The Study Of The Structural Basis Of Spacer-CUB1 Interaction In Metalloprotease ADAMTS13 And Conformational Transition Of ADAMTS13

VWF(von Willebrand Factor)captures platelets to sites of vascular injury and mediates primary hemostasis.The activity of VWF depends on its size.Ultra large VWF(UL-VWF)is hyperactive and induces excessive platelet aggregation and thrombosis formation.ADAMTS13(A disintegrin and metalloproteinase with thrombospondin type 1 motifs,member 13)specially cleaves Y1605-M1606 scissile bond in VWF A2 domain to mediate the size and activity of VWF multimers,regulating the function of VWF in hemostasis.However,when autoimmune antibodies against ADAMTS13 were generated in the human body,ADAMTS13 lost its normal function and induced the occurrence of Thrombotic thrombocytopenic purpura(TTP).ADAMTS13 has auto-inhibition effect.TSP8-CUB2 domains at C-terminus can noncovalently bind to domains at N-terminus,covering VWF A2 binding sites on Spacer domain,leaving ADAMTS13 in a “close” conformation with low proteolytic activity.VWF D4-CK can bind to C-terminus of ADAMTS13,inducing it to transform from a “close” conformation to a highly active “open” conformation.The interaction of Spacer-CUB1 plays an important role in ADAMTS13 auto-inhibition.Exosite-3 on Spacer domain is not only a key binding sites for CUB1,but also a mainly epitope for anti-ADAMTS13 antibodies from TTP patients.When exosite-3 was mutated(R660K/F592Y/R568K/Y661F/Y665F),a gain-of-function ADAMTS13(GOF ADAMTS13)with high activity and resistance to autoantibodies binding was obtained.The conformational transition of ADAMTS13 is related to its function and the development of acquired TTP.Because the lack of crystal structure of Spacer-CUB1 complex,the interaction of Spacer and CUB1 has been not clarified.In addition,the conformational transition of ADAMTS13 is estimated by the change of proteolytic activity in present studies,lacking of the direct evidence to support the mechanism of ADAMTS13 conformational activation.Therefore,we used homologous modeling and molecular docking to construct Spacer-CUB1 complex model,and screened out a stable complex in accordance of previous studies to analysis the interaction of Spacer and CUB1 via molecular dynamics simulation.We used HEK293 T cell to express a wild-type ADAMTS13 with special tag at both its terminus.And then purified protein used to explore its conformational transition by atomic force microscopy(AFM).We successfully obtained a stable Spacer-CUB1 complex in accordance of previous studies and found that there were multiple binding sites between Spacer and CUB1.Binding sites of Spacer could be divided into three regions: exosite-3,exosite-4 and G607-S610.D1259,L1258,T1261,E1231 and R1251 were key residues in CUB1.When these five residues were mutated to Ala,the binding of Spacer and CUB1 was markedly attenuated and easier to disassociation.AFM data shown that the contour length of ADAMTS13 was about 27.37±8.70 nm,which was close to 23 nm reported by previous study.The force required for the conformational transition of ADAMTS13 was about 21.22±10.32 p N.The length increment of unfolded ADAMTS13 was 7.59±2.84 nm,which was much less than 27 nm predicted by previous study,suggesting that ADAMTS13 may have an intermediate conformation.Our study on Spacer-CUB1 complex helps to further understand the structural basis of ADAMTS13 auto-inhibition and predicts the key residues on the interface for reference,which advance the development of potential drug for TTP treatment.Our study on the conformational transition of ADAMTS13 provides further insight into the mechanism of ADAMTS13 conformational activation and reveals the process of conformational transition of ADAMTS13,which help to provide new strategy for TTP treatment.