Conformational transitions in human translin enable nucleic acid binding

Abstract

Translin is a highly conserved RNA- and DNA-binding protein that plays essential roles in eukaryotic cells. Human translin functions as an octamer, but in the octameric crystallographic structure, the residues responsible for nucleic acid binding are not accessible. Moreover, electron microscopy data reveal very different octameric configurations. Consequently, the functional assembly and the mechanism of nucleic acid binding by the protein remain unclear. Here, we present an integrative study combining small-angle X-ray scattering (SAXS), site-directed mutagenesis, biochemical analysis and computational techniques to address these questions. Our data indicate a significant conformational heterogeneity for translin in solution, formed by a lesser-populated compact octameric state resembling the previously solved X-ray structure, and a highly populated open octameric state that had not been previously identified. On the other hand, our SAXS data and computational analyses of translin in complex with the RNA oligonucleotide (GU)12 show that the internal cavity found in the octameric assemblies can accommodate different nucleic acid conformations. According to this model, the nucleic acid binding residues become accessible for binding, which facilitates the entrance of the nucleic acids into the cavity. Our data thus provide a structural basis for the functions that translin performs in RNA metabolism and transport.

Figure 1.

Structural models of translin according to SAXS data. (a) Experimental fit of the structural models of full-length translin (green) and best MES dynamic ensemble (red) to the experimental SAXS curve (black circles). The structure of the crystallographic translin yielded a fitting to SAXS data of χ_i = 5.01. The best ensemble of translin conformers was found to be formed by a compact and an ensemble of three open conformations (with individual relative populations of 45.8, 23.3 and 13.3%), with a fitting to SAXS data of χ_i = 1.67. Point-by-point deviations for each model are displayed at the bottom of the plot with the same color code. Although the full-length protein has been used in both fittings, flexible N-terminal histidine-tag and C- terminal regions are not shown here for the sake of clarity. (b) The RNA/DNA binding residues according to OPRA (49) prediction (previously validated in S.Pombe (28)) are shown in red for the two major configurations of translin.

Figure 2.

Glycerol gradient centrifugation of wild-type and mutant translin variants. (a) Sedimentation profiles of recombinant wild-type translin and the mutant translin variants listed in Table 1. Vertical arrows indicate the peaks of the major configuration of each of the translin mutant variants. (b) The two adjacent substitutions I6A and F7A of the mutant Nt-1 break the octamer in tetramers (a tetramer is shown in orange in the compact and open translin models, with the corresponding mutated residues in red CPK). The insert shows a hydrophobic pocket in which I6 and F7 of one monomer (in cyan) interact with the opposing residues of the second monomer (in orange). (c) The E206A substitution of Ct-1 mutant or the R153A substitution of Ct-2 break the octamer into dimers (one dimer is shown in orange in the compact and open translin models, with the corresponding mutated residues E206 in red CPK and R153 in blue CPK). The insert shows an ion bridge (indicated with two straight lines) between E206 in one monomer (in cyan) and R153 of the second monomer (in orange). (d) The Nt+Ct mutations (I6A, F7A and E206A substitutions) break the translin octamer into monomers (a monomer is shown in orange, with the corresponding mutated residues in CPK). (e) The Equatorial mutant (R182G/L183V/R129G/Y85A) has no effect in the octameric structure. Interestingly, according to the crystallographic structure, these mutants in a given monomer (in orange) are interacting with other monomers, while in the open conformation, these mutants are not located at the oligomer interface, which explains the incapability of this mutant to break up the octamer (see Discussion).

Figure 3.

Structural and dynamic models of translin–ssRNA complexes according to SAXS data. (a) Experimental fit of the best single-conformation of the complex (in green) and the dynamic structural model of translin–RNA interaction (in red) to the experimental SAXS curve (black circles). Point-by-point deviations for each model are displayed at the bottom with the same color code. (b) The best equilibrium of conformers was found to be formed by an ensemble of compact and open translin forms bound to structurally different RNA molecules, with a fitting to SAXS data of χ_i = 1.62, notably better than the one found for the best single conformation (χ_i = 2.44). Translin is shown in gray ribbon, while the docked RNA conformations are shown in ribbon and surface, colored according to their population within the global ensemble (red is higher population; blue is lower). Two different views are displayed.