Banci Lucia, Bertini Ivano, Cantini Francesca, Migliardi Manuele, Natile Giovanni, Nushi Fiorentin, Rosato Antonio
Magnetic Resonance Center (CERM)-University of Florence, Via L. Sacconi 6, 50019 Sesto Fiorentino, Italy.
Biochemistry. 2009 Aug 25;48(33):7849-55. doi: 10.1021/bi901003k.
ATP7A and ATP7B are two human P(1B)-type ATPases that have a crucial role in maintaining copper(I) homeostasis. Among the various domains of these enzymes, one, called the Actuator or A-domain, has a regulatory function and is required for the phosphatase step of the catalytic cycle (dephosphorylation of the intermediate formed during ATP hydrolysis). Here we report the solution structures of the A-domain of both proteins, solved by heteronuclear NMR spectroscopy and a characterization of the dynamics of the A-domain of ATP7A. We observed that the catalytically important TGE loop protrudes from the structure ready for interaction with the phosphorylated site in the ATP-binding domain. The loop is rigid, suggesting that the catalytic step does not require substantial structural flexibility or rearrangements. The present structures were useful to rationalize the molecular effects of disease-causing mutations. In particular, it can be concluded that mutations occurring in the A-domain either destabilize the fold of the domain (such as Gly860Val in ATP7A) or affect the network of communication within the domain (such as Leu873Arg in ATP7A) or with the other domains of the enzyme (such as Gly853Arg in ATP7A).
ATP7A和ATP7B是两种人类P(1B)型ATP酶，在维持铜(I)稳态中起关键作用。在这些酶的各个结构域中，有一个被称为“促动器”或“A结构域”的结构域具有调节功能，是催化循环中磷酸酶步骤（ATP水解过程中形成的中间体的去磷酸化）所必需的。在此，我们报告了通过异核核磁共振光谱法解析的这两种蛋白质A结构域的溶液结构，并对ATP7A的A结构域动力学进行了表征。我们观察到，具有催化重要性的TGE环从结构中突出，准备与ATP结合结构域中的磷酸化位点相互作用。该环是刚性的，这表明催化步骤不需要大量的结构灵活性或重排。目前的结构有助于阐明致病突变的分子效应。特别是，可以得出结论，发生在A结构域中的突变要么会使该结构域的折叠不稳定（如ATP7A中的Gly860Val），要么会影响该结构域内（如ATP7A中的Leu873Arg）或与酶的其他结构域（如ATP7A中的Gly853Arg）之间的通讯网络。
