Yu Linliang, Fang Jun, Wei Yinan
Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055, USA.
Biochemistry. 2009 Mar 17;48(10):2099-108. doi: 10.1021/bi801662s.
While a rich collection of bacterium-like regulating proteins has been identified in the archaeal genome, few of them have been studied at the molecular level. In this study, we characterized the ligand and DNA binding properties of a putative regulator ST1710 from the archaeon Sulfolobus tokodaii. ST1710 is homologous to the multiple-antibiotic resistance repressor (MarR) family bacterial regulators. The protein consists of a ligand binding site, partially overlapping with a winged helix-turn-helix DNA binding site. We characterized the interactions between ST1710 and three ligands, salicylate, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and ethidium, which bind to bacterial MarRs. The binding affinities of the ligands for ST1710 were comparable to their affinities for the bacterial MarRs. The ligand binding was temperature sensitive and caused conformational changes in ST1710. To investigate the effect of ligand binding on the interaction between ST1710 and DNA, we fluorescently labeled a 47mer dsDNA (ST1) containing a putative ST1710 recognition site and determined the dissociation constant between ST1 and ST1710 using the fluorescence polarization method. The binding affinity almost doubled from 10 degrees C (Kd = 618 +/- 34 nM) to 30 degreesC (Kd = 334 +/- 15 nM), and again from 30 to 50 degrees C (Kd = 189 +/- 9 nM). This result suggests that under the natural living condition (80 degrees C) of S. tokodaii, the binding affinity might increase even further. The presence of CCCP and salicylate suppressed ST1710-ST1 interaction, indicating that ST1710 functioned as a repressor.
虽然在古菌基因组中已鉴定出大量类似细菌的调控蛋白，但对其中很少一部分进行过分子水平的研究。在本研究中，我们对来自嗜热栖热菌的假定调控因子ST1710的配体结合和DNA结合特性进行了表征。ST1710与多抗生素抗性阻遏物（MarR）家族细菌调控因子同源。该蛋白由一个配体结合位点组成，部分与一个带翼的螺旋-转角-螺旋DNA结合位点重叠。我们表征了ST1710与三种配体（水杨酸、羰基氰化物间氯苯腙（CCCP）和溴化乙锭）之间的相互作用，这些配体可与细菌MarR结合。这些配体对ST1710的结合亲和力与其对细菌MarR的亲和力相当。配体结合对温度敏感，并导致ST1710发生构象变化。为了研究配体结合对ST1710与DNA相互作用的影响，我们对包含假定的ST1710识别位点的47聚体双链DNA（ST1）进行了荧光标记，并使用荧光偏振法测定了ST1与ST1710之间的解离常数。结合亲和力从10℃（Kd = 618±34 nM）到30℃（Kd = 334±15 nM）几乎翻倍，从30℃到50℃（Kd = 189±9 nM）再次翻倍。该结果表明，在嗜热栖热菌的自然生存条件（80℃）下，结合亲和力可能会进一步增加。CCCP和水杨酸的存在抑制了ST1710-ST1相互作用，表明ST1710起到了阻遏物的作用。
