The Characteristics Of The Interactions Of ADAMTS13 C-terminal Domains And The Spacer Domain Revealed By Molecular Dynamics Simulation

By specifically cleaving VWF multimers,ADAMTS13(A disintegrin and metalloproteinase with a thrombospondin type 1 motif,member 13)mediates platelet-rich thrombus formation.The wild-type(WT)ADAMTS13 adopts a closed conformation with the interactions between its C-terminal TSP8-Linker-CUB1(TLC)domains and its N-terminal Spacer domain.This autoinhibition structure prevents its off-target proteolysis or recognition by non-specific hydrolases in flow,and also hides the key binding sites of autoantibodies produced by patients with acquired thrombotic thrombocytopenic purpura.Therefore,it is necessary to investigate the interactions between ADAMTS13 C-terminal domains and the Spacer domain.Using molecular dynamics simulations,we investigated the thermal stabilization of the TSP8/Spacer docking complex and the TLC/Spacer docking complex.In addition,we also mutated the key residues in the Spacer or C-terminal domains of these two complexes to study the effect of mutations on the complexes.The similarity between the WT TSP8/Spacer and WT TLC/Spacer complexes was that both complexes shared the robust interactions of Arg1075 and Arg1095 in the TSP8 domain with Arg635 in the Spacer domain.Specific mutations in Spacer domain exosite 3(R660K/F592Y/R568K/ Y661F/Y665F)enhanced the thermal stability of the hydrogen bonds formed by Arg1075-Asp635 and Arg1095-Asp635,whereas mutations in the predicted residues of the C-terminal domains abolished the hydrogen bonds of both residue pairs.The biggest difference between the WT TSP8/Spacer and WT TLC/Spacer complex was that the binding sites of exosite 3 were different.In the former,exosite 3 was mainly bound to Asp1090 and Cys1125 in the TSP8 domain;however,in the latter,the loop Lys1256-Asp1259 of CUB1 domain occupied exosite 3.In the WT TLC/spacer complex,a dense net of hydrogen bonds and salt bridges was formed between exosite 3 and Lys1256-Asp1259,allowing the TLC and Spacer domain to bind tightly.Mutations in either the Spacer domain or the C-terminal domains significantly reduced these interactions.