Assignment of the Zinc Ligands in RsrA, a Redox-Sensing ZAS Protein from Streptomyces coelicolor

Abstract

ZAS proteins are widespread bacterial zinc-containing anti-sigma factors that regulate the activity of sigma factors in response to diverse cues. One of the best characterized ZAS proteins is RsrA from Streptomyces coelicolor, which responds to disulfide stress. Zn-RsrA binds and represses the transcriptional activity of σ R in the reducing environment of the cytoplasm but undergoes reversible, intramolecular disulfide bond formation during oxidative stress. This expels the single metal ion and causes dramatic structural changes in RsrA that result in its dissociation from σ R , leaving the sigma factor free to activate the transcription of antioxidant genes. We showed recently that Zn 2+ serves a critical role in modulating the redox activity of RsrA thiols but uncertainty remains as to how the metal ion is coordinated in RsrA and related ZAS proteins. Using a combination of random and site-specific mutagenesis with zinc K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy, we have assigned unambiguously the metal ligands in RsrA, thereby distinguishing between the different ligation models that have been proposed. The data show that the zinc site in RsrA is comprised of Cys11, His37, Cys41, and Cys44. Three of these residues are part of a conserved ZAS-specific sequence motif (H 37 xxxC 41 xxC 44 ), with the fourth ligand, Cys11, found in a subset of ZAS proteins. Cys11 and Cys44 form the trigger disulfide in RsrA, explaining why the metal ion is expelled during oxidation. We discuss these data in the context of redox sensing by RsrA and the sensory mechanisms of other ZAS proteins.

Zinc-Responsive Regulation of Alternative Ribosomal Protein Genes inStreptomyces coelicolorInvolves Zur and σR

“…R is controlled posttranslationally by RsrA, a member of the ZAS (zinc binding anti-sigma) family of anti-sigma factors (37). Although zinc-free RsrA can bind to R , its affinity may be decreased and all zinc ligands are essential for RsrA activity in vivo (3,26,37,53). Therefore, under conditions of zinc deprivation, RsrA may accumulate in a zinc-free state, with reduced affinity for R , allowing R to interact with RNA polymerase and activate members of the R regulon.…”

Biosynthesis and Functions of Mycothiol, the Unique Protective Thiol of Actinobacteria

“…A complex is formed between R and the reduced form of the R regulator. Exposure of the oxidized disulfide form of RsrA to thiols generates RsrA red , and thiols generated allow it to bind Zn and form RsrA(Zn) red (161). RsrA(Zn) red binds R , preventing it from activating its target genes.…”

Redox Active Thiol Sensors of Oxidative and Nitrosative Stress

“…The zinc metal is ligated by His 37 , Cys 41 , and Cys 44 that form a characteristic HX 3 CX 2 C zinccontaining antisigma (ZAS) motif (49,76,110). Cys 11 completes the tetrahedral coordination of zinc by RsrA (110). Genetic studies indicate that Cys 11 , Cys 41 , and Cys 44 are essential for the antisigma activity of RsrA (76).…”