Dynamic, Energetic, and Kinetic Determinants of Ribosomal Translocation: Microsecond All-Atom Simulations of Hybrid Cryoem/X-Ray Structural Substates

Translocation of tRNAs through the ribosome is accompanied by large-scale highly concerted conformational motions. We obtained 23 near-atomic resolution structures of translocation conformational substates by combining cryo-EM densities (Fischer, Nature, 2010) with high-resolution X-ray structures. For 13 structures, we carried out extensive molecular dynamics simulations of the fully solvated 70S ribosome, totaling 1.5μs. The obtained structural ensemble, together with the 23 static structures, offers a most complete all-atom picture of ribosomal translocation dynamics. The simulations captured sufficient conformational dynamics to estimate free energy barriers between states, suggesting a hierarchy of timescales for motions of the L1-stalk, tRNAs, and intersubunit rotations. Interaction energies derived from the simulations allowed us to characterize molecular driving forces; e.g., the L1-stalk actively pulls the tRNA from the P- to the E-site, rather than being pushed or passively co-translocated. Addressing the question of how the affinity between the two ribosomal subunits is fine-tuned despite rotations of more than 20 degrees, intersubunit contacts were found to fall into two classes: i) contacts which persist throughout the different states, independent of intersubunit rotations. ii) contacts that are specific to certain states. Persisting contacts are seen close to the axis of rotation and contribute to the baseline of intersubunit interaction energy. In contrast, contacts of residues situated on the periphery are found to be mostly state-specific. The rupture and formation of state-specific contacts entails low changes of the overall interaction energy, allowing the subunits to remain assembled. Key contacts in the periphery predict and explain the decreased amplitude of the intersubunit rotations seen by cryo-EM in the absence of tRNAs. These results suggest further mutations that should stabilize or destabilize specific intermediate states of ribosomal translocation.