Mechano-regulation Mechanism And Molecular Structural Basis On Interaction Of Integrin α5β1 And Mac-1 With Their Respective Ligands CD40L And GPIbα

Once the vascular is damaged,platelets will accumulate and adhesion to achieve the hemostasis.At the same time,these activated platelets capture circulating leukocytes and interact with them to initiate the inflammatory response process.The interaction between platelets and leukocytes is mediated by cytokines,adhesion molecules,chemokines,which also may be regulated by the flow environment.The activation of integrin is an essential event for leukocyte’s immune response.As a critical molecule,CD40 L interaction with α5β1 integrin in the mechanical environment on how to mediate the adhesion process of leukocytes is not clear.In this paper,we used parallel plate flow chamber experiments found that the immobilized monomer CD40 L interacts with α5β1 integrin on U937 cells induced cell adhesion behavior.Under continuous mechanical stimulation,the affinity state of α5β1 integrin changed.AFM experiments showed that divalent ions regulate the integrin affinity state mediated by CD40 L monomers.Moreover,α5β1 activated by Mn2+,the adhesion frequency,and molecular bond lifetime significantly increased,and the molecular bond behavior was catch slip bond.These results indicate that force regulates CD40L-α5β1 interaction.The Mac-1-GPIbα interaction is a process of mechanochemical coupling,in which the binding is in the flow environment.Unfortunately,the crystal structure of the two complexes has not yet resolved.The standard methods for proteins without crystal structure are homology modeling or molecular docking.Unfortunately,regardless of rigid or flexible docking,the predicting results are often not ideal.In this study,we proposed a conformation screening method called Force-ramp + Spring-back based on flexible docking,which successfully screened Mac-1-GPIbα complex.The strategy includes three steps: 1)Balanced molecular dynamics of the docking complex;2)Simulation of constant speed stretching and constant force stretching of the docked conformation after equilibrium;3)Reconfiguration of the configuration after relaxation Balance,and then get a new conformation.We used the ramp-clamp method found that the critical residues confirmed by mutation experiments ASP222,ARG218.Moreover,the force clamp results indicate that Mac-1 and GPIbα exhibit a biphasic force dependence.When a mechanical force is applied,the binding affinity of Mac-1 / GPIbα first increases("Catch bond")and then decreases("slip bond");and the optimal force is 25 pN.Together,our results provide a new insight for the mechanical regulation mechanism of CD40L-α5β1 integrin,the structural basis mechanism of Mac-1-GPIbα complex.These maybe provide a new insight for the relevant signal transduction in different cell biological processes.It may also provide new targets for the design of new drugs and the treatment of related diseases.