Shewchuk L M, Verdine G L, Nash H, Walsh C T
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Biochemistry. 1989 Jul 25;28(15):6140-5. doi: 10.1021/bi00441a002.
Bacterial resistance to mercury(II) compounds is controlled by the metalloregulatory MerR protein, a transcriptional repressor and a mercuric ion dependent activator of the mer operon. Site-directed mutagenesis of all four cysteine residues in the Tn501 MerR protein has led to the specific replacement of C82, C115, and C117 with alanine and of C126 with serine. Mutation of C82 and C126 abolishes transcriptional activation in vivo while mutation of C115 and C117 leads to a slight increase and dramatic decrease in transcriptional activation, respectively. All four mutants are competent, to varying degrees, to repress mer transcription. Characterization of the four purified mutant proteins in vitro demonstrates that only the C126S MerR mutant is most notably deficient in stoichiometric Hg(II) binding. All four mutant proteins possess similar DNA binding properties, and the C82 mutant is most affected in the ability to form stable dimers. Given an observed stoichiometry of one Hg(II) per MerR dimer, it is likely that the transcriptionally activating MerR species is a metal-bridged dimer. It is most likely that one C126 per subunit provides high-avidity bidentate ligation to Hg(II), but it remains possible that C82 may be a secondary Hg(II) ligand (e.g., in a tetracoordinate thiol ligation array).
细菌对汞（II）化合物的抗性由金属调节蛋白MerR控制，MerR是一种转录阻遏物，也是mer操纵子的汞离子依赖性激活剂。对Tn501 MerR蛋白中所有四个半胱氨酸残基进行定点诱变，导致C82、C115和C117被丙氨酸特异性取代，C126被丝氨酸取代。C82和C126的突变消除了体内转录激活，而C115和C117的突变分别导致转录激活略有增加和显著降低。所有四个突变体都有不同程度的抑制mer转录的能力。对四种纯化的突变蛋白进行体外表征表明，只有C126S MerR突变体在化学计量的Hg（II）结合方面最为明显地缺乏。所有四种突变蛋白都具有相似的DNA结合特性，C82突变体在形成稳定二聚体的能力方面受到的影响最大。鉴于观察到每个MerR二聚体有一个Hg（II）的化学计量，转录激活的MerR物种很可能是一种金属桥连二聚体。最有可能的是，每个亚基中的一个C126为Hg（II）提供高亲和力的双齿连接，但C82仍有可能是次要的Hg（II）配体（例如，在四配位硫醇连接阵列中）。
