Structure of the 70S ribosome from human pathogen Staphylococcus aureus

Published online 18 October 2016 Nucleic Acids Research, 2016, Vol. 44, No. 21 10491–10504 doi: 10.1093/nar/gkw933 Structure of the 70S ribosome from human pathogen Staphylococcus aureus Iskander Khusainov1,2,† , Quentin Vicens3,† , Anthony Bochler3 , François Grosse3 , Alexander Myasnikov1 , Jean-François Ménétret1 , Johana Chicher3 , Stefano Marzi3 , Pascale Romby3 , Gulnara Yusupova1 , Marat Yusupov1,* and Yaser Hashem3,* 1 Département de Biologie et de Génomique Structurales, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS UMR7104, INSERM U964, Université de Strasbourg, Illkirch 67400, France, 2 Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan 420008, Russia and 3 Architecture et Réactivité de l’ARN, Université de Strasbourg, Institut de Biologie Moléculaire et Cellulaire du CNRS, Strasbourg 67084, France ABSTRACT Comparative structural studies of ribosomes from various organisms keep offering exciting insights on how species-specific or environment-related structural features of ribosomes may impact translation specificity and its regulation. Although the importance of such features may be less obvious within more closely related organisms, their existence could account for vital yet species-specific mechanisms of translation regulation that would involve stalling, cell survival and antibiotic resistance. Here, we present the first full 70S ribosome structure from Staphylococcus aureus, a Gram-positive pathogenic bacterium, solved by cryo-electron microscopy. Comparative analysis with other known bacterial ribosomes pinpoints several unique features specific to S. aureus around a conserved core, at both the protein and the RNA levels. Our work provides the structural basis for the many studies aiming at understanding translation regulation in S. aureus and for designing drugs against this often multi-resistant pathogen. INTRODUCTION Over the past decade, the stupendous developments in structural biology, including more recently in cryo-electron microscopy (cryo-EM; (1–3)), have extended our structural view of translation in profound ways. Ribosome structures from various species and cellular compartments have been solved, often in complex with multiple partners (see (4–10) for selected recent examples). These provide the basis for understanding how ribosomes vary in composition across species and cellular compartments, but also how they dynamically adjust to growth and stress conditions (11–14). Although we now have structural evidence for a common ribosomal core (15), species-specific features include paralogous ribosomal proteins (r-proteins) (16,17), functionally distinct ribosomal RNAs (rRNA) (18,19), differential rRNA or protein modifications (14) and rRNA expansion segments (20–22). Such variations may be found even within bacteria. As an example of the structural adaptability of ribosomes, diverse bacterial species may carry different versions of the same r-protein, which may have evolved distinct functions as a result. For instance, bL25 contains one (as in Escherichia coli) or several domains (as in Bacillus subtilis), while a gene coding for bL25 is not present in many members of the Bacilli class (23). In E. coli, the N-terminal domain of bL25 binds to 5S rRNA to form part of the central protuberance (24–26). In Thermus thermophilus, the Cterminal domain of bL25 is involved in tRNA proofreading (27). Another characteristic r-protein is bS1, which in E. coli is essential for translation initiation of canonical mRNAs (28,29). bS1 comprises six OB-fold RNA binding domains in E. coli, but only four in S. aureus and other Grampositive bacteria with low-GC content (30). Noteworthy, the N-terminal domain of bS1, which is missing in S. aureus, is responsible for binding to the small subunit (SSU) via bS2 in E. coli (28,31). Finally, a differential number of bL12 proteins (also called bL7 in its acetylated form) is bound to the ribosome according to the length of the 8th alpha helix of uL10. This interaction promotes the recruitment of various translation factors and stimulates GTP hydrolysis (32). Together, these examples illustrate how ribosome composition varies across bacteria, and how this may affect translation. Ribosome composition may also be modulated in response to the environment. For example, the bS1 protein * To whom correspondence should be addressed. Tel: +33 388 41 70 83; Fax: +33 388 60 22 18; Email: Correspondence may also be addressed to Marat Yusupov. Tel: +33 388 65 33 01; Fax: +33 388 65 32 01; Email: † These authors contributed equally to this work as the first authors.  C The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Downloaded from http://nar.oxfordjournals.org/ at University of Pennsylvania on December 13, 2016 Received August 26, 2016; Revised October 02, 2016; Editorial Decision October 06, 2016; Accepted October 06, 2016 10492 Nucleic Acids Research, 2016, Vol. 44, No. 21 Considering how much remains to be discovered about ribosome diversity among bacteria and its impact on ribosome function, we solved at a resolution of 3.8 Å a cryoEM structure of the 70S ribosome from human pathogen S. aureus. Our structure not only confirms the species-specific structural features identified previously in the LSU (51), but it reveals similarly variable features in the SSU, at both the rRNA and protein levels. The comparison of ribosomal structures from four different species of bacteria pinpoints variable regions in bacterial ribosomes, highlighting several species-specific features and suggesting their possible functional roles. Specifically, we discuss the fold of bL31 type B, which is the only bL31 paralog encoded by the S. aureus genome. On the rRNA side, we describe several rRNA insertions and particularities that might be involved in translation modulation. Most importantly, our work offers the fourth structure of a complete ribosome from any bacteria at near-atomic resolution, and the second of a Gram-positive bacterium. As S. aureus is an opportunistic pathogen that may cause significant skin, tissue and systemic infections (54–56), we anticipate that our structure will provide a solid basis for future studies of protein synthesis, and of its regulation for making virulence factors. Our work therefore represents an initial structural framework for developing effective strategies in order to combat infections. MATERIALS AND METHODS Bacterial growth and harvesting In this study, we employed the RN6390 strain of Staphylococcus aureus, which derives from strain NCTC8325. This strain carries a delet (...truncated)