采用分子动力学模拟方法探究Ca~(2+)对VWF-A2结构域稳定性的影响
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摘要
目的探究Ca~(2+)对VWF-A2结构域稳定性的影响。方法 A2和A2/Ca~(2+)的晶体结构取自PDB数据库。通过恒力拉伸分子动力学模拟,比较分析Ca~(2+)结合引起的构象变化、解折叠路径的差异以及酶切位点的暴露程度。结果 A2结构域的解折叠路径和酶切位点的暴露过程是力依赖的。Ca~(2+)结合不影响A2结构域的前期解折叠,但由于α3β4-环链局部构象重排所致柔性降低,约束了β1-β4-β5片层的运动,导致进一步解折叠受阻而停留在中间稳态,影响酶切位点的充分暴露。结论力可诱导A2结构域中β5片层的解折叠使酶切位点暴露,而Ca~(2+)的结合则通过稳定疏水核心结构,阻碍酶切位点的暴露,最终降低ADAMTS13的酶切效率。研究结果有助于加深对ADAMTS13酶切VWF-A2结构域进而调控VWF止血能力过程的理解,并为相关抗血栓药物设计提供指导。
Objective To investigate the effect of calcium on the stability of VWF-A2 domain. Methods The crystal structures of A2( not containing calcium) and A2/Ca~(2+)( with calcium bound) were downloaded from protein data bank.For A2 domain,the conformational changes,unfolding pathway differences and the exposure degree variance of cleavage sites caused by calcium binding were observed and analyzed by steered Molecular Dynamics simulations under constant force. Results The unfolding pathway of A2 domain and exposure process of cleavage sites were force-dependent. Calcium binding did not affect the unfolding process of A2 in the early stage. As the conformational rearrangement of α3β4-loop reduced its localized dynamic properties,the movement among β1-β4-β5 strands was restrained,which suppressed its further unfolding to stay in the intermediate steady state and delayed the cleavage-site exposure.Conclusions Stretch force could induce β5 strand of A2 unfolding and the cleavage-site exposure,while calcium binding inhibited ADAMTS13 proteolysis efficiency through stabilizing A2 hydrophobic core and covering its cleavage sites.These results may help to understand how ADAMTS13 cleavages the VWF-A2 domain and regulates the hemostatic potential of VWF,and further provide useful guidance on the design of related anti-thrombus drugs.
Objective To investigate the effect of calcium on the stability of VWF-A2 domain. Methods The crystal structures of A2( not containing calcium) and A2/Ca~(2+)( with calcium bound) were downloaded from protein data bank.For A2 domain,the conformational changes,unfolding pathway differences and the exposure degree variance of cleavage sites caused by calcium binding were observed and analyzed by steered Molecular Dynamics simulations under constant force. Results The unfolding pathway of A2 domain and exposure process of cleavage sites were force-dependent. Calcium binding did not affect the unfolding process of A2 in the early stage. As the conformational rearrangement of α3β4-loop reduced its localized dynamic properties,the movement among β1-β4-β5 strands was restrained,which suppressed its further unfolding to stay in the intermediate steady state and delayed the cleavage-site exposure.Conclusions Stretch force could induce β5 strand of A2 unfolding and the cleavage-site exposure,while calcium binding inhibited ADAMTS13 proteolysis efficiency through stabilizing A2 hydrophobic core and covering its cleavage sites.These results may help to understand how ADAMTS13 cleavages the VWF-A2 domain and regulates the hemostatic potential of VWF,and further provide useful guidance on the design of related anti-thrombus drugs.
引文
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[2]LENTING PJ,CHRISTOPHE OD,DENIS CV.Von Willebrand factor biosynthesis,secretion,and clearance:Connecting the far ends[J].Blood,2015,125(13):2019-2028.
[3]SPRINGER TA.Von Willebrand factor,Jedi knight of the blood stream[J].Blood,2014,124(9):1412-1425.
[4]CRAWLEY JTB,GROOT R,XIANG Y,et al.Unraveling the scissile bond:How ADAMTS13 recognizes and cleaves von Willebrand factor[J].Blood,2011,118(12):3212-3221.
[5]WU T,LIN JG,CRUZ MA,et al.Force-induced cleavage of single VWFA1A2A3 tri-domains by ADAMTS13[J].Blood,2010,115(2):370-378.
[6]ZHANG X,HALVORSEN K,ZHANG CZ,et al.Mechanoenzymatic cleavage of the ultralarge vascular protein von Willebrand factor[J].Science,2009,324(5932):1330-1334.
[7]TSAI HM.Is severe deficiency of ADAMTS13 specific for thrombotic thrombocytopenic purpura?Yes[J].J Thromb Haemost,2003,1(4):625-631.
[8]ZHANG Q,ZHOU YF,ZHANG CZ,et al.Structural specializations of A2,a force-sensing domain in the ultralarge vascular protein von Willebrand factor[J].Proc Natl Acad Sci U S A,2009,106(23):9226-9231.
[9]LUKEN BM,WINN LYN,EMSLEY J,et al.The importance of vicinal cysteines,C1669 and C1670,for von Willebrand factor A2 domain function[J].Blood,2010,115(23):4910-4913.
[10]LYNCH CJ,LANE DA,LUKEN BM.Control of VWF A2 domain stability and ADAMTS13 access to the scissile bond of full-length VWF[J].Blood,2014,123(16):2585-2592.
[11]MCKINNON TA,CHION AC,MILLINGTON AJ,et al.N-linked glycosylation of VWF modulates its interaction with ADAMTS13[J].Blood,2008,111(6):3042-3049.
[12]LYNCH CJ,LANE DA.N-linked glycan stabilization of the VWF A2 domain[J].Blood,2016,127(13):1711-1718.
[13]ZHOU M,DONG X,BALDAUF C,et al.A novel calcium-binding site of von Willebrand factor A2 domain regulates its cleavage by ADAMTS13[J].Blood,2011,117(17):4623-4631.
[14]JAKOBI AJ,MASHAGHI A,TANS SJ,et al.Calcium modulates force sensing by the von Willebrand factor A2domain[J].Nat Commun,2011,2:385.
[15]XU AJ,SPRINGER TA.Mechanisms by which von Willebrand disease mutations destabilize the A2 domain[J].J Biol Chem,2013,288(9):6317-6324.
[16]XU AJ,SPRINGER TA.Calcium stabilizes the von Willebrand factor A2 domain by promoting refolding[J].Proc Natl Acad Sci U S A,2012,109(10):3742-3747.
[17]CHEN W,LOU JZ,ZHU C.Molecular dynamics simulated unfolding of von Willebrand factor A domains by force[J].Cell Mol Bioeng,2009,2(1):75-86.
[18]SVENJA L,MATTHIAS R,TOBIAS O,et al.Shear-induced unfolding and enzymatic cleavage of full-length VWF multimers[J].Biophys J,2016,110(3):545-554.
[19]HUMPHREY W,DALKE A,SCHULTEN K.VMD-Visual molecular dynamics[J].J Molec Graphics,1996,14(1):33-38.
[20]PHILLIPS JC,BRAUN R,WANG W,et al.Scalable molecular dynamics with NAMD[J].J Comput Chem,2005,26(16):1781-1802.
[21]窦甜甜,吴建华,刘广建,等.一种基于分子动力学模拟来识别GPIbα与VWF-A1结合面上重要残基的方法[J].医用生物力学,2013,28(6):606-614.DOU TT,WU JH,LIU GJ,et al.novel approach for identifying the critical amino acid residues on binding site of GPIbαand v WF-A1 domain through molecular dynamics simulation[J].J Med Biomech,2013,28(6):606-614.
[22]李红,刘文平,刘广建,等.采用分子动力学模拟探究VWF-A1突变体G561S的亲和力变化机制[J].医用生物力学,2015,30(5):433-439.LI H,LIU GP,LIU GJ,et al.Mechanism of affinity alterations in VWF-A1 domain mutant G561S studied by molecular dynamics simulation[J].J Med Biomech,2015,30(5):433-439.
[23]LIU GJ,FANG Y,WU JH.A mechanism for localized dynamics-driven affinity regulation of the binding of von Willebrand factor to platelet glycoprotein Ibα[J].J Biol Chem,2013,288(37):26658-26667.
[24]方翔.抗血栓抗体6B4、假性血友病因子及其蛋白酶ADAMTS13的分子动力学模拟[D].广州:华南理工大学博士学位论文,2012.
[25]宁志龙,刘文平,代春月,等.采用分子动力学模拟研究胶原激活VWF机制[J].医用生物力学,2015,30(S):139.