Showcasing an integrated approach to structural biology
For many years, we have been interested in the translation factor IF2 which participates in translation initiation in bacteria. Using an integrated approach to structural determination, my group and collaborators have succeeded in describing with precision this enzyme at structural and functional levels. In particular, we have brought to light a domain of its structure important for the assembly mechanisms of the ribosome subunits, unveiling a very different functioning from its eukaryote counterpart.
Decyphering IF2 structureIF2 is a multidomain protein (see figure below). Combining results from crystallography, small-angle X-ray scattering (SAXS) and cryo electron microscopy, we have now achieved the determination of the entire 3D structure of IF2, both in solution and in its state bound to the ribosome, in which it stabilizes the initiator transfer RNA that carries the first amino acid of the future protein to be synthetized. We combined the structural studies with functional analysis of IF2 by fast kinetics and single molecule fluorescence (in collaboration with the teams of Claudio Gualerzi, Camerino, and Jody Puglisi, Stanford), analyzing dynamics of the complex in presence and absence of the N-terminal domain, and showing its crucial role in the formation of a ribosome ready for beginning protein synthesis. Notably, we observed that its absence blocks the correct assembly of the ribosome and therefore obstruct its activity. For this study we also set up a collaboration with Thomas Steitz’s team from Yale University (USA) which allowed showing that the mechanism of ribosome assembly catalyzed by the N-terminal extremity of IF2 is rather different from the one known for its eukaryote counterpart, eIF5B. These results are providing key information about the molecular mechanism of translation initiation, a decisive step for the regulation of protein synthesis. Altogether, this work culminated in two articles published on September 24th in Proceedings of the National Academy of Sciences.
The multiapproach technique showcased in this study was possible by the access toInstruct France Centre 1 infrastructure which is also available to all Instruct members.
A. Simonetti, S. Marzi, A. G. Myasnikov, A. Fabbretti, G. Yusupova, M. Yusupov, C. O. Gualerzi & B. P. Klaholz. Structure of the 30S translation initiation complex. Nature, 2008, 455, 416-420.
A. Simonetti, S. Marzi, A. Fabbretti, I. Hazemann, L. Jenner, A. Urzhumtsev, C. O. Gualerzi & B. P. Klaholz. Structure of the protein core of translation initiation factor 2 in apo, GTP-bound and GDP-bound forms. Acta Cryst., 2013, D69, 925–933.
Simonetti A, Marzi S, Billas IM, Tsai A, Fabbretti A, Myasnikov AG, Roblin P, Vaiana AC, Hazemann I, Eiler D, Steitz TA, Puglisi JD, Gualerzi CO, Klaholz BP. Involvement of protein IF2 N domain in ribosomal subunit joining revealed from architecture and function of the full-length initiation factor. Proc Natl Acad Sci U S A Sept. 24, 2013; 110:15656-61.Eiler D, Lin J, Simonetti A, Klaholz BP, Steitz TA. Initiation factor 2 crystal structure reveals a different domain organization from eukaryotic initiation factor 5B and mechanism among translational GTPases. Proc Natl Acad Sci U S A Sept. 24, 2013