Lars V. Bock

The pathway to GTPase activation of elongation factor SelB on the ribosome.

In all domains of life, selenocysteine (Sec) is delivered to the ribosome by selenocysteine-specific tRNA (tRNASec) with the help of a specialized translation factor, SelB in bacteria. Sec-tRNASec recodes a UGA stop codon next to a downstream mRNA stem–loop. Here we present the structures of six intermediates on the pathway of UGA recoding in Escherichia coli by single-particle cryo-electron microscopy. The structures explain the specificity of Sec-tRNASec binding by SelB and show large-scale rearrangements of Sec-tRNASec. Upon initial binding of SelB–Sec-tRNASec to the ribosome and codon reading, the 30S subunit adopts an open conformation with Sec-tRNASec covering the sarcin–ricin loop (SRL) on the 50S subunit. Subsequent codon recognition results in a local closure of the decoding site, which moves Sec-tRNASec away from the SRL and triggers a global closure of the 30S subunit shoulder domain. As a consequence, SelB docks on the SRL, activating the GTPase of SelB. These results reveal how codon recognition triggers GTPase activation in translational GTPases.

A combined cryo-EM and molecular dynamics approach reveals the mechanism of ErmBL-mediated translation arrest

Nascent polypeptides can induce ribosome stalling, regulating downstream genes. Stalling of ErmBL peptide translation in the presence of the macrolide antibiotic erythromycin leads to resistance in Streptococcus sanguis. To reveal this stalling mechanism we obtained 3.6-Å-resolution cryo-EM structures of ErmBL-stalled ribosomes with erythromycin. The nascent peptide adopts an unusual conformation with the C-terminal Asp10 side chain in a previously unseen rotated position. Together with molecular dynamics simulations, the structures indicate that peptide-bond formation is inhibited by displacement of the peptidyl-tRNA A76 ribose from its canonical position, and by non-productive interactions of the A-tRNA Lys11 side chain with the A-site crevice. These two effects combine to perturb peptide-bond formation by increasing the distance between the attacking Lys11 amine and the Asp10 carbonyl carbon. The interplay between drug, peptide and ribosome uncovered here also provides insight into the fundamental mechanism of peptide-bond formation.

Chemomechanical regulation of SNARE proteins studied with molecular dynamics simulations.

SNAP-25B is a neuronal protein required for neurotransmitter (NT) release and is the target of Botulinum Toxins A and E. It has two SNARE domains that form a four-helix bundle when combined with syntaxin 1A and synaptobrevin. Formation of the three-protein complex requires both SNARE domains of SNAP-25B to align parallel, stretching out a central linker. The N-terminal of the linker has four cysteines within eight amino acids. Palmitoylation of these cysteines helps target SNAP-25B to the membrane; however, these cysteines are also an obvious target for oxidation, which has been shown to decrease SNARE complex formation and NT secretion. Because the linker is only slightly longer than the SNARE complex, formation of a disulfide bond between two cysteines might shorten it sufficiently to reduce secretion by limiting complex formation. To test this idea, we have carried out molecular dynamics simulations of the SNARE complex in the oxidized and reduced states. Indeed, marked conformational differences and a reduction of helical content in SNAP-25B upon oxidation are seen. Further differences are found for hydrophobic interactions at three locations, crucial for the helix-helix association. Removal of the linker induced different conformational changes than oxidation. The simulations suggest that oxidation of the cysteines leads to a dysfunctional SNARE complex, thus downregulating NT release during oxidative stress.

Dynamic contact network between ribosomal subunits enables rapid large-scale rotation during spontaneous translocation

During ribosomal translation, the two ribosomal subunits remain associated through intersubunit bridges, despite rapid large-scale intersubunit rotation. The absence of large barriers hindering rotation is a prerequisite for rapid rotation. Here, we investigate how such a flat free-energy landscape is achieved, in particular considering the large shifts the bridges undergo at the periphery. The dynamics and energetics of the intersubunit contact network are studied using molecular dynamics simulations of the prokaryotic ribosome in intermediate states of spontaneous translocation. Based on observed occupancies of intersubunit contacts, residues were grouped into clusters. In addition to the central contact clusters, peripheral clusters were found to maintain strong steady interactions by changing contacts in the course of rotation. The peripheral B1 bridges are stabilized by a changing contact pattern of charged residues that adapts to the rotational state. In contrast, steady strong interactions of the B4 bridge are ensured by the flexible helix H34 following the movement of protein S15. The tRNAs which span the subunits contribute to the intersubunit binding enthalpy to an almost constant degree, despite their different positions in the ribosome. These mechanisms keep the intersubunit interaction strong and steady during rotation, thereby preventing dissociation and enabling rapid rotation.

Structural Basis for Polyproline-Mediated Ribosome Stalling and Rescue by the Translation Elongation Factor EF-P

Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P. The structures suggest that the favored conformation of the polyproline-containing nascent chain is incompatible with the peptide exit tunnel of the ribosome and leads to destabilization of the peptidyl-tRNA. Binding of EF-P stabilizes the P-site tRNA, particularly via interactions between its modification and the CCA end, thereby enforcing an alternative conformation of the polyproline-containing nascent chain, which allows a favorable substrate geometry for peptide bond formation.

Molecular simulations of the ribosome and associated translation factors

The ribosome is a macromolecular complex which is responsible for protein synthesis in all living cells according to their transcribed genetic information. Using X-ray crystallography and, more recently, cryo-electron microscopy (cryo-EM), the structure of the ribosome was resolved at atomic resolution in many functional and conformational states. Molecular dynamics simulations have added information on dynamics and energetics to the available structural information, thereby have bridged the gap to the kinetics obtained from single-molecule and bulk experiments. Here, we review recent computational studies that brought notable insights into ribosomal structure and function.

Dynamics and Energetics of Elongation Factor SelB in the Ternary Complex and the Ribosome

SelB is an elongation factor specialized to deliver the selenocysteine (Sec) tRNA to the ribosome by recoding the UGA stop codon on the mRNA. Initially the tRNA is in complex with selB and GTP forming the ternary complex (TC). High-resolution cryo-EM structures of intermediates of the Sec incorporation pathway uncover large-scale conformational changes of the ribosome and the TC. To complement the structural information with energetics and rapid dynamics, we performed extensive all-atom molecular dynamics simulations of the ribosome with bound TC as well as of the free TC in solution. The simulations of the free TC were started after extracting the TC from the ribosome-bound cryo-EM structures. The TC was found to rapidly interconvert between the different conformations allowing us to construct the free-energy landscape of the involved motions. This free-energy landscape indicates that the intrinsic large-scale conformational changes of the tRNA and SelB during the delivery to the ribosome are not rate-limiting to the process. In simulations of the free TC started from the GTPase-activated ribosome-bound conformation, the TC rapidly transitions into an inactivated conformation, showing that the GTPase-activated state is strongly stabilized by the ribosome. The simulations of the full ribosome with bound TC in the intermediate states allow us to identify the motions that are rate-limiting to the process of tRNA delivery and to identify the molecular mechanism of the domain closure of small ribosomal subunit upon tRNA decoding.

Molecular Dynamics Simulation of the Nascent Peptide Chain in the Ribosomal Exit Tunnel

The ribosome is a large macromolecular complex which synthesizes all proteins in the cell according to the genetic code and thereby the central nanomachine of life. Nascent peptide chains are polymerized by the ribosome and exit through a tunnel in the large subunit. This tunnel is an important target for antibiotic action hindering the movement of the peptide chain. The recently solved structure of the ribosome led to a better understanding of the tunnel’s geometry and the binding sites of the antibiotics. Yet the dynamics of the polypeptide’s conformation and the interactions with the large subunit and the antibiotics remain unclear. We approach these questions with molecular dynamics simulations of the large subunit and the nascent chain in explicit water under physiological conditions. This allows us to investigate the dynamics the ribosome and the polypeptide at an atomistic level, which will lead to a huge improvement in the understanding of the translation process and the antibiotic action.