Molecular Dynamics Simulations Of Antithrombotic Antibody6B4, Von Willebrand Factor And Its Protease ADAMTS13

As the most common cause of death in the developed countris, acute arterial thrombosiscan lead to fatal myocardial infarction (heart attack) and stroke. Adhesion of platelets to thesite of vascular injury is a key process for physiological hemostasis and pathologicalthrombosis. Platelet adhension is a multi-step cascade including tethering, rolling and firmadhesion, which are mediated by various interactions between distinct receptors and ligands.Among them, the interaction between platelet glycoprotein GPIbα and A1domain of vonWillebrand factor (vWF) tethers platelets from the context of high shear stress to vWFimmoblized on the collagen of subendothelium matrix, playing an initial and critical role inplatelet adhesion. Inhibiting binding of GPIbα to A1can prevent pathological thrombosis butnot obviously interfere physiological hemostasis. Thus, GPIbα becomes a noteworthy targetfor antithrombotic antibodies.On the other hand, as an improtant multimeric plasma glycoprotein, vWF contains threetandem A domains. A1domain interacts with GPIbα; vWF-A2domain hosts cleavage sitefor metalloprotease ADAMTS13; A3domain can bind to type III collagen. ADAMTS13(Adisintegrin and metalloprotease with a thrombospondin type1motifs13) cleaves a singlepeptide bond (Tyr1605-Met1606) in the central A2domain and turns Ultralarge vWF intosmaller and less reactive plasma vWF. Congenital or acquired deficiency of ADAMTS13causes the accumulation of ULvWF multimers, leading to thrombotic thrombocytopenicpurpura (TTP), a life-threatening disease. Conversely, some mutations in vWF-A2domainincreases proteolysis of vWF. Excessive cleavage leads to type2A von Willebrand disease(vWD), a qualitative bleeding disorder.Of various antithrombotic monoclonal antibodies targeting human GPIbα,6B4is apotent one to inhibit the interaction between GPIbα and vWF-A1under static and flowconditions. Mapping paratope to epitope with mutagenesis experiments, a traditional route inresearches of these antithrombotic mAbs, is usually expensive and time-consuming. Here, we suggested a novel computational procedure, which combines with homology modeling,rigid body docking, free and steered molecular dynamics (SMD) simulations, to identify keyparatope residues on6B4and their partners on GPIbα, with hypothesis that the stablehydrogen bonds and salt bridges are the important linkers between paratope and epitoperesidues. Based on a best constructed model of6B4bound with GPIbα, the survival ratiosand rupture times of all detected hydrogen bonds and salt bridges in binding site wereexamined via free and steered MD simulations and regarded as indices of thermal andmechanical stabilizations of the bonds, respectively. Five principal paratope residues withtheir partners were predicted with their high survival ratios and/or long rupture times ofinvolved hydrogen bonds, or with their hydrogen bond stabilization indices ranked in top5.Exciting, the present results were in good agreement with previous mutagenesis experimentdata, meaning a wide application prospect of our novel computational procedure onresearches of molecular of basis of ligand-receptor interactions, various antithromboticmAbs and other antibodies as well as theoretically design of biomolecular drugs.To investigate the structural property of vWF-A2under physiological condition, freedynamics simulations are performed on A1, A2and A3domains of vWF. The result indicatedthat A2became loose due to absence of disulfide bond linking N and C termini. Besides, A3is more flexible than A1and A2. We also studied unfolding detail of A2via low-velocitySMD simulation. Besides, to understand the interactions between A1and A2, an A1-A2complex model was constructed by flexible docking method. Conformation of the model isconsistent with known experimental results. In this model we detected eight intermolecularH-bonds and salt bridges, from which the key residues for binding are predicted.Spacer domain of ADAMTS13contains an important exosite, which binds to α6helix ofvWF-A2domain. However, no interaction can proceed until A2is unfolded since α6helix iscryptic in native A2. To understand the molecular mechanism underlying association betweenspacer and α6, we developed two novel SMD-docking methods (first docking then SMD andfirst SMD then docking) to predict spacer-α6complex models, with hypothesis that the extentof unfolding (or extension) of α6might have a significant impact on binding strength. The results indicated a biphasic pattern characteristic: The binding strength first increased andthen decreased after reaching an optimal at0.25nm extension as the length of α6increased.Changes of interface area and intermolecular salt bridge might serve as the molecular basisfor this characteristic. Thus, upon binding, spacer may favor a partially unfolded (extended)α6, which may contribute to optimal contact and proteolysis.In conclusion, we mapped paratope residues on6B4and epitope residues on GPIbα viacombination of bioinformatic analysis and molecular dynamics simulations. The residues areconsistent with those identified with mutagenesis experiments. Furthermore, we introducedtwo novel SMD-docking methods to investigate interactions between spacer domain and α6helix. The change of the binding affinity is also in accordance with that proposed by previousresearch. These work may provide insights for mutagenesis experiments and design ofantithrombotic drugs.