Nucleotide-dependent movement of the epsilon subunit between alpha and beta subunits in the Escherichia coli F1F0-type ATPase
Nucleotide-dependent movement of the epsilon subunit between alpha and beta subunits in the Escherichia coli F1F0-type ATPase
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DOI：
10.1074/jbc.271.23.13888
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发表时间：
1996-06-07
影响因子：
4.8
通讯作者：
Capaldi, RA
中科院分区：
文献类型：
--
作者：
Aggeler, R;Capaldi, RA
产生了ECF(1)-ATPase的突变体，在以下一个或多个位置含有半胱氨酸残基：Alpha Ser-411、βGlu-381和epsilon Ser-108，之后通过CuCl2诱导的氧化可以在α和epsilon、β和epsilon、α和Gamma、β和Gamma(内源性Cys-87)以及α和β之间高效地建立二硫键桥。所有这些交联物都会抑制ATP水解酶的活性。在两个双突变体中，epsilon Ser-108上的半胱氨酸与β亚基的脱落区(Glu-381)或α区的同源区(Ser-411)一起存在，与epsilon亚基的交联形成对核苷酸有明显的依赖性。在βE381C/epsilon S108C中，β-epsilon交联物优先与Mg2+和ADP+P-I(添加MgCl2+ATP)，而在αS411C/epsilon S108C突变体中，在Mg2+ATP态(添加MgCl2+5‘-腺苷-β，γ-亚胺二磷酸)中，α-epsilon交联物强烈优先。在三个突变体αS411C/βE381C/epsilon S108C中，epsilon亚基与镁离子-ADP中的β亚基和镁离子-三磷酸腺苷中的α亚基结合，表明该亚基发生了显著的移动。在Mg(2+)-ADP中，γ与β亚基的交联率比在Mg(2+)-ADP中高，并且在可能的情况下，即在三重突变体中，总是优先选择与β亚基的相互作用而不是α亚基。
Mutants of ECF(1)-ATPase were generated, containing cysteine residues in one or more of the following positions: alpha Ser-411, beta Glu-381, and epsilon Ser-108, after which disulfide bridges could be created by CuCl2 induced oxidation in high yield between alpha and epsilon, beta and epsilon, alpha and gamma, beta and gamma (endogenous Cys-87), and alpha and beta. All of these cross-links lead to inhibition of ATP hydrolysis activity. In the two double mutants, containing a cysteine in epsilon Ser-108 along with either the DELSEED region of beta (Glu-381) or the homologous region in alpha (Ser-411), there was a clear nucleotide dependence of the cross-link formation with the epsilon subunit. In beta E381C/epsilon S108C the beta-epsilon cross-link was obtained preferentially with Mg2+ and ADP + P-i (addition of MgCl2 + ATP) was present, while the alpha-epsilon cross-link product was strongly favored in the alpha S411C/epsilon S108C mutant in the Mg2+ ATP state (addition of MgCl2 + 5'-adenylyl-beta,gamma-imidodiphosphate). In the triple mutant alpha S411C/beta E381C/epsilon S108C, the epsilon subunit bound to the beta subunit in Mg2+-ADP and to the alpha subunit in Mg2+-ATP, indicating a significant movement of this subunit. The gamma cross-linked to the beta subunit in higher yield in Mg2+-ATP than in Mg(2+)-ADP, and when possible, i.e. in the triple mutant, always preferred the interaction with the beta over the alpha subunit.