Kimura T, Nakatani M, Kawabe T, Yamaguchi A
Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Japan.
Biochemistry. 1998 Apr 21;37(16):5475-80. doi: 10.1021/bi973188g.
Seven arginine residues are conserved in all the tetracycline/H+ antiporters of Gram-negative bacteria. Four (Arg67, -70, -71, and -127) of them are located in the putative cytoplasmic loop regions and three (Arg31, -101, and -238) in the putative periplasmic loop regions [Eckert, B., and Beck, C. F. (1989) J. Biol. Chem. 264, 11663-11670]. These arginine residues were replaced by alanine, lysine, or cysteine one by one through site-directed mutagenesis. None of the mutants showed significant alteration of the protein expression level. The mutants resulting in the replacement of Arg31, Arg67, Arg71, and Arg238 with either Ala, Cys, or Lys retained tetracycline resistance levels comparable to that of the wild type. Among them, only the Arg238 --> Ala mutant showed very low transport activity in everted membrane vesicles, probably due to the instability of the mutant protein. The replacement of Arg70 and Arg127 with Ala or Cys resulted in a drastic decrease in the drug resistance and almost complete loss of the transport activity, while the Lys replacement mutants retained significant resistance and transport activity, indicating that the positively charged side chains at these positions conferred the transport function. On the other hand, neither the Ala, Cys, nor Lys replacement mutant of Arg101 exhibited any drug resistance or transport activity. As for the reactivity of the Cys replacement mutants, only two (Arg71 --> Cys and Arg101 --> Cys) were not reactive with NEM, the other five mutants being highly or moderately reactive. The reactivity of the cysteine-scanning mutants around Arg101 with NEM revealed that Arg101 is located in transmembrane helix IV. It is not likely that Arg101 confers the protein folding through a salt bridge with a transmembrane acidic residue because no double mutants involving Arg101 --> Ala and the replacement of one of three transmembrane acidic residues (Asp15, Asp84, and Asp285) showed the recovery of any tetracycline resistance or transport activity. The effect of tetracycline on the [14C]NEM binding to the combined mutants S65C/R101A and L97C/R101A suggests that Arg101 may cause a substrate-induced conformational change of the putative exit gate of TetA(B).
革兰氏阴性菌的所有四环素/H⁺反向转运蛋白中都有7个精氨酸残基保守存在。其中4个（Arg67、-70、-71和-127）位于假定的胞质环区域，3个（Arg31、-101和-238）位于假定的周质环区域[埃克特，B.，和贝克，C. F.（1989年）《生物化学杂志》264卷，11663 - 11670页]。通过定点诱变，这些精氨酸残基被逐个替换为丙氨酸、赖氨酸或半胱氨酸。没有一个突变体显示出蛋白质表达水平有显著改变。用丙氨酸、半胱氨酸或赖氨酸替换Arg31、Arg67、Arg71和Arg238所得到的突变体保留了与野生型相当的四环素抗性水平。其中，只有Arg238→Ala突变体在翻转膜囊泡中显示出非常低的转运活性，这可能是由于突变蛋白的不稳定性。用丙氨酸或半胱氨酸替换Arg70和Arg127导致耐药性急剧下降且转运活性几乎完全丧失，而用赖氨酸替换的突变体保留了显著的抗性和转运活性，这表明这些位置带正电荷的侧链赋予了转运功能。另一方面，Arg101的丙氨酸、半胱氨酸或赖氨酸替换突变体均未表现出任何耐药性或转运活性。至于半胱氨酸替换突变体的反应性，只有两个（Arg71→Cys和Arg101→Cys）不与N - 乙基马来酰亚胺（NEM）反应，其他五个突变体反应性高或中等。围绕Arg101的半胱氨酸扫描突变体与NEM的反应性表明Arg101位于跨膜螺旋IV中。Arg101不太可能通过与跨膜酸性残基形成盐桥来赋予蛋白质折叠功能，因为涉及Arg101→Ala以及三个跨膜酸性残基（Asp15、Asp84和Asp285）之一被替换的双突变体均未显示出任何四环素抗性或转运活性的恢复。四环素对[¹⁴C]NEM与组合突变体S65C/R101A和L97C/R101A结合的影响表明，Arg101可能导致TetA(B)假定出口门的底物诱导构象变化。
