采用分子动力学模拟方法探究Ca2+对VWF-A2结构域稳定性的影响

目的 探究Ca2+对VWF-A2结构域稳定性的影响。方法 A2和A2/Ca2+的晶体结构取自PDB数据库。通过恒力拉伸分子动力学模拟，比较分析Ca2+结合引起的构象变化、解折叠路径的差异以及酶切位点的暴露程度。结果 A2结构域的解折叠路径和酶切位点的暴露过程是力依赖的。Ca2+结合不影响A2结构域的前期解折叠，但由于α3β4-环链局部构象重排所致柔性降低，约束了β1-β4-β5片层的运动，导致进一步解折叠受阻而停留在中间稳态，影响酶切位点的充分暴露。结论 力可诱导A2结构域中β5片层的解折叠使酶切位点暴露，而Ca2+的结合则通过稳定疏水核心结构，阻碍酶切位点的暴露，最终降低ADAMTS13的酶切效率。研究结果有助于加深对ADAMTS13酶切VWF-A2结构域进而调控VWF止血能力过程的理解，并为相关抗血栓药物设计提供指导。
Objective To investigate the effect of calcium on the stability of VWF-A2 domain. Methods The crystal structures of A2 (not containing calcium) and A2/Ca2+ (with calcium bound) were downloaded from protein data bank. For A2 domain, the conformational changes, unfolding pathway differences and the exposure degree variance of cleavage sites caused by calcium binding were observed and analyzed by steered Molecular Dynamics simulations under constant force. Results The unfolding pathway of A2 domain and exposure process of cleavage sites were force-dependent. Calcium binding did not affect the unfolding process of A2 in the early stage. As the conformational rearrangement of α3β4-loop reduced its localized dynamic properties, the movement among β1-β4-β5 strands was restrained, which suppressed its further unfolding to stay in the intermediate steady state and delayed the cleavage-site exposure. Conclusions Stretch force could induce β5 strand of A2 unfolding and the cleavage-site exposure, while calcium binding inhibited ADAMTS13 proteolysis efficiency through stabilizing A2 hydrophobic core and covering its cleavage sites. These results way help to understand how ADAMTS13 cleavages the VWF-A2 domain and regulates the hemostatic potential of VWF, and further provide useful guidance on the design of related anti-thrombus drugs.