K-064. The Structure of a DsbA-Substrate Complex: Implications for Substrate Recognition by Oxidoreductases

J. J. Paxman1, N. A. Borg1, J. Horne1, J. Rossjohn1, P. Thompson1, S. Piek2, C. M. Kahler2, H. Sakellaris2, M. J. Scanlon1;
1Monash Univ., Melbourne, VIC, AUSTRALIA, 2Univ. of Western Australia, Perth, WA, AUSTRALIA.

Background: Oxidoreductases (DsbA) introduce covalent disulphide bonds between thiol groups of two cysteine residues, thus contributing to the stability and function of many proteins. To understand how DsbA interacts with diverse substrates, we have produced the first crystal structure of a DsbA-substrate peptide complex. Methods: The Shigella autotransporter, SigA, contains a disulphide bond whose formation is catalysed by DsbA. A nine residue peptide encompassing the sequence around one of the cysteines in SigA was synthesised with an acetylated N-terminus, a C-terminal amide and with a homoserine residue in place of the cysteine (Ac-PIPFL-Hse-QKD-NH2). The hydroxyl of the homoserine was substituted with bromine to generate the homobromoalanine analog, which was reacted with C30 of DsbA to form a stable covalent thioether complex. Diffraction quality crystals of the complex were formed by soaking the homobromoalanine-peptide into pre-formed crystals of reduced DsbA. The structure of the complex was solved by molecular replacement to a resolution of 1.9Å (R-work = 21.7%, R-free = 25.2%). Results: The peptide substrate binds at the interface between the alpha-helical and thioredoxin domains of DsbA, and not the hydrophobic groove below the active site. To further test this observation, we constructed chimeric DsbA enzymes consisting of alpha domains from two meningococcal oxidoreductases inserted into the E. coli thioredoxin domain and analysed their function by complementation of motility and DTT sensitivity in an E. coli DsbA mutant. Exchange of the alpha-domains had no affect on the ability of DsbA to complement motility or DTT resistance. Changes in the amino acid composition and length of the hinge regions between the alpha domain and the thioredoxin domain, however, prevented the restoration of motility whilst retaining oxidoreductase function, as determined by restoration of DTT resistance to the E. coli DsbA mutant. Conclusions: This work provides important insights into the structural basis for the interaction of DsbA with proteins and provides a rationale for potential substrate discrimination based upon interdomain flexibility.