Mapping Paratope To Epitope Residues Through Residue Interaction Index And The Mechanical Mechanism To Regulate Activity Of ERM Proteins And LFA-1

Atherosclerosis and its related thrombotic diseases are major causes of heart attacks and strokes.Platelets are critical for atherosclerosis and subsequent thrombosis formation because they have special abilities to adhere to the injury sites of blood vessel and trigger the activition of other platelets.With the understanding of VWF mediated platelets adhesion and thrombosis formation,a series of VWF inhibitors have been applied to thrombotic diseases stroke treatment.Among them,monoclonal antibodies play important roles.Antibodies generated by hybridomas and phage-display technology are usually difficult to meet clinical demands,whose efficacy are needed to be increased by improving their properties.During that progress,mapping paratope to epitope is an essential step and becomes the hotspot of molecular biology,bioinformatics and drug design.Different from traditional biophysical methods and functional approaches,our lab has recently developed a novel computational procedure,named hydrogen bond stabilization index,to identify key paratope residues and their partners.That computational procedure succeeded in mapping paratope to epitope of 6B4/GPIba complex with high agreement with mutagenesis experiments.On the other side,the recruitment of leucocytes at sites of tissue inflammation are our primary internal defence against the invasion of harmful bacteria,viruses and micro-organisms.Initial attachment of leukocytes to the endothelium is mainly mediated by the interaction of selectins with their ligands due to their high on-and off-rates,such as P-selectin and P-selectin glycoprotein ligand 1(PSGL-1).That progress relies on the polarization and redistribution of PSGL-1 on the tips of leukocyte microvilli,or in another word,the binding of PSGL-1 to the activated ezrin/radixin/moesin(ERM)proteins and actin.During the rolling of leucocytes,G-protein coupled receptors will interact with chemokines specifically,which leads to integrin activation and subsequent firm leukocyte arrest.Besides,force generated by the bloodstream has been shown as an allosteric effector to stabilize the integrin in an extended,high-affinity conformation through ―Outside-In‖ signaling.After firm adhesion,leukocytes move slowly over the endothelial cell surface and migrate in the extracellular matrix to sites of tissue inflammation finally.Although hydrogen bond stabilization index shows great advantages on mapping paratope to epitope residues,there still needs some improvements.However,we noticed that a residue might form more than one hydrogen bond,and there would be a great loss of interaction information during the interface if only the hydrogen bond with highest survival ratio was taken into consideration.To overcome that,we here proposed a new index,termed residue interaction index,to evaluate the importance of the residues within the interface.Results showed that residue interaction index had higher sensitivity and lower FDR on 82D6A3/VWF-A3,6B4/GPIba and VWF-A1/GPIba complexes,compared with hydrogen bond stabilization inde.And we also conducted a further discussion of the criterion(top percentage)selection.The results showed that top percentage turned out to be a good standard for both indices,as the choice of top 30%~25% residues was the best in statistics.Besides,rangkings of residues were optimized by RII and we found that it had an impressive advantage in dealing with the complex with medium-sized interface.As the linkers between membrane and actin cytoskeleton,ERMs play a great role in leukocyte capture by selectins,as well as the leukocyte rolling stability.But there are two questions about their activation: how to overcome the sterically hindered effect of phosphorylation site at C-terminal,and what is the role of ERM oligomers? To answer those questions,we here carried out a model composed of PSGL-1-an asymmetric dimer of ERMs-F-actin.Molecular dynamic simulations were used to explore whether the force transmitted through the cytoplasmic tail of PSGL-1 could induce the exposure of the conserved threonine residue in the C-terminal domain of ERM proteins.The results showed that the unbinding force of PSGL-1 and raxidin was higher than the force to expose the buried phosphorylation site in asymmetric dimers of moesin.That mechanical pathway not only solved the sterically hindered effect of phosphorylation site,but also proved the meaning of asymmetric dimer of ERMs,which keeps balance between the inactive monomers in cytosol and the active monomers at membrane and would be activated under force.Besides,it was proved that force exerted on integrin LFA-1 would induce an extended,high-affinity conformation of its aI domain.Here,we applied molecular dynamic simulation to the intermediate affinity aI domain/ICAM complex.Through the equilibriums,we found that aI domain tended to adopt low affinity state with the outward movement of α1 helix and the exposure of the hydrophobic region among α1,β4 and β5.When the force applied on the complex in steered molecular dynamic simulations,we observed the downward movement of α7 helix accompanied with the tightening of the hydrophobic region among α1,β4 and β5 to its intermediate affinity state.Besides,through the systemic analysis of the interface,we found that the β5α6 loop of LFA-1 could form hydrogen bonds with ICAM-1 indeed,which was regulated by the hydrophobic region among α1,β4 and β5.