Integrin activation,the transition from a low to a high affinity state,regulates the numerous cellular responses consequent to integrin engagement by extracellular matrix proteins.Kindlin proteins,play crucial roles i...Integrin activation,the transition from a low to a high affinity state,regulates the numerous cellular responses consequent to integrin engagement by extracellular matrix proteins.Kindlin proteins,play crucial roles in the integrin-signaling pathway by directly interacting with and activating integrins,which mediate the cell-extracellular matrix adhesion and signaling.As a widely distributed PTB domain protein and a major member of the kindlin family,kindlin2 interacts withβ3-tail,bridges talin-activated integrins to promote integrin aggregation,and enhances talin-induced integrin activation.Thus,kindlin2 is identified as a coactivator of integrins.Unlike talins,kindlin2 cannot directly alter the conformation of the integrin transmembrane helix and fail to activate integrin alone.Nevertheless,although it is widely accepted that kindlins and talins synergistically promote integrin activation,the underlying mechanism is unclear.Thus,the study of the force dissociation of the kindlin2/β3-tail complex and the conformation stabilization under different mechanical micro-environments should be of great significance for the further understanding of the structural basis of its synergistically activation of integrin.To reveal the molecular dynamics mechanism of interaction between kindlin2 andβ3-tail,we perform molecular dynamics(MD)simulations for this complex with different computing strategies interaction.In MD simulations,the available crystal structures of Kindlin-2/β3-tail complex(Protein Data Bank code 5XQ1)was downloaded from the PDB database.Two software packages,VMD for visualization and modeling and NAMD 2.13 for energy minimizations and MD simulations,were used here.The steadystate conformation of the complex was obtained from the equilibrium simulation.The dissociation event was observed by the constant velocity simulation,and the mechanical stability of the complex was observed by the constant force simulation.Our results showed that,during the equilibrium of the kindlin2-F3/β34ail complex,the residue MET612,LYS613 and TRP615 on the F3 domain of kindlin2 contributed to hydrogen-bonding with the corresponding residues onβ3 integrin.These bonds exhibit moderate or strong stability through steered molecular dynamics(SMD)simulation.During the constant velocity simulation,the complex exhibits a variety of unfolding pathways against tension applications,which are mainly distinguished by the disruption of hydrogen-bonds between the F3 domain a1/a2 helixes andβ1/β2 sheets.During the constant force simulation,the different phases of the composite force dissociation have different dissociation probabilities,which shows the biphasic force-dependent characteristics.And,the key residues in the pulling were recognized according not only to the number of interacting residue pairs,but also to their bond strength.Using molecular dynamics simulation,we showed the steady state of the kindlin2-F3/β3-tail complex under different tensile forces,and observe the dynamic process of molecular interaction.A possible underlying biophysical mechanism is that,the dissociation of Kindlin2-F3/β3-tail complex is biphasic force-dependent,and the conformations under different stretching states have different binding affinities.This study not only provides insights into the structural basis and mechanical regulation mechanisms of the kindlin/integrin interaction,in understanding in kindlin/integrin-related signaling in different cellular biological processes,but also provides new ideas for novel drug design and the treatment of related diseases.展开更多
基金supported by the National Natural Science Foundation of China ( 116272109, 11432006)
文摘Integrin activation,the transition from a low to a high affinity state,regulates the numerous cellular responses consequent to integrin engagement by extracellular matrix proteins.Kindlin proteins,play crucial roles in the integrin-signaling pathway by directly interacting with and activating integrins,which mediate the cell-extracellular matrix adhesion and signaling.As a widely distributed PTB domain protein and a major member of the kindlin family,kindlin2 interacts withβ3-tail,bridges talin-activated integrins to promote integrin aggregation,and enhances talin-induced integrin activation.Thus,kindlin2 is identified as a coactivator of integrins.Unlike talins,kindlin2 cannot directly alter the conformation of the integrin transmembrane helix and fail to activate integrin alone.Nevertheless,although it is widely accepted that kindlins and talins synergistically promote integrin activation,the underlying mechanism is unclear.Thus,the study of the force dissociation of the kindlin2/β3-tail complex and the conformation stabilization under different mechanical micro-environments should be of great significance for the further understanding of the structural basis of its synergistically activation of integrin.To reveal the molecular dynamics mechanism of interaction between kindlin2 andβ3-tail,we perform molecular dynamics(MD)simulations for this complex with different computing strategies interaction.In MD simulations,the available crystal structures of Kindlin-2/β3-tail complex(Protein Data Bank code 5XQ1)was downloaded from the PDB database.Two software packages,VMD for visualization and modeling and NAMD 2.13 for energy minimizations and MD simulations,were used here.The steadystate conformation of the complex was obtained from the equilibrium simulation.The dissociation event was observed by the constant velocity simulation,and the mechanical stability of the complex was observed by the constant force simulation.Our results showed that,during the equilibrium of the kindlin2-F3/β34ail complex,the residue MET612,LYS613 and TRP615 on the F3 domain of kindlin2 contributed to hydrogen-bonding with the corresponding residues onβ3 integrin.These bonds exhibit moderate or strong stability through steered molecular dynamics(SMD)simulation.During the constant velocity simulation,the complex exhibits a variety of unfolding pathways against tension applications,which are mainly distinguished by the disruption of hydrogen-bonds between the F3 domain a1/a2 helixes andβ1/β2 sheets.During the constant force simulation,the different phases of the composite force dissociation have different dissociation probabilities,which shows the biphasic force-dependent characteristics.And,the key residues in the pulling were recognized according not only to the number of interacting residue pairs,but also to their bond strength.Using molecular dynamics simulation,we showed the steady state of the kindlin2-F3/β3-tail complex under different tensile forces,and observe the dynamic process of molecular interaction.A possible underlying biophysical mechanism is that,the dissociation of Kindlin2-F3/β3-tail complex is biphasic force-dependent,and the conformations under different stretching states have different binding affinities.This study not only provides insights into the structural basis and mechanical regulation mechanisms of the kindlin/integrin interaction,in understanding in kindlin/integrin-related signaling in different cellular biological processes,but also provides new ideas for novel drug design and the treatment of related diseases.
