AFM Imaging Reveals Multiple Conformational States of ADAMTS13
AFM Imaging Reveals Multiple Conformational States of ADAMTS13
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AFM 成像揭示 ADAMTS13 的多种构象状态
DOI：
10.1186/s13036-018-0102-y
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发表时间：
2019-01-22
影响因子：
5.6
通讯作者：
Lin, Jiangguo
中科院分区：
文献类型：
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作者：
Yu, Shanshan;Liu, Wang;Lin, Jiangguo
背景ADAMTS 13（A disintegrin and metalloprotease with a thrombospondin type 1 motif 13）通过切割血管性血友病因子（Von Willebrand factor，VWF）来调节其大小，从而防止异常血小板聚集和血栓形成。由基因突变或针对ADAMTS 13的抑制性自身抗体引起的ADAMTS 13缺陷导致血栓性血小板减少性紫癜（TTP）。最近，ADAMTS 13被报道采用具有较低活性的“闭合”构象和由于VWF D4-CK结构域或抗体与ADAMTS 13的远端结构域的接合或其间隔区结构域的突变而导致的“开放”构象。这些接合或突变使ADAMTS 13活性增加约2.5倍。结果野生型ADAMTS 13（WT-ADAMTS 13）和间隔区有5个突变（R568 K/F592 Y/R660 K/Y 661 F/Y 665 F）的功能获得性变体（GOF-ADAMTS 13）在pH 6和pH 7.5下用原子力显微镜（AFM）成像。数据显示，在pH 6和pH 7.5时，WT-ADAMTS 13采用两种不同的构象状态（状态I和状态II），而在GOF-ADAMTS 13中观察到另一种状态（状态III）。在本研究中，我们提出，状态I是“封闭”的构象，状态III是“开放”的，状态II是一个中间的。与pH7.5相比，在pH 6时，WT-ADAMTS 13的状态II和GOF-ADAMTS 13的状态III的百分比增加，WT-ADAMTS 13的状态I和GOF-ADAMTS 13的状态I和状态II的百分比减少，结论WT-和GOF-ADAMTS 13均存在多种构象状态，较低的pH值可能会改变ADAMTS 13的三级结构，或破坏结构域内相互作用，增加ADAMTS 13分子的灵活性。
BackgroundADAMTS13 (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.ResultsWild 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.ConclusionBoth 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.