A monovalent cation acts as structural and catalytic cofactor in translational GTP

Translational GTPases are universally conserved GTP hydrolyzing enzymes, critical for fidelity and speed of ribosomal protein biosynthesis. Despite their central roles, the mechanisms of GTP‐dependent conformational switching and GTP hydrolysis that govern the function of trGTPases remain poorly understood. Here, we provide biochemical and high‐resolution structural evidence that eIF5B and aEF1A/EF‐Tu bound to GTP or GTPγS coordinate a monovalent cation (M$^+$) in their active site. Our data reveal that M$^+$ ions form constitutive components of the catalytic machinery in trGTPases acting as structural cofactor to stabilize the GTP‐bound “on” state. Additionally, the M$^+$ ion provides a positive charge into the active site analogous to the arginine‐finger in the Ras‐RasGAP system indicating a similar role as catalytic element that stabilizes the transition state of the hydrolysis reaction. In sequence and structure, the coordination shell for the M$^+$ ion is, with exception of eIF2γ, highly conserved among trGTPases from bacteria to human. We therefore propose a universal mechanism of M$^+$‐dependent conformational switching and GTP hydrolysis among trGTPases with important consequences for the interpretation of available biochemical and structural data

Ribosome formation from subunits studied by stopped-flow and Rayleigh light scattering

Light scattering and standard stopped-flow techniques were used to monitor rapid association of ribosomal subunits during initiation of eubacterial protein synthesis. The effects of the initiation factors IF1, IF2, IF3 and buffer conditions on subunit association were studied along with the role of GTP in this process. The part of light scattering theory that is essential for kinetic measurements is high-lighted in the main text and a more general treatment of Rayleigh scattering from macromolecules is given in an appendix

eIF5B employs a novel domain release mechanism to catalyze ribosomal subunit joining

eIF5B is a eukaryal translational GTPase that catalyzes ribosomal subunit joining to form elongation‐competent ribosomes. Despite its central role in protein synthesis, the mechanistic details that govern the function of eIF5B or its archaeal and bacterial (IF2) orthologs remained unclear. Here, we present six high‐resolution crystal structures of eIF5B in its apo, GDP‐ and GTP‐bound form that, together with an analysis of the thermodynamics of nucleotide binding, provide a detailed picture of the entire nucleotide cycle performed by eIF5B. Our data show that GTP binding induces significant conformational changes in the two conserved switch regions of the G domain, resulting in the reorganization of the GTPase center. These rearrangements are accompanied by the rotation of domain II relative to the G domain and release of domain III from its stable contacts with switch 2, causing an increased intrinsic flexibility in the free GTP‐bound eIF5B. Based on these data, we propose a novel domain release mechanism for eIF5B/IF2 activation that explains how eIF5B and IF2 fulfill their catalytic role during ribosomal subunit joining