Kirby Tara L, Karim Christine B, Thomas David D
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Biochemistry. 2004 May 18;43(19):5842-52. doi: 10.1021/bi035749b.
We used EPR spectroscopy to probe directly the interaction between phospholamban (PLB) and its regulatory target, the sarcoplasmic reticulum Ca-ATPase (SERCA). Synthetic monomeric PLB was prepared with a single cytoplasmic cysteine at residue 11, which was then spin labeled. PLB was reconstituted into membranes in the presence or absence of SERCA, and spin label mobility and accessibility were measured. The spin label was quite rotationally mobile in the absence of SERCA, but became more restricted in the presence of SERCA. SERCA also decreased the dependence of spin label mobility on PLB concentration in the membrane, indicating that SERCA reduces PLB-PLB interactions. The spin label MTSSL, attached to Cys11 on PLB by a disulfide bond, was stable at position 11 in the absence of SERCA. In the presence of SERCA, the spin label was released and a covalent bond was formed between PLB and SERCA, indicating direct interaction of one or more SERCA cysteine residues with Cys11 on PLB. The accessibility of the PLB-bound spin label IPSL to paramagnetic agents, localized in different phases of the membrane, indicates that SERCA greatly reduces the level of interaction of the spin label with the membrane surface. We propose that the cytoplasmic domain of PLB associates with the lipid surface, and that association with SERCA induces a major conformational change in PLB in which the cytoplasmic domain is drawn away from the lipid surface by SERCA.
我们使用电子顺磁共振光谱法直接探测受磷蛋白（PLB）及其调节靶点——肌浆网Ca-ATP酶（SERCA）之间的相互作用。合成的单体PLB在第11位残基处含有一个单一的胞质半胱氨酸，然后对其进行自旋标记。在有或没有SERCA的情况下，将PLB重组到膜中，并测量自旋标记的流动性和可及性。在没有SERCA的情况下，自旋标记具有相当大的旋转流动性，但在有SERCA的情况下，其流动性受到更大限制。SERCA还降低了自旋标记流动性对膜中PLB浓度的依赖性，表明SERCA减少了PLB-PLB相互作用。通过二硫键连接到PLB上第11位半胱氨酸的自旋标记MTSSL，在没有SERCA的情况下在第11位是稳定的。在有SERCA的情况下，自旋标记被释放，并且在PLB和SERCA之间形成了共价键，表明一个或多个SERCA半胱氨酸残基与PLB上的第11位半胱氨酸直接相互作用。与定位在膜不同相中的顺磁性试剂结合的PLB自旋标记IPSL的可及性表明，SERCA大大降低了自旋标记与膜表面的相互作用水平。我们提出，PLB的胞质结构域与脂质表面结合，并且与SERCA的结合会诱导PLB发生重大构象变化，其中胞质结构域被SERCA从脂质表面拉开。
