Saba R I, Goormaghtigh E, Ruysschaert J M, Herchuelz A
Laboratoire de Pharmacodynamie et de Thérapeutique, Faculté de Médecine, Bât. GE, 808 route de Lennik, B-1070, Brussels, Belgium.
Biochemistry. 2001 Mar 20;40(11):3324-32. doi: 10.1021/bi0010672.
The 120-kDa Na+/Ca2+ exchanger was purified and reconstituted into lipid vesicles. The secondary structure composition of the exchanger was 39% alpha-helices, 20% beta-sheets, 25% beta-turns, and 16% random coils, as analyzed by Fourier transform infrared attenuated total reflection spectroscopy. The secondary structure composition of the COOH-terminal portion of the protein was compatible with a topology model containing 4-6 transmembrane segments. Furthermore, the secondary structure of the NH2-terminal portion of the cytoplasmic loop was analyzed and found to be different from that of the COOH-terminal portion. Ca2+ and/or the exchange inhibitory peptide (XIP) failed to affect the secondary structure of the 120-kDa protein. Tertiary structure modifications induced by Ca2+ and XIP were analyzed by monitoring the hydrogen/deuterium exchange rate for the reconstituted exchanger. In the absence of ligand, 51% of the protein was accessible to solvent. Ca2+ decreased accessibility to 40%, implicating the shielding of at least 103 amino acids. When both Ca2+ and XIP were added, accessibility increased to 66%. No modification was obtained when XIP was added alone. Likewise, in the presence of Ca2+, XIP failed to modify the tertiary structure of the 70-kDa protein, suggesting that XIP acts at the level of the COOH-terminal portion of the intracellular loop. The present data describe, for the first time, conformational changes of the Na+/Ca2+ exchanger induced by Ca2+ and XIP, compatible with an interaction model where regulatory Ca2+ and inhibitory XIP bind to distinct sites, and where XIP binding requires the presence of Ca2+.
120 kDa的钠钙交换蛋白被纯化并重组到脂质小泡中。通过傅里叶变换红外衰减全反射光谱分析，该交换蛋白的二级结构组成为39%的α-螺旋、20%的β-折叠、25%的β-转角和16%的无规卷曲。该蛋白COOH末端部分的二级结构组成与包含4 - 6个跨膜片段的拓扑模型相符。此外，对胞质环NH2末端部分的二级结构进行分析，发现其与COOH末端部分不同。钙离子和/或交换抑制肽（XIP）未能影响120 kDa蛋白的二级结构。通过监测重组交换蛋白的氢/氘交换率，分析了钙离子和XIP诱导的三级结构修饰。在没有配体的情况下，51%的蛋白可与溶剂接触。钙离子使其可及性降低至40%，这意味着至少103个氨基酸被屏蔽。当同时加入钙离子和XIP时，可及性增加到66%。单独加入XIP时未获得修饰。同样，在有钙离子存在的情况下，XIP未能改变70 kDa蛋白的三级结构，这表明XIP作用于细胞内环的COOH末端部分水平。本数据首次描述了由钙离子和XIP诱导的钠钙交换蛋白的构象变化，这与一种相互作用模型相符，在该模型中，调节性钙离子和抑制性XIP结合到不同位点，且XIP的结合需要钙离子的存在。
