Structural and Biochemical Bases for the Redox Sensitivity of Mycobacterium tuberculosis RslA

Abstract

An effective transcriptional response to redox stimuli is of particular importance for Mycobacterium tuberculosis, as it adapts to the environment of host alveoli and macrophages. The M. tuberculosis σ factor σL regulates the expression of genes involved in cell-wall and polyketide syntheses. σL interacts with the cytosolic anti-σ domain of a membrane-associated protein, RslA. Here we demonstrate that RslA binds Zn2+ and can sequester σL in a reducing environment. In response to an oxidative stimulus, proximal cysteines in the CXXC motif of RslA form a disulfide bond, releasing bound Zn2+. This results in a substantial rearrangement of the σL/RslA complex, leading to an 8-fold decrease in the affinity of RslA for σL. The crystal structure of the − 35-element recognition domain of σL, σ4L, bound to RslA reveals that RslA inactivates σL by sterically occluding promoter DNA and RNA polymerase binding sites. The crystal structure further reveals that the cysteine residues that coordinate Zn2+ in RslA are solvent exposed in the complex, thus providing a structural basis for the redox sensitivity of RslA. The biophysical parameters of σL/RslA interactions provide a template for understanding how variations in the rate of Zn2+ release and associated conformational changes could regulate the activity of a Zn2+-associated anti-σ factor.

A Novel P1B-type Mn2+-transporting ATPase Is Required for Secreted Protein Metallation in Mycobacteria

“…We observed little or no sensitivity of the ctpC::hyg cells to various metal stressors, except for the described lack of tolerance to Zn 2ϩ (11 (27), and it is possible that high Zn 2ϩ concentrations might mimic Fe 2ϩ deficiency. Alternatively, a link between Zn 2ϩ and factors has been proposed in M. tuberculosis (64), and reactive oxygen species have been shown to affect the signaling pathways involved in the activation of multiple transcription factors (65). Considering the observation that ctpC::hyg cells are also sensitive to redox stressors, it is tempting to speculate that the sensitivity to Zn 2ϩ is related to alteration in Fe 2ϩ or redox homeostasis rather than to an increase in cytosolic Zn 2ϩ .…”

Oxidant Sensing by Reversible Disulfide Bond Formation

“…Another strategy is employed by redox-regulated prokaryotic anti-sigma factors and eukaryotic inhibitors. Their disulfidemediated degradation causes the release and activation of the respective transcriptional regulators, resulting in the activation of gene expression (77,78).…”

Crystallization and preliminary X-ray crystallographic analysis of carboxyl-terminal region 4 of SigR fromStreptomyces coelicolorA3(2)

“…Based on the fragments identified by the MS/MS data, we believe that residues 134-223 of region 4 are contained in the crystal. Although a single peptide of R region 2 was detected ( (Lane & Darst, 2006;Thakur et al, 2007Thakur et al, , 2010.…”