Effect of metal ions on the structure and function of PerR in group A streptococcus

• Main Authors: Sheng-HueiLinchung, 林張聖彙
• Other Authors: Shu-Ying Wang
• Format: Others
• Language: en_US
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/69622561820568307493

碩士 === 國立成功大學 === 微生物及免疫學研究所 === 100 === Group A streptococcus (GAS, Streptococcus pyogenes), an important Gram-positive human pathogen, is responsible for a wide spectrum of human diseases which ranges from minor to lethal. GAS is a catalase-deficient bacterium, but it can survive in an oxidative environment. Previous studies have reported that GAS regulates oxidative stress and metal homeostasis through peroxide response regulator, PerR. However, the molecular mechanism of PerR regulation is not clear. The crystal structure of PerR which can bind DNA has been solved. PerR structure is a homodimer with a zinc ion binding to structural and regulatory sites in each monomer. It was speculated that binding of metals to the regulatory site decides PerR function. To test the hypothesis, we purified PerR protein without metal-binding to regulatory site and examined its function and structure. In this study, we overexpressed PerR in metal-limited M9 medium (M9 PerR) and obtained a protein with less metal content compared with LB broth-expressed PerR (LB PerR). M9 PerR showed no DNA-binding ability to dpr promoter on EMSA gel. The binding ability was restored in the presence of various concentrations of zinc ion. Addition of metal ions enhanced M9 PerR protein stability as judged by the metling temperature in the differential scanning calorimetry (DSC) experiments. Characterization of mutant H99A further confirmed the critical role of regulatory site in functional PerR. Based on these findings, we have demonstrated that zinc ions binding to GAS PerR determines protein stability and DNA-binding function.