Structural and biochemical characterization of the essential DsbA-like disulfide bond forming protein from Mycobacterium tuberculosis

Nicholas Chim,Celia W Goulding,Christine A Harmston,David J Guzman

doi:10.1186/1472-6807-13-23

Abstract

BackgroundBacterial Disulfide bond forming (Dsb) proteins facilitate proper folding and disulfide bond formation of periplasmic and secreted proteins. Previously, we have shown that Mycobacterium tuberculosis Mt-DsbE and Mt-DsbF aid in vitro oxidative folding of proteins. The M. tuberculosis proteome contains another predicted membrane-tethered Dsb protein, Mt-DsbA, which is encoded by an essential gene.ResultsHerein, we present structural and biochemical analyses of Mt-DsbA. The X-ray crystal structure of Mt-DsbA reveals a two-domain structure, comprising a canonical thioredoxin domain with the conserved CXXC active site cysteines in their reduced form, and an inserted α-helical domain containing a structural disulfide bond. The overall fold of Mt-DsbA resembles that of other DsbA-like proteins and not Mt-DsbE or Mt-DsbF. Biochemical characterization demonstrates that, unlike Mt-DsbE and Mt-DsbF, Mt-DsbA is unable to oxidatively fold reduced, denatured hirudin. Moreover, on the substrates tested in this study, Mt-DsbA has disulfide bond isomerase activity contrary to Mt-DsbE and Mt-DsbF.ConclusionThese results suggest that Mt-DsbA acts upon a distinct subset of substrates as compared to Mt-DsbE and Mt-DsbF. One could speculate that Mt-DsbE and Mt-DsbF are functionally redundant whereas Mt-DsbA is not, offering an explanation for the essentiality of Mt-DsbA in M. tuberculosis.

Highlights

Bacterial Disulfide bond forming (Dsb) proteins facilitate proper folding and disulfide bond formation of periplasmic and secreted proteins
The crystal structure of the soluble form of Mt-DsbA was solved by molecular replacement utilizing the model of Bs-BdbD (PDB ID: 3EU3 [39]), which shares 20% sequence identity with Mt-DsbA, combined with the location of two selenium atoms determined from the anomalous data
Neither Mycobacterium tuberculosis (Mtb)-DsbE nor Mt-DsbF can catalyze the refolding of scRNaseA to produce active RNaseA, and do not appear to have isomerase activity under the conditions tested (Figure 4C). These results demonstrate that Mt-DsbA has protein disulfide isomerase activity, while Mt-DsbE and Mt-DsbF do not possess this activity on the substrates tested in this study

Summary

Introduction

Bacterial Disulfide bond forming (Dsb) proteins facilitate proper folding and disulfide bond formation of periplasmic and secreted proteins. Correct folding and disulfide bond formation is essential for the function of many secreted proteins including bacterial toxins, and their formation is facilitated by disulfide bond forming (Dsb) oxidoreductase proteins, which usually contain a conserved thioredoxin (TRX) fold [1]. The Dsb proteins of Escherichia coli are the best characterized, and reside in its periplasm to correctly fold disulfide bond containing secreted and cell-wall proteins [4]. E. coli DsbA (Ec-DsbA) catalyzes the oxidation of disulfide bonds in reduced, unfolded proteins [5,6], and is reoxidized by ubiquinone via E. coli DsbB (Ec-DsbB), an inner membrane transmembrane protein, which in turn is oxidized by the electron transport pathway [7,8]. E. coli DsbE (Ec-DsbE) is a reductant involved in cytochrome c maturation [12], its redox partner is proposed to be Ec-

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