Assignment of the zinc ligands in RsrA, a redox-sensing ZAS protein from Streptomyces coelicolor.
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 sigmaR 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 sigmaR, leaving the sigma factor free to activate the transcription of antioxidant genes. We showed recently that Zn2+ 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 (H37xxxC41xxC44), 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.
Zdanowski
Konrad
K
Department of Biology (Area 10), University of York, P.O. Box 373, York YO10 5YW, UK.
Doughty
Phillip
P
Jakimowicz
Piotr
P
O'Hara
Liisa
L
Buttner
Mark J
MJ
Paget
Mark S B
MS
Kleanthous
Colin
C
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D008024:Ligands; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics; D015032:Zinc / Q000737:chemistry*
D001426:Bacterial Proteins; D008024:Ligands; C121147:RsrA protein, Streptomyces coelicolor; D014157:Transcription Factors; D006639:Histidine; D015032:Zinc; D003545:Cysteine