Concerted conformational effects of Ca2+ and ATP are required for activation of sequential reactions in the Ca2+ ATPase (SERCA) catalytic cycle
Concerted conformational effects of Ca2+ and ATP are required for activation of sequential reactions in the Ca2+ ATPase (SERCA) catalytic cycle
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DOI：
10.1021/bi061255d
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发表时间：
2006-11-21
期刊：
影响因子：
2.9
通讯作者：
Prasad, Anand
中科院分区：
文献类型：
--
作者：
Inesi, Giuseppe;Lewis, David;Prasad, Anand
我们将溶液行为与Ca2+ atp酶（SERCA）的晶体结构联系起来。我们发现，即使在没有Ca2+和Mg2+的情况下，通过腺苷部分与N结构域的相互作用，或者通过thapsigargin （TG）稳定的封闭构象，核苷酸结合也以高亲和力发生。那么为什么Ca2+对ATP的利用至关重要呢？腺苷5'-（β， γ -亚甲基）三磷酸（AMPPCP）， Ca2+和Mg2+对蛋白质水解消化模式的影响，根据已知的晶体结构进行解释，表明atp酶头的Ca2+依赖构象是核苷酸结合诱导的进一步转变所必需的。这包括打开耳机，这反过来又允许“A”域的倾斜和“P”域的弯曲。因此，结合ATP的磷酸链获得了一个扩展的结构，允许γ -磷酸到达Asp351，形成包括Mg2+的复合物。我们通过Asp351突变证明，由于磷酸化位点的静电排斥，这种底物-酶复合物的“生产性”构象是不稳定的。然而，这种构象随后通过产生磷酸酶中间体的γ -磷酸共价接合而稳定下来。我们还证明，ADP产物与过渡态复合物保持高亲和力结合，但随着磷酸酶进一步发生构象变化（即从E1-P到E2-P转变），ADP产物会以较低的亲和力游离。最后，我们测量了低亲和力ATP与稳定的磷酸酶类似物的结合，表明ATP与磷酸酶结合以换取ADP加速了E1-P到E2-P的转变和酶的周转。
We relate solution behavior to the crystal structure of the Ca2+ ATPase ( SERCA). We find that nucleotide binding occurs with high affinity through interaction of the adenosine moiety with the N domain, even in the absence of Ca2+ and Mg2+, or to the closed conformation stabilized by thapsigargin ( TG). Why then is Ca2+ crucial for ATP utilization? The influence of adenosine 5'-(beta, gamma- methylene) triphosphate ( AMPPCP), Ca2+, and Mg2+ on proteolytic digestion patterns, interpreted in the light of known crystal structures, indicates that a Ca2+-dependent conformation of the ATPase headpiece is required for a further transition induced by nucleotide binding. This includes opening of the headpiece, which in turn allows inclination of the "A" domain and bending of the "P" domain. Thereby, the phosphate chain of bound ATP acquires an extended configuration allowing the gamma-phosphate to reach Asp351 to form a complex including Mg2+. We demonstrate by Asp351 mutation that this "productive" conformation of the substrate-enzyme complex is unstable because of electrostatic repulsion at the phosphorylation site. However, this conformation is subsequently stabilized by covalent engagement of the gamma-phosphate yielding the phosphoenzyme intermediate. We also demonstrate that the ADP product remains bound with high affinity to the transition state complex but dissociates with lower affinity as the phosphoenzyme undergoes a further conformational change ( i.e., E1-P to E2-P transition). Finally, we measured low-affinity ATP binding to stable phosphoenzyme analogues, demonstrating that the E1-P to E2-P transition and the enzyme turnover are accelerated by ATP binding to the phosphoenzyme in exchange for ADP.