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.
Highlights
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, elongation and termination[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
Amide residual dipolar couplings (RDCs) (DNH) were determined for ribosome-bound bL12 by measurement of 15N frequency differences between amide resonances in 1H, 15N HSQC and TROSY spectra (Fig. 1b)[32]. We found that this approach provided the greatest precision in values of RDCs when compared with alternative methods such as the in-phase/anti-phase approach
Summary
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]. Further measurements of NMR relaxation and small-angle x-ray scattering (SAXS) were combined with molecular modelling to create an optimized structural ensemble of the bL12 dimer, which indicated that the hinge region was not as flexible as would be expected from a random coil, and that the orientations of the NTD and CTD were instead partially coupled, with an order parameter of S2 = 0.1727. RDCs can be measured under weakly anisotropic solution conditions and report on both the local structure of the protein and the overall orientation of domains with respect to a laboratory frame of reference[28,29] As such, they are useful sources of distance-independent structural information that are highly complementary to chemical shifts, scalar couplings and NOEs. Anisotropy can be induced by a variety of steric and electrostatic methods including liquid crystals, bicelles, bacteriophage and stretched polyacrylamide gels[30]
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