tRNA Dissociation from EF-Tu after GTP Hydrolysis: Primary Steps and Antibiotic Inhibition

Abstract

In each round of ribosomal translation, the translational GTPase elongation factor Tu (EF-Tu) delivers a transfer RNA (tRNA) to the ribosome. After successful decoding, EF-Tu hydrolyzes GTP, which triggers a conformational change that ultimately results in the release of the tRNA from EF-Tu. To identify the primary steps of these conformational changes and how they are prevented by the antibiotic kirromycin, we employed all-atom explicit-solvent molecular dynamics simulations of the full ribosome-EF-Tu complex. Our results suggest that after GTP hydrolysis and P i release, the loss of interactions between the nucleotide and the switch 1 loop of EF-Tu allows domain D1 of EF-Tu to rotate relative to domains D2 and D3 and leads to an increased flexibility of the switch 1 loop. This rotation induces a closing of the D1-D3 interface and an opening of the D1-D2 interface. We propose that the opening of the D1-D2 interface, which binds the CCA tail of the tRNA, weakens the crucial EF-Tu-tRNA interactions, which lowers tRNA binding affinity, representing the first step of tRNA release. Kirromycin binds within the D1-D3 interface, sterically blocking its closure, but does not prevent hydrolysis. The resulting increased flexibility of switch 1 explains why it is not resolved in kirromycin-bound structures.

Effects of cryo-EM cooling on structural ensembles

“…During the simulation, the deviation from the starting structure measured by the root mean square deviation (rmsd) approaches 5 Å (Fig. S1a), which is similar to the rmsd obtained from 2-µs simulations of the same system at 300 K presented earlier 69 . The observation that, at 300 K, similar rmsd values are reached in shorter time suggests that the dynamics on the timescales of the simulations is markedly slowed down by the lower temperature, as expected.…”

“…To generate an ensemble of structures of the ribosome·EF-Tu·kirromycin complex at the temperature before cooling (277.15 K), we used all-atom explicit-solvent molecular dynamics (MD) simulations starting from a high-resolution cryo-EM structure 30 . We used the same system setup and pre-equilibration protocol as described earlier for MD simulations of the same system 69 , except that the temperature was set to 277.15 K and that the production simulation was extended to 3.5 μ s. Briefly, the simulations were carried out using GROMACS 5.1 81 , with the amber99sb force field 82 , the SPC/E water model 83 , and K + Cl − ion parameters by Joung et al 84 . Bond lengths were constrained using the LINCS algorithm 85 .…”

“…Virtual site constraints for hydrogens 86 allowed a 4-fs integration step. For more details, refer to Warias et al 69 .…”

Folding of VemP into translation-arresting secondary structure is driven by the ribosome exit tunnel

“…For simulations of the RNC, we used the Amber ff12SB force field ( 38 ) for the solute with the parameter set of non-canonical nucleotides based on ff99SB ( 39 ), the SPC/E water model ( 40 ), and Joung and Cheatham ion parameters ( 41 ). This setup has proven reliable in simulation studies of the ribosome ( 16 , 42 ). For VemP-1 in solution, we used two force-fields of different families.…”

Folding of VemP into translation-arresting secondary structure is driven by the ribosome exit tunnel

“…31 This setup has proven reliable in several simulation studies including those of the ribosome. 15,32 For VemP constructs in solution, we used two force-fields of different families. First, the identical force field as for the RNC complexes, namely ff12SB and SPC/E.…”