scientifique, F-91128 Palaiseau cedex, France
Nucleic Acids Res. 2015 Mar 11;43(5):2946-57. doi: 10.1093/nar/gkv053. Epub 2015 Feb 17.
Eukaryotic and archaeal translation initiation processes involve a heterotrimeric GTPase e/aIF2 crucial for accuracy of start codon selection. In eukaryotes, the GTPase activity of eIF2 is assisted by a GTPase-activating protein (GAP), eIF5. In archaea, orthologs of eIF5 are not found and aIF2 GTPase activity is thought to be non-assisted. However, no in vitro GTPase activity of the archaeal factor has been reported to date. Here, we show that aIF2 significantly hydrolyses GTP in vitro. Within aIF2γ, H97, corresponding to the catalytic histidine found in other translational GTPases, and D19, from the GKT loop, both participate in this activity. Several high-resolution crystal structures were determined to get insight into GTP hydrolysis by aIF2γ. In particular, a crystal structure of the H97A mutant was obtained in the presence of non-hydrolyzed GTP. This structure reveals the presence of a second magnesium ion bound to GTP and D19. Quantum chemical/molecular mechanical simulations support the idea that the second magnesium ion may assist GTP hydrolysis by helping to neutralize the developing negative charge in the transition state. These results are discussed in light of the absence of an identified GAP in archaea to assist GTP hydrolysis on aIF2.
真核生物和古细菌的翻译起始过程涉及一种异源三聚体GTP酶e/aIF2，它对起始密码子选择的准确性至关重要。在真核生物中，eIF2的GTP酶活性由一种GTP酶激活蛋白（GAP）eIF5协助。在古细菌中，未发现eIF5的直系同源物，并且认为aIF2的GTP酶活性是无辅助的。然而，迄今为止尚未报道过该古细菌因子的体外GTP酶活性。在此，我们表明aIF2在体外能显著水解GTP。在aIF2γ内，对应于其他翻译GTP酶中发现的催化组氨酸的H97以及来自GKT环的D19都参与了这一活性。我们测定了几个高分辨率晶体结构，以深入了解aIF2γ对GTP的水解作用。特别地，在非水解GTP存在的情况下获得了H97A突变体的晶体结构。该结构揭示了存在与GTP和D19结合的第二个镁离子。量子化学/分子力学模拟支持这样一种观点，即第二个镁离子可能通过帮助中和过渡态中发展的负电荷来协助GTP水解。鉴于古细菌中不存在已鉴定的协助aIF2上GTP水解的GAP，我们对这些结果进行了讨论。
