Wilkinson Steven P, Grove Anne
Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA.
J Mol Biol. 2005 Jul 22;350(4):617-30. doi: 10.1016/j.jmb.2005.05.027.
Members of the MarR family of winged helix transcriptional regulators have been shown to regulate multidrug and oxidative stress response, pathogenesis, and catabolism of aromatic compounds. Many respond to anionic lipophilic compounds in their capacity to bind DNA, and the co-crystal structure of MarR bound to salicylate revealed two ligand-binding pockets, SAL-A and SAL-B. The MarR homolog, HucR, from Deinococcus radiodurans has been shown to repress expression of a predicted uricase, and DNA-binding by HucR is antagonized by uric acid, the substrate of uricase. We provide a biochemical investigation of DNA-binding and uric acid-binding by HucR. Equilibrium analytical ultracentrifugation indicates that HucR exists as a dimer. Intrinsic fluorescence spectra suggest that the association of the HucR dimer with its cognate DNA involves conformational flexibility in the globular interior and/or dimerization domain of the protein, and near-UV circular dichroism spectra indicate a concomitant change in the helical twist of the DNA duplex. DNA-binding affinity, measured by electrophoretic mobility-shift assays, for HucR mutants bearing single amino acid substitutions suggests the importance of the beta-hairpin "wing" in DNA binding. Analysis of intrinsic fluorescence spectra demonstrates that uric acid induces conformational changes in HucR and binds with an apparent K(d)=11.6(+/-3.7)muM and a Hill coefficient of 0.7+/-0.1, indicating negative cooperativity. Fluorescence and DNA-binding properties of the HucR variants indicate that SAL-A is a low-affinity, uric acid-binding site and that negative cooperativity exists between homologous, high-affinity sites. The conservation of residues comprising site SAL-A suggests that it is a low-affinity, ligand-binding site in MarR homologs. Mechanistic considerations suggest that HucR is regulated by uric acid to maintain optimal cellular levels of this scavenger of free radicals in response to oxidative stress and DNA damage.
有翼螺旋转录调节因子的MarR家族成员已被证明可调节多药和氧化应激反应、发病机制以及芳香族化合物的分解代谢。许多成员在结合DNA的能力方面对阴离子亲脂性化合物有反应，与水杨酸盐结合的MarR的共晶体结构揭示了两个配体结合口袋，即SAL-A和SAL-B。来自耐辐射球菌的MarR同源物HucR已被证明可抑制一种预测的尿酸酶的表达，并且尿酸（尿酸酶的底物）可拮抗HucR与DNA的结合。我们对HucR的DNA结合和尿酸结合进行了生化研究。平衡分析超速离心表明HucR以二聚体形式存在。内在荧光光谱表明，HucR二聚体与其同源DNA的结合涉及蛋白质球状内部和/或二聚化结构域的构象灵活性，近紫外圆二色光谱表明DNA双链的螺旋扭曲同时发生变化。通过电泳迁移率变动分析测量的HucR单氨基酸取代突变体的DNA结合亲和力表明β-发夹“翼”在DNA结合中的重要性。对内在荧光光谱的分析表明，尿酸可诱导HucR的构象变化，并以表观解离常数K(d)=11.6(±3.7)μM和希尔系数0.7±0.1结合，表明存在负协同性。HucR变体的荧光和DNA结合特性表明，SAL-A是一个低亲和力的尿酸结合位点，并且同源高亲和力位点之间存在负协同性。构成位点SAL-A的残基的保守性表明它是MarR同源物中的一个低亲和力配体结合位点。机制上的考虑表明，HucR受尿酸调节，以在氧化应激和DNA损伤时维持这种自由基清除剂的最佳细胞水平。
