Structural basis for catalysis and substrate specificity of a 3C-like cysteine protease from a mosquito mesonivirus

Manuel Kanitz; Sandra Blanck; Andreas Heine; Anastasia A Gulyaeva; Alexander E Gorbalenya; John Ziebuhr; Wibke E Diederich

doi:10.1016/j.virol.2019.05.001

Structural basis for catalysis and substrate specificity of a 3C-like cysteine protease from a mosquito mesonivirus

Virology. 2019 Jul:533:21-33. doi: 10.1016/j.virol.2019.05.001. Epub 2019 May 2.

Authors

Manuel Kanitz¹, Sandra Blanck², Andreas Heine³, Anastasia A Gulyaeva⁴, Alexander E Gorbalenya⁵, John Ziebuhr⁶, Wibke E Diederich⁷

Affiliations

¹ Center for Tumor Biology and Immunology, Philipps University, Marburg, Germany; Institute of Pharmaceutical Chemistry, Philipps University, Marburg, Germany.
² Institute of Medical Virology, Justus Liebig University, Giessen, Germany.
³ Institute of Pharmaceutical Chemistry, Philipps University, Marburg, Germany.
⁴ Department of Medical Microbiology, Leiden University Medical Center, Leiden, the Netherlands.
⁵ Department of Medical Microbiology, Leiden University Medical Center, Leiden, the Netherlands; Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia; Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, Russia.
⁶ Institute of Medical Virology, Justus Liebig University, Giessen, Germany. Electronic address: john.ziebuhr@viro.med.uni-giessen.de.
⁷ Center for Tumor Biology and Immunology, Philipps University, Marburg, Germany; Institute of Pharmaceutical Chemistry, Philipps University, Marburg, Germany. Electronic address: wibke.diederich@staff.uni-marburg.de.

Abstract

Cavally virus (CavV) is a mosquito-borne plus-strand RNA virus in the family Mesoniviridae (order Nidovirales). We present X-ray structures for the CavV 3C-like protease (3CL^pro), as a free enzyme and in complex with a peptide aldehyde inhibitor mimicking the P4-to-P1 residues of a natural substrate. The 3CL^pro structure (refined to 1.94 Å) shows that the protein forms dimers. The monomers are comprised of N-terminal domains I and II, which adopt a chymotrypsin-like fold, and a C-terminal α-helical domain III. The catalytic Cys-His dyad is assisted by a complex network of interactions involving a water molecule that mediates polar contacts between the catalytic His and a conserved Asp located in the domain II-III junction and is suitably positioned to stabilize the developing positive charge of the catalytic His in the transition state during catalysis. The study also reveals the structural basis for the distinct P2 Asn-specific substrate-binding pocket of mesonivirus 3CL^pros.

Keywords: 3C-like protease; Active site of chymotrypsin-like proteases; Coronavirus; Crystal structure; Invertebrate RNA virus; Mesonivirus.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Motifs
Amino Acid Sequence
Animals
Binding Sites
Catalysis
Crystallography, X-Ray
Culicidae / virology*
Cysteine Proteases / chemistry*
Cysteine Proteases / genetics
Cysteine Proteases / metabolism*
Nidovirales / chemistry
Nidovirales / enzymology*
Nidovirales / genetics
Sequence Alignment
Substrate Specificity
Viral Proteins / chemistry*
Viral Proteins / genetics
Viral Proteins / metabolism*

Substances

Viral Proteins
Cysteine Proteases