Crystallographic analysis of birnavirus VP4 proteases.

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Enzyme mechanisms
Serine-lysine catalytic dyad mechanism

Birnaviruses have a bi-segmented double-stranded RNA genome residing within a single-shelled non-enveloped icosahedral particle. There are economic reasons to study birnaviruses and prevent their propagation as many of the birnaviruses are pathogenic to species that are consumed by humans (salmon, yellowtail fish, chicken, and clam) and some are commercially farmed. Protease VP4 cleaves the polyprotein (NH2-pVP2-VP4-VP3-COOH) of birnavirus into components required for virion assembly. It utilizes a serine-lysine (Ser/Lys) catalytic dyad that is less characterized than the Ser/His/Asp classical catalytic triad; thus, there are also scientific interests in studying its mechanism. The crystal structure of Infectious pancreatic necrosis virus (IPNV) revealed acyl-enzyme complexes suggesting that VP4 proteases could be used to trap different stages of the reaction mechanism. Here, I present the results of the crystallography analysis on VP4 proteases from Tellina virus 1 (TV-1), and Yellowtail ascites virus (YAV). These structures provided insights on how VP4 proteases interact with the substrates and how the polyprotein is cleaved; thus, will aid in the design of anti- birnavirus compounds. TV-1 was first isolated from the sand dwelling marine bivalve mollusk Tellina tenuis (clam). Manifestations of the disease include a thinner and chalkier shell as well as a pale yellow digestive gland. The structure of TV-1 VP4 was solved to 2.1 Å resolution revealing an intramolecular (cis) acyl-enzyme complex which demonstrates how the enzyme recognizes its own carboxy-terminus during the VP4/VP3 cleavage event. To our knowledge, this is the first time that an intramolecular acyl-enzyme has been observed within a protease crystal structure. YAV infection leads to ascites in yellowtail fish (Seriola quinqueradiata), which is popular in sushi. The existence of a previously proposed internal cleavage site within VP4 was confirmed using protein and fluorometric peptide cleavage assays as well as capturing it in two acyl-enzyme structures. The native active site structure (2.5 Å resolution) revealed both the acyl-enzyme and product bound states. The lysine mutant structure (2.3 Å resolution) revealed the acyl-enzyme and empty binding site states of VP4, which allows for the observation of structural changes upon substrate or product binding.

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Dr. Mark Paetzel
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.