Crystal structure of vaccinia virus mRNA capping enzyme provides insights into the mechanism and evolution of the capping apparatus

[EN] Vaccinia virus capping enzyme is a heterodimer of D1 (844 aa) and D12 (287 aa) polypeptides that executes all three steps in m(7)GpppRNA synthesis. The D1 subunit comprises an N-terminal RNA triphosphatase (TPase)-guanylyltransferase (GTase) module and a C-terminal guanine-N7-methyltransferase (MTase) module. The D12 subunit binds and allosterically stimulates the MTase module. Crystal structures of the complete D1.D12 heterodimer disclose the TPase and GTase as members of the triphosphate tunnel metalloenzyme and covalent nucleotidyltransferase superfamilies, respectively, albeit with distinctive active site features. An extensive TPase-GTase interface clamps the GTase nucleotidyltransferase and OB-fold domains in a closed conformation around GTP. Mutagenesis confirms the importance of the TPase-GTase interface for GTase activity. The D1.D12 structure complements and rationalizes four decades of biochemical studies of this enzyme, which was the first capping enzyme to be purified and characterized, and provides new insights into the origins of the capping systems of other large DNA viruses.We are grateful for access to platforms of the Grenoble Partnership for Structural Biology, especially the High Throughput Crystallization (HTX) laboratory of the European Molecular Biology Laboratory (EMBL) for robotic crystallization. We thank the staff of the European Synchrotron Radiation Facility (ESRF)-EMBL Joint Structural Biology Group for help with data collection on beamlines BM14, ID14-4, ID14-3, and ID23-1. We acknowledge the help of Dr. Heinz Gut in setting up the cross-crystal averaging. This work was supported by National Institutes of Health grant GM42498 (to S.S.). S.S. is an American Cancer Society Research ProfessorKyrieleis, OJP.; Chang, J.; La Peña Del Rivero, MD.; Shuman, S.; Cusack, S. (2014). Crystal structure of vaccinia virus mRNA capping enzyme provides insights into the mechanism and evolution of the capping apparatus. Structure. 22(3):452-465. https://doi.org/10.1016/j.str.2013.12.014S45246522

Crystal structures of the tricorn interacting factor F3 from thermoplasma acidophilum, a zinc aminopeptidase in three different conformations

The tricorn interacting factor F3 is an 89 kDa zinc aminopeptidase from the archaeon Thermoplasma acidophilum. Together with the tricorn interacting factors F1 and F2, F3 degrades the tricorn protease products and thus completes the proteasomal degradation pathway by generating free amino acids. Here, we present the crystal structures of F3 in three different conformations at 2.3 Å resolution. The zinc aminopeptidase is composed of four domains: an N-terminal saddle-like β-structure domain; a thermolysin-like catalytic domain; a small barrel-like β-structure domain; and an α-helical C-terminal domain, the latter forming a deep cavity at the active site. Three crystal forms provide snapshots of the molecular dynamics of F3 where the C-terminal domain can adapt to form an open, an intermediate and a nearly closed cavity, respectively. With the conserved Zn2+-binding motifs HEXXH and NEXFA as well as the N-terminal substrate-anchoring glutamate residues, F3 together with the leukotriene A4 hydrolase, represents a novel gluzincin subfamily of aminoproteases. We discuss the functional implications of these structures with respect to the underlying catalytic mechanism, substrate recognition and processing, and possible component interactions