Domain dissection of large polyproteins as a tool for structure-function studies: Examples from bacterial polyketide synthases and viral replicases

Todd William Geders, Purdue University

Abstract

Polyproteins are large proteins with multiple functional domains. Many are difficult to study biochemically due to their size and multiple enzymatic functions. Here I present examples of a domain dissection methodology used to excise functional domains from larger polyproteins for structure-function analysis. Examples are given from bacterial polyketide synthases and plus-stranded RNA viral replicases. The first domain dissection example is of the loading domain of CurA, a 2311 amino acid polyprotein involved in polyketide chain initiation of curacin A biosynthesis. One domain excised from CurA, termed GNATL, was demonstrated to possess an unprecedented decarboxylase and S-acyltransferase activity. The GNATL-domain structure was determined and revealed a GCN5-related N-acetyltransferase (GNAT) fold. The structure, in conjunction with functional analysis and site-directed mutagenesis, identified two catalytically important active site residues, Thr355 and His389. A new GNAT-like strategy for polyketide chain initiation was identified and proposed. The second domain dissection example is of the ECH2 domain of CurF, a 3195 amino acid polyprotein involved in curacin A biosynthesis. The N-terminal ECH 2 domain of CurF is a decarboxylase and the last component of a five protein HMG-CoA synthase-containing "HCS cassette" that is used to introduce a β-branch into polyketides. The structure of the ECH2 domain of CurF was determined, revealed a crotonase fold, and identified a number of potential active site residues. Modeling of the substrate into the active site aided the selection of three residues for site-directed mutagenesis. In a coupled ECH1/ECH2 dehydration/decarboxylase assay, two catalytically important residues, His240 and Lys 86, were identified and their roles in decarboxylation proposed. The structure-function analysis of CurF ECH2 has provided insight into how β-branching is controlled in "HCS cassettes". The third domain dissection example is from the alphavirus nsP2 and nsP3 components of the ∼2500 amino acid nonstructural polyprotein genome replicase. Here I present results from high-throughput domain dissection experiments designed to obtain soluble protein for structure-function studies. The protease domain of Sindbis nsP2 was produced and demonstrated to possess site-specific protease activity in two in vitro protease assays, an unexpected RNA binding activity, and a previously unrecognized methyltransferase-like subdomain.

Degree

Ph.D.

Advisors

Chen, Purdue University.

Subject Area

Molecular biology|Biochemistry|Virology

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