Lenoir Guillaume, Jaxel Christine, Picard Martin, le Maire Marc, Champeil Philippe, Falson Pierre
Unité de Recherche Associée 2096, CNRS, and Service de Biophysique des Fonctions Membranaires, CEA, DBJC, CEA Saclay, 91191 Gif-sur-Yvette, France.
Biochemistry. 2006 Apr 25;45(16):5261-70. doi: 10.1021/bi0522091.
By analyzing, after expression in yeast and purification, the intrinsic fluorescence properties of point mutants of rabbit Ca(2+)-ATPase (SERCA1a) with alterations to amino acid residues in Ca(2+)-binding site I (E(771)), site II (E(309)), in both sites (D(800)), or in the nucleotide-binding domain (W(552)), we were able to follow the conformational changes associated with various steps in the ATPase catalytic cycle. Whereas Ca(2+) binding to purified wild-type (WT) ATPase in the absence of ATP leads to the rise in Trp fluorescence expected for the so-called E2 --> E1Ca(2) transition, the Ca(2+)-induced fluorescence rise is dramatically reduced for the E(309)Q mutant. As this purified E(309)Q mutant retains the ability to bind Ca(2+) at site I (but not at site II), we tentatively conclude that the protein reorganization induced by Ca(2+) binding at site II makes the major contribution to the overall Trp fluorescence changes observed upon Ca(2+) binding to both sites. Judging from the fluorescence response of W(552)F, similar to that of WT, these changes appear to be primarily due to membranous tryptophans, not to W(552). The same holds for the fluorescence rise observed upon phosphorylation from P(i) (the so-called E2 --> E2P transition). As for WT ATPase, Mg(2+) binding in the absence of Ca(2+) affects the fluorescence of the E(309)Q mutant, suggesting that this Mg(2+)-dependent fluorescence rise does not reflect binding of Mg(2+) to Ca(2+) sites; instead, Mg(2+) probably binds close to the catalytic site, or perhaps near transmembrane span M3, at a location recently revealed by Fe(2+)-catalyzed oxidative cleavage. Mutation of W(552) hardly affects ATP-induced fluorescence changes in the absence of Ca(2+), which are therefore mostly due to membranous Trp residues, demonstrating long-range communication between the nucleotide-binding domain and the membranous domain.
通过分析兔钙（2 +）-ATP酶（SERCA1a）的点突变体在酵母中表达并纯化后，其钙结合位点I（E（771））、位点II（E（309））、两个位点（D（800））或核苷酸结合结构域（W（552））中的氨基酸残基发生改变后的内在荧光特性，我们能够追踪与ATP酶催化循环中各个步骤相关的构象变化。在没有ATP的情况下，钙（2 +）与纯化的野生型（WT）ATP酶结合会导致色氨酸荧光升高，这是所谓的E2→E1Ca（2）转变所预期的，而对于E（309）Q突变体，钙（2 +）诱导的荧光升高则显著降低。由于这种纯化的E（309）Q突变体保留了在位点I结合钙（2 +）的能力（但在位点II不能），我们初步得出结论，在位点II结合钙（2 +）所诱导的蛋白质重组对钙（2 +）结合到两个位点时观察到的总体色氨酸荧光变化起主要作用。从W（552）F的荧光响应来看，与WT相似，这些变化似乎主要是由于膜上的色氨酸，而不是W（552）。从无机磷酸（P（i））磷酸化时观察到的荧光升高情况也是如此（所谓的E2→E2P转变）。对于WT ATP酶，在没有钙（2 +）的情况下镁（2 +）结合会影响E（309）Q突变体的荧光，这表明这种依赖镁（2 +）的荧光升高并不反映镁（2 +）与钙（2 +）位点的结合；相反，镁（2 +）可能结合在靠近催化位点的位置，或者可能靠近跨膜片段M3，处于最近通过铁（2 +）催化的氧化裂解所揭示的位置。在没有钙（2 +）的情况下，W（552）的突变几乎不影响ATP诱导的荧光变化，因此这些变化主要是由于膜上的色氨酸残基，这表明核苷酸结合结构域与膜结构域之间存在长程通讯。
