Cytosolic Modulation of Plasma Membrane Ion Transport

描述(申请人提供)：P型ATPase，包括高度同源的NalK泵和肌浆/内质网钙质泵(SERCA)，是负责维持电解质平衡的酶，对细胞功能至关重要。这些泵使用来自ATP水解的能量来驱动离子的上行跨膜运动。它们的主要特征是将磷酸盐从三磷酸腺苷转移到泵中，形成高能的磷中间体。SERCA最近在两种构象中的高分辨结构为理解P型ATPase的结构与功能关系提供了重要的新信息。然而，这两种结构都不包含这类离子泵独特的磷中间形式。这笔拨款集中在Na，K泵上，因为它为阐明离子运动和ATP水解之间的分子耦合提供了重要的优势。钠泵的优势是K是SERCA的反阳离子对H。无论是在结晶学上还是在生物化学上，跟随K离子都比跟随运输的H离子容易得多。也就是说，几乎所有的K效应都发生在阳离子转运部位，而质子效应除了在转运部位结合外，还可能是几种氨基酸滴定的结果。在没有质子的情况下研究SERCA是不可能的，而在没有K的情况下人们可以很容易地研究Na泵。P型泵有三个主要的细胞质结构域：N结构域结合核苷酸，P结构域包含天冬氨酸残基接受形成催化磷中间体(EP)，以及A结构域对催化磷酸化和去磷酸化都是重要的。当Na泵在Asp369上被催化磷酸化时，N和P结构域必须紧密接触，才能使Asp369攻击ATP上的末端磷酸。在其他酶的构象过程中，N和P结构域在哪里？我们的目标是使用基于生化和荧光的分析方法来跟踪这些结构域在整个运输周期中的相对运动。我们的目的是确定N结构域在泵周期中的运动，确定磷中间体在泵周期中的状态，再次关注细胞外结合和释放步骤，并检查N结构域配体对跨膜结构域状态的影响。

DESCRIPTION (provided by applicant): P-type ATPases, which include the highly homologous NalK pump and sarco/endoplasmic reticulum caldum pump (SERCA), are the enzymes responsible for maintaining electrolyte homeostasis critical for cell function. These pumps use the energy from ATP hydrolysis to power the uphill transmembrane movement of ions. Their defining feature is the transfer of a phosphate from ATP to the pump to form a high-energy phosphointermediate. The recent high-resolution structure of SERCA in two conformations provides important new information for understanding the structure-function relationship of P-type ATPases. However, neither structure contains the unique phosphointermediate form of this class of ion pumps. This grant focuses on the Na,K pump because it offers an important advantage for elucidating the molecular coupling between ion movements and ATP hydrolysis. The Na pump advantage is that K is the counter cation vs. H for SERCA. Both crystallographically and biochemically, following the K ion is much easier than following the transported H. "That is, almost all K effects occur at the cation transport site, whereas proton effects can be the result of several amino acid titrations in addition to binding at the transport sites. It is impossible to study SERCA in the absence of protons, whereas one can easily study the Na pump in the absence of K. P-type pumps have three major cytoplasmic domains: the N domain which binds nucleotides, the P domain which contains the aspartic acid residue that accepts the forms the catalytic phosphointermediate (EP), and the A domain which is important for both catalytic phosphorylation and dephosphorylation. When the Na pump is catalytically phosphorylated on Asp369, the N and P domains must necessarily be in close contact to allow Asp369 to attack the terminal phosphate on ATP. Where are the N and P domains during other enzyme conformations? Our aim is to track the relative motion of these domains throughout the transport cycle using biochemical and fluorescence-based assays. Our aims are to determine the movements of the N domain during the pump cycle, determine the status of the phosphointermediate during the pump cycle, again focusing on the extracellular binding and release steps, and to examine the influence of N domain ligands on the status of the transmembrane domains.