He Z, Dunker A K, Wesson C R, Trumble W R
Department of Bacteriology and Biochemistry, University of Idaho, Moscow 83843.
J Biol Chem. 1993 Nov 25;268(33):24635-41.
Calsequestrin is an intermediate affinity, high capacity Ca(2+)-binding protein found in the lumen of the sarcoplasmic reticulum of both skeletal and cardiac muscle cells. Previous sequence analysis suggested that calsequestrin may contain a hydrophobic binding site for the drug trifluoperazine, a site shared by the calmodulin family and shown to play a role in calmodulin/calmodulin receptor interaction. Previous studies showed that, upon Ca2+ binding, calsequestrin undergoes a conformational change, burying the trifluoperazine-binding site, folding into a more compact structure that is trypsin-resistant, and increasing the negative ellipticity of the circular dichroism spectrum. In this study, the structural and functional roles of the trifluoperazine-binding site in the Ca(2+)-induced conformational change of calsequestrin are further studied using the calmodulin antagonists trifluoperazine and melittin. If trifluoperazine or melittin is added to calsequestrin prior to Ca2+ addition, then Ca(2+)-induced folding is inhibited as determined by the changes in circular dichroism spectra and protein sensitivity to trypsin digestion. If, however, Ca2+ is added prior to trifluoperazine or melittin, calsequestrin remains resistant to trypsin digestion, just as if the calmodulin antagonists are not present, suggesting that the conformational change is not affected. Aggregates of calsequestrin that exhibit high Ca2+ binding capacity have previously been shown to occur at high Ca2+ and calsequestrin concentrations. By preventing a prerequisite folding step, trifluoperazine or melittin also prevents the Ca(2+)-induced aggregation of calsequestrin, thus decreasing the maximal Ca2+ binding by calsequestrin. These data suggest that the trifluoperazine-binding site is critically involved in the Ca(2+)-induced intramolecular folding step required for the intermolecular interactions leading to high capacity Ca(2+)-binding by calsequestrin.
肌集钙蛋白是一种中等亲和力、高容量的Ca(2+)结合蛋白，存在于骨骼肌和心肌细胞的肌浆网腔中。先前的序列分析表明，肌集钙蛋白可能含有与药物三氟拉嗪结合的疏水位点，这是钙调蛋白家族共有的位点，且已证明该位点在钙调蛋白/钙调蛋白受体相互作用中发挥作用。先前的研究表明，在结合Ca2+后，肌集钙蛋白会发生构象变化，掩埋三氟拉嗪结合位点，折叠成更紧凑的、对胰蛋白酶有抗性的结构，并增加圆二色光谱的负椭圆率。在本研究中，使用钙调蛋白拮抗剂三氟拉嗪和蜂毒素进一步研究了三氟拉嗪结合位点在Ca(2+)诱导的肌集钙蛋白构象变化中的结构和功能作用。如果在添加Ca2+之前将三氟拉嗪或蜂毒素添加到肌集钙蛋白中，那么通过圆二色光谱的变化和蛋白质对胰蛋白酶消化的敏感性测定，Ca(2+)诱导的折叠会受到抑制。然而，如果在添加三氟拉嗪或蜂毒素之前添加Ca2+，肌集钙蛋白仍然对胰蛋白酶消化有抗性，就好像不存在钙调蛋白拮抗剂一样，这表明构象变化不受影响。先前已证明，在高Ca2+和肌集钙蛋白浓度下会出现具有高Ca2+结合能力的肌集钙蛋白聚集体。通过阻止一个必要的折叠步骤，三氟拉嗪或蜂毒素也会阻止Ca(2+)诱导的肌集钙蛋白聚集，从而降低肌集钙蛋白的最大Ca2+结合量。这些数据表明，三氟拉嗪结合位点在分子间相互作用导致肌集钙蛋白高容量Ca(2+)结合所需的Ca(2+)诱导的分子内折叠步骤中起关键作用。
