Structure and dynamics in solution of the stop codon decoding N‐terminal domain of the human polypeptide chain release factor eRF1

Abstract

The high-resolution NMR structure of the N-domain of human eRF1, responsible for stop codon recognition, has been determined in solution. The overall fold of the protein is the same as that found in the crystal structure. However, the structures of several loops, including those participating in stop codon decoding, are different. Analysis of the NMR relaxation data reveals that most of the regions with the highest structural discrepancy between the solution and solid states undergo internal motions on the ps-ns and ms time scales. The NMR data show that the Ndomain of human eRF1 exists in two conformational states. The distribution of the residues having the largest chemical shift differences between the two forms indicates that helices a2 and a3, with Abbreviations: CBCA(CO)NH, 3D experiment correlating the amide NH with the Ca and Cb signals of the preceding amino acid; CeRF1 or C-domain, C-terminal domain (or domain 3) of class-1 polypeptide chain release factor; eRF1, eukaryotic class-1 polypeptide chain release factor; eRF3, eukaryotic class-2 polypeptide chain release factor 3; HNCACB, 3D experiment correlating the amide NH with the Ca and Cb signals; HNCO, 3D experiment correlating the amide NH with the C 0 signal of the preceding amino acid; HSQC, heteronuclear single quantum coherence spectroscopy; M-eRF1 or M-domain, eRF1 middle domain (or domain 2); N-eRF1 or N-domain, N-terminal domain (or domain 1) of class-1 polypeptide chain release factor; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser enhancement spectroscopy; RDC, residual dipolar coupling; R 1 , longitudinal or spin-lattice relaxation rate; R 2 , transverse or spin-spin relaxation rate; R ex , conformational exchange contribution to R 2 ; RF, release factor; RMSD, root-meansquare deviation; S 2 , order parameter reflecting the amplitude of ps-ns bond vector dynamics.

Optimal Translational Termination Requires C4 Lysyl Hydroxylation of eRF1

“…Hydroxylation can modulate protein function via intra- or intermolecular hydrogen-bonds or electronic effects ( Coleman et al., 2007; Hon et al., 2002; Loenarz and Schofield, 2011 ). Recent studies suggest that the loop containing the NIKS motif and the helical extension surrounding it have some inherent plasticity ( Polshakov et al., 2012 ). An interaction between hydroxylated-K63 and a neighboring eRF1 residue could modulate this flexibility and optimize the conformation of this domain during stop codon recognition and/or subsequent steps of termination.…”