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Probing the dynamic stalk region of the ribosome using solution NMR

Abstract : 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 process 1,2. In parallel, NMR-based observations of nascent polypeptide chains emerging from the ribosome are providing unique structural and mechanistic insights into co-translational folding processes 3-5. 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 proteasome 6-8. 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 mutagenesis 10-14. 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 electrostatics 14. The CTD is mobile in solution, being separated from the NTD by a flexible
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Xiaoling Wang, John Kirkpatrick, Helene Launay, Alfonso de Simone, Daniel Häussinger, et al.. Probing the dynamic stalk region of the ribosome using solution NMR. Scientific Reports, Nature Publishing Group, 2019, 9 (1), ⟨10.1038/s41598-019-49190-1⟩. ⟨hal-02466067⟩

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