Metalloprotease ADAMTS13 specifically cleaves VWF (von Willebrand Factor) to prevent excessive platelet aggregation and thrombus formation at the sites of vascular injury. To avoid non-specific cleavage, ADAMTS13 has the auto-inhibition effect in which the Spacer domain in N-terminal interacts with the CUB1 domain in C-terminal, resulting in decreased proteolytic activity. Previous studies reported that exosite-3 in the Spacer domain was a key binding site in the Spacer-CUB1 interaction. When exosite-3 was mutated (R660K/F592Y/R568K/Y661F/Y665F, GOF), the auto-inhibition of ADAMTS13 was disrupted and the enzymatic activity was markedly increased. However, the characteristics of the Spacer-CUB1 interaction is not fully understood. Here, we constructed the model of Spacer-CUB1 complex by homologous modeling and molecular docking to characterize the Spacer-CUB1 binding and predict key amino acid residues via molecular dynamics simulation. Our data showed that G607-S610 was a non-reported potential binding site in the Spacer domain; GOF mutation attenuated the formation of hydrogen bond between exosite-3 and the CUB1 domain; Residues E1231, R1251, L1258, D1259 and T1261 in the CUB1 domain might play an important role in the Spacer-CUB1 interaction. Our study advances the understanding of the structural basis of the auto-inhibition of ADAMTS13 and provides information about the key residues in the binding interface.

金属蛋白酶 ADAMTS13 特异性裂解 VWF（von Willebrand 因子）以防止血管损伤部位的过度血小板聚集和血栓形成。为了避免非特异性切割，ADAMTS13 具有自动抑制作用，其中 N 端的 Spacer 域与 C 端的 CUB1 域相互作用，导致蛋白水解活性降低。先前的研究报告称，Spacer 域中的 exosite-3 是 Spacer-CUB1 相互作用中的关键结合位点。当 exosite-3 突变（R660K/F592Y/R568K/Y661F/Y665F，GOF）时，ADAMTS13 的自动抑制被破坏，酶活性显着增加。然而，尚未完全了解 Spacer-CUB1 相互作用的特征。这里，我们通过同源建模和分子对接构建了 Spacer-CUB1 复合物的模型，以表征 Spacer-CUB1 结合并通过分子动力学模拟预测关键氨基酸残基。我们的数据显示 G607-S610 是 Spacer 域中未报告的潜在结合位点；GOF突变减弱了exosite-3和CUB1结构域之间氢键的形成；CUB1 结构域中的 E1231、R1251、L1258、D1259 和 T1261 残基可能在 Spacer-CUB1 相互作用中起重要作用。我们的研究促进了对 ADAMTS13 自动抑制的结构基础的理解，并提供了有关结合界面中关键残基的信息。我们的数据显示 G607-S610 是 Spacer 域中未报告的潜在结合位点；GOF突变减弱了exosite-3和CUB1结构域之间氢键的形成；CUB1 结构域中的 E1231、R1251、L1258、D1259 和 T1261 残基可能在 Spacer-CUB1 相互作用中起重要作用。我们的研究促进了对 ADAMTS13 自动抑制的结构基础的理解，并提供了有关结合界面中关键残基的信息。我们的数据显示 G607-S610 是 Spacer 域中未报告的潜在结合位点；GOF突变减弱了exosite-3和CUB1结构域之间氢键的形成；CUB1 结构域中的 E1231、R1251、L1258、D1259 和 T1261 残基可能在 Spacer-CUB1 相互作用中起重要作用。我们的研究促进了对 ADAMTS13 自动抑制的结构基础的理解，并提供了有关结合界面中关键残基的信息。