Tang C, Capaldi R A
Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229, USA.
J Biol Chem. 1996 Feb 9;271(6):3018-24. doi: 10.1074/jbc.271.6.3018.
The interaction faces of the gamma and epsilon subunits in the Escherichia coli F1-ATPase have been explored by a combination of cross-linking and chemical modification experiments using several mutant epsilon subunits as follows: epsilonS10C, epsilonH38C, epsilonT43C, epsilonS65C, epsilonS108C, and epsilonM138C, along with a mutant of the gamma subunit, gammaT106C. The replacement of Ser-10 by a Cys or Met-138 by a Cys reduced the inhibition of ECF1 by the epsilon subunit, while the mutation S65C increased this inhibitory effect. Modification of the Cys at position 10 with N-ethylmaleimide or fluoroscein maleimide further reduced the binding affinity of, and the maximal inhibition by, the epsilon subunit. Similar chemical modification of the Cys at position 43 of the epsilon subunit (in the mutant epsilonT43C) and a Cys at position 106 of the gamma subunit (gammaT106C) also affected the inhibition of ECF1 by the epsilon subunit. The various epsilon subunit mutants were reacted with TFPAM3, and the site(s) of cross-linking within the ECF1 complex was determined. Previous studies have shown cross-linking from the Cys at positions 10 and 38 with the gamma subunit and from a Cys at position 108 to an alpha subunit (Aggeler, R., Chicas-Cruz, K., Cai, S. X., Keana, J. F. W., and Capaldi, R. A. (1992) Biochemistry 31, 2956-2961; Aggeler, R., Weinreich, F., and Capaldi, R. A. (1995) Biochim. Biophys. Acta 1230, 62-68). Here, cross-linking was found from a Cys at position 43 to the gamma subunit and from the Cys at position 138 to a beta subunit. The site of cross-linking from Cys-10 of epsilon to the gamma subunit was localized by peptide mapping to a region of the gamma subunit between residues 222 and 242. Cross-linking from a Cys at position 38 and at position 43 was with the C-terminal part of the gamma subunit, between residues 202 and 286. ECF1 treated with trypsin at pH 7.0 still binds purified epsilon subunit, while enzyme treated with the protease at pH 8.0 does not. This identifies sites around residue 70 and/or between 202 and 212 of the gamma subunit as involved in epsilon subunit binding.
通过交联和化学修饰实验相结合的方法，利用几种突变的ε亚基，即εS10C、εH38C、εT43C、εS65C、εS108C和εM138C，以及γ亚基的突变体γT106C，对大肠杆菌F1 - ATP酶中γ和ε亚基的相互作用界面进行了研究。将Ser - 10替换为Cys或Met - 138替换为Cys会降低ε亚基对ECF1的抑制作用，而S65C突变则增强了这种抑制作用。用N - 乙基马来酰亚胺或荧光素马来酰亚胺对第10位的Cys进行修饰，进一步降低了ε亚基的结合亲和力和最大抑制作用。对ε亚基第43位的Cys（在突变体εT43C中）和γ亚基第106位的Cys（γT106C）进行类似的化学修饰，也影响了ε亚基对ECF1的抑制作用。使各种ε亚基突变体与TFPAM3反应，并确定了ECF1复合物内的交联位点。先前的研究表明，第10位和第38位的Cys与γ亚基交联，第108位的Cys与α亚基交联（Aggeler, R., Chicas - Cruz, K., Cai, S. X., Keana, J. F. W., and Capaldi, R. A. (1992) Biochemistry 31, 2956 - 2961; Aggeler, R., Weinreich, F., and Capaldi, R. A. (1995) Biochim. Biophys. Acta 1230, 62 - 68）。在此，发现第43位的Cys与γ亚基交联，第138位的Cys与β亚基交联。通过肽图谱分析将ε的Cys - 10与γ亚基的交联位点定位到γ亚基222至242位残基之间的区域。第38位和第43位的Cys与γ亚基C末端部分（202至286位残基之间）交联。在pH 7.0用胰蛋白酶处理的ECF1仍能结合纯化的ε亚基，而在pH 8.0用蛋白酶处理的酶则不能。这表明γ亚基70位残基周围和/或202至212位之间的位点参与了ε亚基的结合。
