Yu Shanshan, Liu Wang, Fang Jinhua, Shi Xiaozhong, Wu Jianhua, Fang Ying, Lin Jiangguo
Institute of Biomechanics, School of Biosciences and Bioengineering, South China University of Technology, Guangzhou, 510006 China.
J Biol Eng. 2019 Jan 22;13:9. doi: 10.1186/s13036-018-0102-y. eCollection 2019.
ADAMTS13 (A disintegrin and metalloprotease with a thrombospondin type 1 motif 13) cleaves Von Willebrand factor (VWF) to regulate its size, thereby preventing aberrant platelet aggregation and thrombus. Deficiency of ADAMTS13 caused by either genetic mutations or by inhibitory autoantibodies against ADAMTS13 leads to thrombotic thrombocytopenic purpura (TTP). Recently, ADAMTS13 was reported to adopt a "closed" conformation with lower activity and an "open" one resulting from the engagements of VWF D4-CK domains or antibodies to the distal domains of ADAMTS13, or mutations in its spacer domain. These engagements or mutations increase ADAMTS13 activity by ~ 2.5-fold. However, it is less known whether the conformation of ADAMTS13 is dynamic or stable.
Wild type ADAMTS13 (WT-ADAMTS13) and the gain-of-function variant (GOF-ADAMTS13) with five mutations (R568K / F592Y / R660K / Y661F / Y665F) in spacer domain were imaged by atomic force microscopy (AFM) at pH 6 and pH 7.5. The data revealed that at both pH 6 and pH 7.5, WT-ADAMTS13 adopted two distinct conformational states (state I and state II), while an additional state (state III) was observed in GOF-ADAMTS13. In the present study, we propose that state I is the "closed" conformation, state III is the "open" one, and state II is an intermediate one. Comparing to pH 7.5, the percentages of state II of WT-ADAMTS13 and state III of GOF-ADAMTS13 increased at pH 6, with the decrease in the state I for WT-ADAMTS13 and state I and state II for GOF-ADAMTS13, suggesting lower pH extended the conformation of ADAMTS13.
Both WT- and GOF-ADAMTS13 exist multiple conformational states and lower pH might alter the tertiary structure and/or disrupt the intra-domain interactions, increasing the flexibility of ADAMTS13 molecules.
ADAMTS13（含Ⅰ型血小板反应蛋白基序的解聚素样金属蛋白酶13）可裂解血管性血友病因子（VWF）以调节其大小，从而防止异常的血小板聚集和血栓形成。由基因突变或针对ADAMTS13的抑制性自身抗体引起的ADAMTS13缺乏会导致血栓性血小板减少性紫癜（TTP）。最近有报道称，ADAMTS13会呈现活性较低的“闭合”构象以及因VWF D4-CK结构域与ADAMTS13远端结构域结合、抗体结合或其间隔区突变而产生的“开放”构象。这些结合或突变使ADAMTS13的活性增加约2.5倍。然而，对于ADAMTS13的构象是动态的还是稳定的，人们了解较少。
在pH 6和pH 7.5条件下，通过原子力显微镜（AFM）对野生型ADAMTS13（WT-ADAMTS13）和间隔区有五个突变（R568K / F592Y / R660K / Y661F / Y665F）的功能获得性变体（GOF-ADAMTS13）进行成像。数据显示，在pH 6和pH 7.5时，WT-ADAMTS13均呈现两种不同的构象状态（状态Ⅰ和状态Ⅱ），而在GOF-ADAMTS13中观察到了另外一种状态（状态Ⅲ）。在本研究中，我们提出状态Ⅰ是“闭合”构象，状态Ⅲ是“开放”构象，状态Ⅱ是中间构象。与pH 7.5相比，WT-ADAMTS13的状态Ⅱ和GOF-ADAMTS13的状态Ⅲ在pH 6时的百分比增加，而WT-ADAMTS13的状态Ⅰ以及GOF-ADAMTS13的状态Ⅰ和状态Ⅱ的百分比降低，这表明较低的pH值扩展了ADAMTS13的构象。
WT-ADAMTS13和GOF-ADAMTS13均存在多种构象状态，较低的pH值可能会改变三级结构和/或破坏结构域内相互作用，增加ADAMTS13分子的灵活性。
