Abstract
BackgroundMycobacterium 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.ResultsUsing 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.ConclusionWe 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.
Highlights
Mycobacterium tuberculosis, an intracellular pathogen encounters redox stress throughout its life inside the host
WhiB1 interacts with GlgB WhiB1, a thioredoxin like protein is likely to function via protein-protein interaction yeast two-hybrid system was employed to identify the potential interacting partner/s of WhiB1
Based upon our earlier report [9] and the present mass spectrometry data, we propose that an intra-molecular disulfide bond is formed between C193 and C617
Summary
Mycobacterium tuberculosis, an intracellular pathogen encounters redox stress throughout its life inside the host. We have earlier reported that WhiB1/Rv3219 is a thioredoxin like protein of M. tuberculosis and functions as a protein disulfide reductase. A large number of cellular processes are mediated through protein-protein interactions These interactions are non-covalent and are outcome of hydrophobic or ionic or both interactions. Trx is a major protein disulfide reductase responsible for maintaining the redox state of cytosol. They are involved in large number of cell processes and regulate the activity of many proteins through reversible reduction of their disulfide bonds. Regulate their biochemical function [4] They often regenerate cellular proteins by reducing non-specific disulfides formed during oxidative stress function as an important antioxidant system. The activation of peroxiredoxins, which facilitates reduction of reactive oxygen species, is dependent on Trxs [7]
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