Cytosolic Modulation of Plasma Membrane Ion Transport

描述（由申请人提供）：p型atp酶，包括高度同源的NalK泵和sarco/内质网钙泵（SERCA），是负责维持细胞功能至关重要的电解质稳态的酶。这些泵利用ATP水解产生的能量为离子上坡跨膜运动提供动力。它们的主要特征是将磷酸从ATP转移到泵中，形成高能磷酸中间体。最近SERCA在两种构象中的高分辨率结构为理解p型atp酶的结构-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的独特的磷中间形式。这项资助的重点是Na，K泵，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。钠泵的优势在于，对于SERCA， K是反阳离子，而H是反阳离子。从晶体学和生物化学的角度来看，跟踪K离子比跟踪输运的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运输位点，而质子效应除了在运输位点结合外，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：结合核苷酸的N结构域，包含接受催化磷酸化中间体（EP）形式的天冬氨酸残基的P结构域，以及对催化磷酸化和去磷酸化都很重要的A结构域。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。其他酶构象中的N和P结构域在哪里？我们的目标是跟踪这些领域的相对运动在整个运输周期使用生化和荧光为基础的分析。我们的目标是确定泵循环中N结构域的运动，确定泵循环中磷中间体的状态，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.