From the Krannert Institute of Cardiology and the Departments of Medicine and.
Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202.
J Biol Chem. 2013 Oct 18;288(42):30181-30191. doi: 10.1074/jbc.M113.501585. Epub 2013 Aug 31.
P-type ATPases are a large family of enzymes that actively transport ions across biological membranes by interconverting between high (E1) and low (E2) ion-affinity states; these transmembrane transporters carry out critical processes in nearly all forms of life. In striated muscle, the archetype P-type ATPase, SERCA (sarco(endo)plasmic reticulum Ca(2+)-ATPase), pumps contractile-dependent Ca(2+) ions into the lumen of sarcoplasmic reticulum, which initiates myocyte relaxation and refills the sarcoplasmic reticulum in preparation for the next contraction. In cardiac muscle, SERCA is regulated by phospholamban (PLB), a small inhibitory phosphoprotein that decreases the Ca(2+) affinity of SERCA and attenuates contractile strength. cAMP-dependent phosphorylation of PLB reverses Ca(2+)-ATPase inhibition with powerful contractile effects. Here we present the long sought crystal structure of the PLB-SERCA complex at 2.8-Å resolution. The structure was solved in the absence of Ca(2+) in a novel detergent system employing alkyl mannosides. The structure shows PLB bound to a previously undescribed conformation of SERCA in which the Ca(2+) binding sites are collapsed and devoid of divalent cations (E2-PLB). This new structure represents one of the key unsolved conformational states of SERCA and provides a structural explanation for how dephosphorylated PLB decreases Ca(2+) affinity and depresses cardiac contractility.
P 型 ATP 酶是一个庞大的酶家族，通过在高（E1）和低（E2）离子亲和力状态之间的相互转换，主动将离子跨生物膜运输；这些跨膜转运蛋白在几乎所有形式的生命中都执行着关键的过程。在横纹肌中，原型 P 型 ATP 酶，SERCA（肌质网 Ca2+-ATP 酶），将收缩依赖性 Ca2+离子泵入肌质网腔，引发肌细胞松弛，并为下一次收缩重新填充肌质网。在心肌中，SERCA 受磷蛋白（PLB）调节，PLB 是一种小的抑制性磷酸蛋白，降低 SERCA 的 Ca2+亲和力并减弱收缩强度。PLB 的 cAMP 依赖性磷酸化逆转了 Ca2+-ATP 酶的抑制作用，具有强大的收缩效果。在这里，我们呈现了长期以来备受追捧的 PLB-SERCA 复合物的晶体结构，分辨率为 2.8-Å。该结构是在没有 Ca2+的情况下，在一种新型去污剂系统中解决的，该系统使用烷基甘露糖苷。该结构显示 PLB 结合到 SERCA 的一种以前未描述的构象中，其中 Ca2+结合位点塌陷，没有二价阳离子（E2-PLB）。这个新结构代表了 SERCA 的一个关键未解决构象状态之一，并为去磷酸化的 PLB 如何降低 Ca2+亲和力和抑制心脏收缩力提供了结构解释。
