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Dynamin oligomerizes into helical filaments on tubular membrane templates and, through constriction, cleaves them in a GTPase-driven way. Structural observations of GTP-dependent cross-bridges between neighboring filament turns have led to the suggestion that dynamin operates as a molecular ratchet motor. However, the proof of such mechanism remains absent. Particularly, it is not known whether a powerful enough stroke is produced and how the motor modules would cooperate in the constriction process. Here, we characterized the dynamin motor modules by single-molecule Förster resonance energy transfer (smFRET) and found strong nucleotide-dependent conformational preferences. Integrating smFRET with molecular dynamics simulations allowed us to estimate the forces generated in a power stroke. Subsequently, the quantitative force data and the measured kinetics of the GTPase cycle were incorporated into a model including both a dynamin filament, with explicit motor cross-bridges, and a realistic deformable membrane template. In our simulations, collective constriction of the membrane by dynamin motor modules, based on the ratchet mechanism, is directly reproduced and analyzed. Functional parallels between the dynamin system and actomyosin in the muscle are seen. Through concerted action of the motors, tight membrane constriction to the hemifission radius can be reached. Our experimental and computational study provides an example of how collective motor action in megadalton molecular assemblies can be approached and explicitly resolved., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

The single G protein of the spliceosome, Snu114, has been proposed to facilitate splicing as a molecular motor or as a regulatory G protein. However, available structures of spliceosomal complexes show Snu114 in the same GTP-bound state, and presently no Snu114 GTPase-regulatory protein is known. We determined a crystal structure of Snu114 with a Snu114-binding region of the Prp8 protein, in which Snu114 again adopts the same GTP-bound conformation seen in spliceosomes. Snu114 and the Snu114-Prp8 complex co-purified with endogenous GTP. Snu114 exhibited weak, intrinsic GTPase activity that was abolished by the Prp8 Snu114-binding region. Exchange of GTP-contacting residues in Snu114, or of Prp8 residues lining the Snu114 GTP-binding pocket, led to temperature-sensitive yeast growth and affected the same set of splicing events in vivo. Consistent with dynamic Snu114-mediated protein interactions during splicing, our results suggest that the Snu114-GTP-Prp8 module serves as a relay station during spliceosome activation and disassembly, but that GTPase activity may be dispensable for splicing., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Selenocysteine tRNAs (tRNA(Sec)) exhibit a number of unique identity elements that are recognized specifically by proteins of the selenocysteine biosynthetic pathways and decoding machineries. Presently, these identity elements and the mechanisms by which they are interpreted by tRNA(Sec)-interacting factors are incompletely understood., Methodology/principal Findings: We applied rational mutagenesis to obtain well diffracting crystals of murine tRNA(Sec). tRNA(Sec) lacking the single-stranded 3'-acceptor end ((ΔGCCA)RNA(Sec)) yielded a crystal structure at 2.0 Å resolution. The global structure of (ΔGCCA)RNA(Sec) resembles the structure of human tRNA(Sec) determined at 3.1 Å resolution. Structural comparisons revealed flexible regions in tRNA(Sec) used for induced fit binding to selenophosphate synthetase. Water molecules located in the present structure were involved in the stabilization of two alternative conformations of the anticodon stem-loop. Modeling of a 2'-O-methylated ribose at position U34 of the anticodon loop as found in a sub-population of tRNA(Sec)in vivo showed how this modification favors an anticodon loop conformation that is functional during decoding on the ribosome. Soaking of crystals in Mn(2+)-containing buffer revealed eight potential divalent metal ion binding sites but the located metal ions did not significantly stabilize specific structural features of tRNA(Sec)., Conclusions/significance: We provide the most highly resolved structure of a tRNA(Sec) molecule to date and assessed the influence of water molecules and metal ions on the molecule's conformation and dynamics. Our results suggest how conformational changes of tRNA(Sec) support its interaction with proteins.

In eukaryotes and Archaea, selenocysteine synthase (SecS) converts O-phospho-L-seryl-tRNA [Ser]Sec into selenocysteyl-tRNA [Ser]Sec using selenophosphate as the selenium donor compound. The molecular mechanisms underlying SecS activity are presently unknown. We have delineated a 450-residue core of mouse SecS, which retained full selenocysteyl-tRNA [Ser]Sec synthesis activity, and determined its crystal structure at 1.65 A resolution. SecS exhibits three domains that place it in the fold type I family of pyridoxal phosphate (PLP)-dependent enzymes. Two SecS monomers interact intimately and together build up two identical active sites around PLP in a Schiff-base linkage with lysine 284. Two SecS dimers further associate to form a homotetramer. The N terminus, which mediates tetramer formation, and a large insertion that remodels the active site set SecS aside from other members of the family. The active site insertion contributes to PLP binding and positions a glutamate next to the PLP, where it could repel substrates with a free alpha-carboxyl group, suggesting why SecS does not act on free O-phospho-l-serine. Upon soaking crystals in phosphate buffer, a previously disordered loop within the active site insertion contracted to form a phosphate binding site. Residues that are strictly conserved in SecS orthologs but variant in related enzymes coordinate the phosphate and upon mutation corrupt SecS activity. Modeling suggested that the phosphate loop accommodates the gamma-phosphate moiety of O-phospho-l-seryl-tRNA [Ser]Sec and, after phosphate elimination, binds selenophosphate to initiate attack on the proposed aminoacrylyl-tRNA [Ser]Sec intermediate. Based on these results and on the activity profiles of mechanism-based inhibitors, we offer a detailed reaction mechanism for the enzyme.