Teriete Peter, Franzin Carla M, Choi Jungyuen, Marassi Francesca M
Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, California 92037, USA.
Biochemistry. 2007 Jun 12;46(23):6774-83. doi: 10.1021/bi700391b. Epub 2007 May 19.
FXYD1 is a major regulatory subunit of the Na,K-ATPase and the principal substrate of hormone-regulated phosphorylation by c-AMP dependent protein kinases A and C in heart and skeletal muscle sarcolemma. It is a member of an evolutionarily conserved family of membrane proteins that regulate the function of the enzyme complex in a tissue-specific and physiological-state-specific manner. Here, we present the three-dimensional structure of FXYD1 determined in micelles by NMR spectroscopy. Structure determination was made possible by measuring residual dipolar couplings in weakly oriented micelle samples of the protein. This allowed us to obtain the relative orientations of the helical segments and information about the protein dynamics. The structural analysis was further facilitated by the inclusion of distance restraints, obtained from paramagnetic spin label relaxation enhancements, and by refinement with a micelle depth restraint, derived from paramagnetic Mn line broadening effects. The structure of FXYD1 provides the foundation for understanding its intra-membrane association with the Na,K-ATPase alpha subunit and suggests a mechanism whereby the phosphorylation of conserved Ser residues, by protein kinases A and C, could induce a conformational change in the cytoplasmic domain of the protein to modulate its interaction with the alpha subunit.
FXYD1是钠钾ATP酶的主要调节亚基，也是心脏和骨骼肌肌膜中由c - AMP依赖性蛋白激酶A和C进行激素调节磷酸化的主要底物。它是一个进化上保守的膜蛋白家族成员，以组织特异性和生理状态特异性的方式调节酶复合物的功能。在此，我们展示了通过核磁共振光谱法在胶束中测定的FXYD1的三维结构。通过测量蛋白质弱取向胶束样品中的残余偶极耦合实现了结构测定。这使我们能够获得螺旋片段的相对取向以及有关蛋白质动力学的信息。通过纳入从顺磁自旋标记弛豫增强获得的距离限制，以及通过使用从顺磁锰线展宽效应推导的胶束深度限制进行精修，进一步促进了结构分析。FXYD1的结构为理解其与钠钾ATP酶α亚基的膜内缔合提供了基础，并提示了一种机制，即蛋白激酶A和C对保守丝氨酸残基的磷酸化可诱导该蛋白质胞质结构域的构象变化，从而调节其与α亚基的相互作用。
