Probing the dynamic stalk region of the ribosome using solution NMR

www.nature.com/scientificreports OPEN Received: 9 April 2019 Accepted: 31 July 2019 Published: xx xx xxxx Probing the dynamic stalk region of the ribosome using solution NMR Xiaolin Wang1, John P. Kirkpatrick 1, Hélène M. M. Launay1, Alfonso de Simone2, Daniel Häussinger 3, Christopher M. Dobson 4, Michele Vendruscolo 4, Lisa D. Cabrita1, Christopher A. Waudby 1 & John Christodoulou1 We describe an NMR approach based on the measurement of residual dipolar couplings (RDCs) to probe the structural and motional properties of the dynamic regions of the ribosome. Alignment of intact 70S ribosomes in filamentous bacteriophage enabled measurement of RDCs in the mobile C-terminal domain (CTD) of the stalk protein bL12. A structural refinement of this domain using the observed RDCs did not show large changes relative to the isolated protein in the absence of the ribosome, and we also found that alignment of the CTD was almost independent of the presence of the core ribosome particle, indicating that the inter-domain linker has significant flexibility. The nature of this linker was subsequently probed in more detail using a paramagnetic alignment strategy, which revealed partial propagation of alignment between neighbouring domains, providing direct experimental validation of a structural ensemble previously derived from SAXS and NMR relaxation measurements. Our results demonstrate the prospect of better characterising dynamical and functional regions of more challenging macromolecular machines and systems, for example ribosome–nascent chain complexes. In recent years, X-ray crystallography and cryo-electron microscopy (cryo-EM) have elucidated the details of high-resolution structures of ribosomes, revealing intricate mechanistic information about their function during the translation process1,2. In parallel, NMR-based observations of nascent polypeptide chains emerging from the ribosome are providing unique structural and mechanistic insights into co-translational folding processes3–5. In order to develop further solution-state NMR spectroscopy as a technique for structural studies of dynamic regions within large complexes, we have explored the measurement of residual dipolar couplings (RDCs) within intact ribosomes, focusing in particular on the mobile bL12 protein from the GTPase-associated region (GAR) of the prokaryotic 70S ribosome. RDCs have been used to characterise other macromolecular machines and assemblies, including HIV-1 capsid protein, bacterial Enzyme I, and the 20S proteasome6–8. These developments are particularly relevant as macromolecular complexes tend to exhibit a wide variety of functional motions that are challenging to characterise by methods such as X-ray crystallography or cryoelectron microscopy. The GAR is a highly conserved region of both prokaryotic and eukaryotic ribosomes, and is so named to reflect its role in both the recruitment and the stimulation of the GTPase activity of several auxiliary factors associated with the key steps of protein synthesis: initiation (initiation factor 2, IF2), elongation (elongation factors EF-Tu and EF-G) and termination (release factor 3, RF3)9. The prokaryotic GAR includes helices 42–44 and 95 of the 23S rRNA, and the ribosomal proteins bL10, bL11 and bL12 (Fig. 1a). bL12, the focus of the present work, is a 120 residue dimeric protein consisting of an N-terminal dimerisation domain (NTD), which binds to the extended bL10 helix of the core ribosome particle, and a C-terminal domain (CTD), which interacts with GTPase substrates to facilitate their recruitment to the ribosome. The bL12 CTD interacts with four major translational GTPases, IF2, EF-Tu, EF-G and RF3, through a highly conserved and positively charged binding site in helices 4 and 5, identified through NMR mapping and mutagenesis10–14. GTPase binding has been found by mutagenesis to occur through the G4–G5 helix in the G domain of IF2 (and, by homology, likely also via the G domain in other GTPases), which presents a complementary negatively charged surface and results in extremely rapid binding driven by favourable electrostatics14. The CTD is mobile in solution, being separated from the NTD by a flexible 1 Institute of Structural and Molecular Biology, UCL and Birkbeck College London, Gower Street, London, WC1E 6BT, UK. 2Department of Life Sciences, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK. 3Department of Chemistry, University of Basel, St. Johannsring 19, 4056, Basel, Switzerland. 4Centre for Misfolding Disease, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK. Correspondence and requests for materials should be addressed to C.A.W. (email: ) or J.C. (email: ) Scientific Reports | (2019) 9:13528 | https://doi.org/10.1038/s41598-019-49190-1 1 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 1. RDC measurement and analysis of ribosome-bound and free bL12 aligned in Pf1 phage. (a) Schematic illustration of the 50S subunit of the bacterial ribosome illustrating the position and composition of the stalk region. (b) Excerpt from 1H, 15N HSQC (red) and TROSY (blue) spectra of the ribosome-bound bL12 CTD in isotropic and aligned conditions (light and dark colouring, respectively), acquired at 700 MHz and 298 K. (c) Correlation plot of measured amide RDCs between free and ribosome-bound bL12. The error bars represent the uncertainties in the measured RDCs, as estimated from the signal-to-noise ratios and linewidths of the spectral peaks. (d) Amide RDCs measured for ribosome-bound and isolated bL12. (e) Refinement of the bL12 template structure (PDB code:1rqu) using observed RDCs for isolated and ribosome-bound bL12. (f) Sanson-Flamsteed projection of the principal axes of the alignment tensor calculated for free and ribosomebound bL12. hinge region, and is absent in structures of ribosomes determined by X-ray crystallography and cryoelectron microscopy. bL12 is unique among ribosomal proteins in being present in multiple copies: typically four or six monomers are present, although some cyanobacteria may contain eight15. In the particular case of the E. coli ribosome studied here, four copies are present16. However, while bL12 is essential for protein synthesis17, not all copies are needed. While variant ribosomes containing only one bL12 dimer show less than 50% of the protein synthesis activity of wild-type ribosomes18, engineered variants in which only one CTD is present per bL12 dimer have approximately 80% activity related to wild-type ribosomes19. These observations suggest therefore that only one CTD may be functioning within each dimer at any one time. Aside from the CTD, the flexible hinge region of bL12 is also essential for ribosome activity20. However, this flexibility, which has been proposed to facilitate the recruitment of translation factors from the cellular space to the 30S/50S interface9,21,22, has precluded the observation and structure (...truncated)