Molecular Mechanism Study Of Vinculin/p130Cas Interaction Via Steered Molecular Dynamics Simulation

Focal adhesion(FA)connects the extracellular matrix(ECM)to the cytoskeleton,and can transform the force generated by intracellular cytoskeleton rearrangement and external mechanical stimuli into biochemical signals,thereby regulates cell morphology and behavior.Vinculin and p130Cas are important adaptive proteins in FA,which interact with each other to regulate the formation and turnover of FA.P130Cas is a force sensor protein in FA,which regulates cell movement through phosphorylation signals under mechanical tension.Stable binding of Vinculin to p130Cas is essential for the transfer of mechanical tension on p130Cas.However,the mechanical stability and molecular dynamics mechanism of the binding between Vinculin and p130Cas are still unclear.Vinculin is a scaffold protein in FA,including a head domain,tail domain and linker in the middle.Sequence 854-870 in Vinculin Linker binds to the N-terminal SH3 domain of p130Cas to recruit and stabilize p130Cas in FA.The Linker in the middle of Vinculin is directly subjected to mechanical stretching from the head and tail domains,but it is not clear how the mechanical tension affects its binding to the p130Cas.The stable conformation of complex Vinculin/SH3was obtained by equilibrium MD simulation.Then SMD simulation was performed to observe the conformational changes of Vinculin and its binding ability to SH3 by fixing the N-terminal of Vinculin sequence and steering the C-terminal.The results showed that when the length of Vinculin approached the limit length,the hydrogen bond between Vinculin and P130cas remained at 3-4 pairs.This stable binding is important for the Vinculin in FA to provide docking sites for p130Cas while bridging Talin and actin.In addition,Vinculin mainly exhibits two conformations under different forces:vin?bent conformation system under low force and vin?extended conformation system under high force.The binding affinity of the vin?bent system is higher than that of vin?extended system.We further used SMD simulation technology to study the dissociation path of vin?bent and vin?extended systems and the change of mechanical stability under constant force.Firstly,the two terminals of Vinculin in vin?bent and vin?extended systems were fixed.Then,C?⁶⁷was stretched at a constant speed along the connecting direction between the midpoint of Vinculin and C?⁶⁷ of SH3 domain.The results show that there will be a two-step dissociation path in the vin?bent system,and the maximum rupture force is determined by the strength of the Glu857-Arg59 interaction of the hydrogen bond and salt bridge in the first step.Then we performed a constant force of 20p N and compared it with the unstressed result.We found that there is a catch bond in both systems,a mechanism that the binding affinity increases with the force.This is essential for the force to be transferred to the SD domain of p130Cas and to maintain its extended state.In addition,we found that force will increase the hydrogen bond formation rate of Tyr12 in the vin?extended system,which will also stabilize the binding of P130cas to Vinculin.In conclusion,we simulated the different stress scenes of Vinculin and p130Cas in FA,and found that force can stabilize the combination between Vinculin and p130Cas.Our results provide ideas for the"durotaxis"behavior of cells in the tumor physical microenvironment and lay a theoretical foundation for future study.