title: Crystal structure of the VP4 protease from infectious pancreatic necrosis virus reveals the acyl-enzyme complex for an intermolecular self-cleavage reaction.
abstract: Infectious pancreatic necrosis virus (IPNV), an aquatic birnavirus that infects salmonid fish, encodes a large polyprotein (NH(2)-pVP2-VP4-VP3-COOH) that is processed through the proteolytic activity of its own protease, VP4, to release the proteins pVP2 and VP3. pVP2 is further processed to give rise to the capsid protein VP2 and three peptides that are incorporated into the virion. Reported here are two crystal structures of the IPNV VP4 protease solved from two different crystal symmetries. The electron density at the active site in the triclinic crystal form, refined to 2.2-A resolution, reveals the acyl-enzyme complex formed with an internal VP4 cleavage site. The complex was generated using a truncated enzyme in which the general base lysine was substituted. Inside the complex, the nucleophilic Ser(633)Ogamma forms an ester bond with the main-chain carbonyl of the C-terminal residue, Ala(716), of a neighboring VP4. The structure of this substrate-VP4 complex allows us to identify the S1, S3, S5, and S6 substrate binding pockets as well as other substrate-VP4 interactions and therefore provides structural insights into the substrate specificity of this enzyme. The structure from the hexagonal crystal form, refined to 2.3-A resolution, reveals the free-binding site of the protease. Three-dimensional alignment with the VP4 of blotched snakehead virus, another birnavirus, shows that the overall structure of VP4 is conserved despite a low level of sequence identity ( approximately 19%). The structure determinations of IPNV VP4, the first of an acyl-enzyme complex for a Ser/Lys dyad protease, provide insights into the catalytic mechanism and substrate recognition of this type of protease.
authors.affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
authors.forename: Jaeyong
authors.initials: J
authors.lastname: Lee
authors.forename: Anat R
authors.initials: AR
authors.lastname: Feldman
authors.forename: Bernard
authors.initials: B
authors.lastname: Delmas
authors.forename: Mark
authors.initials: M
authors.lastname: Paetzel
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004583:Electrons; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012645:Selenomethionine / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D012645:Selenomethionine; D012697:Serine Endopeptidases; C414992:VP4 protease, birnavirus; D000409:Alanine