At the vascular injury sites,the ultra-large (UL) multimeric von willebrand Factor (VWF) is released in response to physiological and pathophysiological stimuli,and mediates platelet adhesion,aggregation,and cross-lin...At the vascular injury sites,the ultra-large (UL) multimeric von willebrand Factor (VWF) is released in response to physiological and pathophysiological stimuli,and mediates platelet adhesion,aggregation,and cross-linking to maintain hemostasis.This UL-VWF is specifically cleaved by ADAMTS13(A Disintegrin And Metalloprotease with a ThromboSpondintype-1 motif,member 13)to prevent microvascular thrombosis.Each VWF monomer consists of five types of repeat domains in the order of D1-D2-D’-D3-A1-A2-A3-D4-C1-C2-C3-C4-C5-C6-CK,in which the A2 domain contains the ADAMTS13cleavage site(Tyr1605-Met1606),exposure of which requires mechanical or chemical stimuli.Under flows,fluid shear stress regulates VWF degradation and size distribution through opening the A2 domain and exposing its cleavage site for ADAMTS13.VWF A2 domain contains a C-terminal vicinal disulfide bond,a calcium binding sites,and a flexibleα4-less-loop.These unique structure features together make A2 more sensitive to mechanical signal than other VWF A subdomains,i.e.A1 and A3 domains.It is believed that A2 is first bound with and then cleaved by ADAMTS13,together with force-induced conformation transformation.To reveal molecular basis of this two-step model of VWF hydrolyzation by ADAMTS13,we here examined stretch-induced unfolding processes of VWF A2 domain in more detail by Steered molecular dynamics(SMD)simulations,with the use of crystal structure of VWF A2(PDB ID 3GXB),and observed that there were multiple quasi-stable conformations of stretched A2 until itsβ4-strand and a3-helix were pulled away the central hydrophobic core and the cleavage sites were fully accessible to solvent.Our MD simulation data showed that,in unfolding,at first,the cleavage site residue Tyr1605 was exposed partially and binding sites for Spacer domain of ADAMTS13 were exposed to a high level whenα6-helix was separated from A2 body;then,withβ6-strand and a5-helix been pulled away,the binding sites for Cysrich domain of ADAMTS13 was exposed completely while the exposure degree of Tyr1605 was not improved;further,separation ofβ5-strand andα4-less-loop made Tyr1605 and Met1606 and the respective binding sites for ADAMTS13 Spacer domain,Cys-rich domain,and Disintegrin-like domain be fully exposed to reach the optimal catalytic state;lastly,withβ4-strand separation,the cleavage sites and binding sites all were overstretched,leading to mismatch of ADAMTS13 and A2 conformation especially in the binding sites.This conformational mismatch may cause reduction of ADAMTS13 hydrolysis efficiency.Furthermore,the data of SMD simulations under constant forces demonstrated that,the stretched A2 conformation had different quasi-stable states,which all had the better mechanical stability within simulation time of 100 ns;and the conformational transformation from one state to another must overcome their respective potential barriers.The hydrolysis efficiency should depend on each state of the stretched A2 conformation,because of the exclusive matched-degree of A2 and ADMATS13.This computer prediction on the mechanical stability and multi-states of stretched A2 provides a novel insight into the mechano-chemical regulation on cleavage of A2 by ADAMTS13.It would be helpful for design of related drug targeting the binding sites on A2 and exosites on ADAMTS13 for the treatment of patients with acquired TTP.展开更多
基金supported by National Natural Science Foundation of China Grants 11672109 ( to Y. F.) and 11432006 ( to J. W.)
文摘At the vascular injury sites,the ultra-large (UL) multimeric von willebrand Factor (VWF) is released in response to physiological and pathophysiological stimuli,and mediates platelet adhesion,aggregation,and cross-linking to maintain hemostasis.This UL-VWF is specifically cleaved by ADAMTS13(A Disintegrin And Metalloprotease with a ThromboSpondintype-1 motif,member 13)to prevent microvascular thrombosis.Each VWF monomer consists of five types of repeat domains in the order of D1-D2-D’-D3-A1-A2-A3-D4-C1-C2-C3-C4-C5-C6-CK,in which the A2 domain contains the ADAMTS13cleavage site(Tyr1605-Met1606),exposure of which requires mechanical or chemical stimuli.Under flows,fluid shear stress regulates VWF degradation and size distribution through opening the A2 domain and exposing its cleavage site for ADAMTS13.VWF A2 domain contains a C-terminal vicinal disulfide bond,a calcium binding sites,and a flexibleα4-less-loop.These unique structure features together make A2 more sensitive to mechanical signal than other VWF A subdomains,i.e.A1 and A3 domains.It is believed that A2 is first bound with and then cleaved by ADAMTS13,together with force-induced conformation transformation.To reveal molecular basis of this two-step model of VWF hydrolyzation by ADAMTS13,we here examined stretch-induced unfolding processes of VWF A2 domain in more detail by Steered molecular dynamics(SMD)simulations,with the use of crystal structure of VWF A2(PDB ID 3GXB),and observed that there were multiple quasi-stable conformations of stretched A2 until itsβ4-strand and a3-helix were pulled away the central hydrophobic core and the cleavage sites were fully accessible to solvent.Our MD simulation data showed that,in unfolding,at first,the cleavage site residue Tyr1605 was exposed partially and binding sites for Spacer domain of ADAMTS13 were exposed to a high level whenα6-helix was separated from A2 body;then,withβ6-strand and a5-helix been pulled away,the binding sites for Cysrich domain of ADAMTS13 was exposed completely while the exposure degree of Tyr1605 was not improved;further,separation ofβ5-strand andα4-less-loop made Tyr1605 and Met1606 and the respective binding sites for ADAMTS13 Spacer domain,Cys-rich domain,and Disintegrin-like domain be fully exposed to reach the optimal catalytic state;lastly,withβ4-strand separation,the cleavage sites and binding sites all were overstretched,leading to mismatch of ADAMTS13 and A2 conformation especially in the binding sites.This conformational mismatch may cause reduction of ADAMTS13 hydrolysis efficiency.Furthermore,the data of SMD simulations under constant forces demonstrated that,the stretched A2 conformation had different quasi-stable states,which all had the better mechanical stability within simulation time of 100 ns;and the conformational transformation from one state to another must overcome their respective potential barriers.The hydrolysis efficiency should depend on each state of the stretched A2 conformation,because of the exclusive matched-degree of A2 and ADMATS13.This computer prediction on the mechanical stability and multi-states of stretched A2 provides a novel insight into the mechano-chemical regulation on cleavage of A2 by ADAMTS13.It would be helpful for design of related drug targeting the binding sites on A2 and exosites on ADAMTS13 for the treatment of patients with acquired TTP.
