Research article

Redox biology of Mycobacterium tuberculosis H37Rv: protein-protein interaction between GlgB and WhiB1 involves exchange of thiol-disulfide

Saurabh Garg^{* 1} , Md Suhail Alam^{* 2} , Richa Bajpai² , KV Radha Kishan³ and Pushpa Agrawal²

¹Department of Environmental and Biomolecular Systems, OGI School of Science and Engineering, Oregon Health and Science University, 20000 NW Walker Road, Beaverton, Oregon, 97006, USA

²Institute of Microbial Technology, CSIR, Sector-39A, Chandigarh, 160036, India

³Informatics, GVK Biosciences, Hyderabad, 500037, India

author email corresponding author email* Contributed equally

BMC Biochemistry 2009, 10:1doi:10.1186/1471-2091-10-1

Published:	5 January 2009

Abstract

Background

Mycobacterium tuberculosis, an intracellular pathogen encounters redox stress throughout its life inside the host. In order to protect itself from the redox onslaughts of host immune system, M. tuberculosis appears to have developed accessory thioredoxin-like proteins which are represented by ORFs encoding WhiB-like proteins. We have earlier reported that WhiB1/Rv3219 is a thioredoxin like protein of M. tuberculosis and functions as a protein disulfide reductase. Generally thioredoxins have many substrate proteins. The current study aims to identify the substrate protein(s) of M. tuberculosis WhiB1.

Results

Using yeast two-hybrid screen, we identified alpha (1,4)-glucan branching enzyme (GlgB) of M. tuberculosis as a interaction partner of WhiB1. In vitro GST pull down assay confirmed the direct physical interaction between GlgB and WhiB1. Both mass spectrometry data of tryptic digests and in vitro labeling of cysteine residues with 4-acetamido-4' maleimidyl-stilbene-2, 2'-disulfonic acid showed that in GlgB, C⁹⁵ and C⁶⁵⁸ are free but C¹⁹³ and C⁶¹⁷ form an intra-molecular disulfide bond. WhiB1 has a C³⁷XXC⁴⁰ motif thus a C⁴⁰S mutation renders C³⁷ to exist as a free thiol to form a hetero-disulfide bond with the cysteine residue of substrate protein. A disulfide mediated binary complex formation between GlgB and WhiB1C⁴⁰S was shown by both in-solution protein-protein interaction and thioredoxin affinity chromatography. Finally, transfer of reducing equivalent from WhiB1 to GlgB disulfide was confirmed by 4-acetamido-4' maleimidyl-stilbene-2, 2'-disulfonic acid trapping by the reduced disulfide of GlgB. Two different thioredoxins, TrxB/Rv1471 and TrxC/Rv3914 of M. tuberculosis could not perform this reaction suggesting that the reduction of GlgB by WhiB1 is specific.

Conclusion

We conclude that M. tuberculosis GlgB has one intra-molecular disulfide bond which is formed between C¹⁹³ and C⁶¹⁷. WhiB1, a thioredoxin like protein interacts with GlgB and transfers its electrons to the disulfide thus reduces the intra-molecular disulfide bond of GlgB. For the first time, we report that GlgB is one of the in vivo substrate of M. tuberculosis WhiB1.