Characterization of the ligand and DNA binding properties of a putative archaeal regulator ST1710.
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.
Yu
Linliang
L
Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055, USA.
Fang
Jun
J
Wei
Yinan
Y
D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004996:Ethidium / Q000737:chemistry / Q000378:metabolism; D005454:Fluorescence Polarization; D008024:Ligands; D008958:Models, Molecular; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D011487:Protein Conformation / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D012980:Sodium Salicylate / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D016944:Sulfolobus / Q000235:genetics; D013696:Temperature
D019843:Archaeal Proteins; D008024:Ligands; D011994:Recombinant Proteins; D012097:Repressor Proteins; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D004247:DNA; D004996:Ethidium; D012980:Sodium Salicylate