Department of Biomedical Engineering, The University of Alabama at Birmingham,Birmingham, Alabama 35294, USA.
Biochemistry. 2010 May 4;49(17):3685-94. doi: 10.1021/bi902067f.
Thrombospondin-1 (TSP1) binding to calreticulin (CRT) on the cell surface stimulates association of CRT with LDL receptor-related protein (LRP1) to signal focal adhesion disassembly and engagement of cellular activities. The structural basis for this phenomenon is unknown. We studied the binding thermodynamics of the TSP1-CRT complex and the conformational changes in CRT induced by binding to TSP1 with combined binding free energy analysis, molecular dynamics simulation, and anisotropic network model restrained molecular dynamics simulation. Results showed that mutations of Lys 24 and Lys 32 in TSP1 to Ala and of amino acids 24-26 and 32-34 in CRT to Ala significantly weakened the binding of TSP1 and CRT, which is consistent with experimental results. Upon validation of the calculated binding affinity changes of the TSP1-CRT complex by mutations in key residues in TSP1 and CRT with the experimental results, we performed conformational analyses to understand the role of TSP1 binding to CRT in the induction of conformational changes in CRT. Conformational analyses showed that TSP1 binding to CRT resulted in a more "open" conformation and a significant rotational change for the CRT N-domain with respect to the CRT P-domain, which could expose the potential binding site(s) in CRT for binding to LRP1 to signal focal adhesion disassembly. Results offer structural insight into the role of TSP1 binding to CRT in CRT-induced focal adhesion disassembly.
血小板反应蛋白-1（TSP1）与细胞表面的钙网蛋白（CRT）结合，刺激 CRT 与 LDL 受体相关蛋白（LRP1）的结合，从而发出信号，使细胞黏附斑解体并激活细胞活动。这种现象的结构基础尚不清楚。我们结合结合自由能分析、分子动力学模拟和各向异性网络模型约束分子动力学模拟研究了 TSP1-CRT 复合物的结合热力学和 CRT 与 TSP1 结合引起的构象变化。结果表明，TSP1 中的 Lys24 和 Lys32 突变为 Ala，CRT 中的氨基酸 24-26 和 32-34 突变为 Ala，显著减弱了 TSP1 和 CRT 的结合，这与实验结果一致。在通过 TSP1 和 CRT 中关键残基的突变验证 TSP1-CRT 复合物计算的结合亲和力变化与实验结果一致后，我们进行了构象分析，以了解 TSP1 与 CRT 结合在 CRT 构象变化诱导中的作用。构象分析表明，TSP1 与 CRT 结合导致 CRT 更“开放”的构象和 CRT N 结构域相对于 CRT P 结构域的显著旋转变化，这可能暴露出 CRT 中与 LRP1 结合的潜在结合位点，从而发出细胞黏附斑解体的信号。结果为 TSP1 与 CRT 结合在 CRT 诱导的细胞黏附斑解体中的作用提供了结构上的见解。
