Real-time kinetic studies of Mycobacterium tuberculosis LexA–DNA interaction

Abstract

Transcriptional repressor, LexA, regulates the ‘SOS’ response, an indispensable bacterial DNA damage repair machinery. Compared with its Escherichia coli ortholog, LexA from Mycobacterium tuberculosis (Mtb) possesses a unique N-terminal extension of additional 24 amino acids in its DNA-binding domain (DBD) and 18 amino acids insertion at its hinge region that connects the DBD to the C-terminal dimerization/autoproteolysis domain. Despite the importance of LexA in ‘SOS’ regulation, Mtb LexA remains poorly characterized and the functional importance of its additional amino acids remained elusive. In addition, the lack of data on kinetic parameters of Mtb LexA–DNA interaction prompted us to perform kinetic analyses of Mtb LexA and its deletion variants using Bio-layer Interferometry (BLI). Mtb LexA is seen to bind to different ‘SOS’ boxes, DNA sequences present in the operator regions of damage-inducible genes, with comparable nanomolar affinity. Deletion of 18 amino acids from the linker region is found to affect DNA binding unlike the deletion of the N-terminal stretch of extra 24 amino acids. The conserved RKG motif has been found to be critical for DNA binding. Overall, the present study provides insights into the kinetics of the interaction between Mtb LexA and its target ‘SOS’ boxes. The kinetic parameters obtained for DNA binding of Mtb LexA would be instrumental to clearly understand the mechanism of ‘SOS’ regulation and activation in Mtb.

Boronic acid derivative inhibits LexA mediated SOS response in Mycobacteria

“…Extrinsic fluorescence-based studies were performed according to our previously published protocol [30]. About 5 μM of the protein in 10 mM HEPES, 50 mM NaCl (pH 7.5), was incubated with varying concentrations of the inhibitor (3-nPBA) for 30 mins at 37°C.…”

“…All constructs generated were confirmed by sequencing. The recombinant WT and its mutant proteins were over-expressed in E. coli BL21(DE3) cells following the published protocol [30].…”

“…Circular dichroism (CD) studies were performed using Jasco J-815 spectropolarimeter according to our previously published protocol [30]. About 5 μM of protein in 10 mM HEPES, 50 mM NaCl (pH 7.5) and 100X molar concentration of the inhibitor was taken for analysis.…”

“…Further insights into the mechanistic aspect needed probing. Presently, no known inhibitors of Mtb LexA have been identified and Mtb LexA differs from its E. coli counterpart in several ways [30]. Mtb LexA harbours additional stretches of amino acids at its N-terminal end and linker region [31].…”

“…Mtb LexA harbours additional stretches of amino acids at its N-terminal end and linker region [31]. Unlike its E. coli counterpart, Mtb LexA binds to different “SOS” box sequences with comparable nanomolar affinity in-vitro [30]. Since boronic acid class of inhibitors have been reported to block the catalytic activity of serine protease family of proteins including Mtb LexA, we hypothesized that this class of inhibitors could exert a similar effect on Mtb LexA [16,32].…”