Nevertheless, we did observe the relevant “ratchet” like movements on residues L289, F292, and L295, which followed the movements of the a7-helix. Residue D239 coordinated directly with the MIDAS metal ion in the closed conformation as observed in the crystal structures. On the other hand, in the open conformation, D239 might not coordinate with metal ion directly but through a water molecule. In our pulling simulation, it seemed that the LEE011 1211441-98-3 strong ionic interaction between D239 and the metal ion constrained the metal ion at its closed position, thus preventing the inward movement from being observed within the short timescale of the simulation. To test this hypothesis, we performed a set of three simulations. These simulations started from the structures generated from the above pulling simulations. The snapshots at 0, 3.7 and 16 ns were taken as the respective new starting points. Among them, the 0 ns configuration represented the “up” position of the a7-helix, the 3.7 ns configuration represented the “middle” position and the 16 ns one represented the “down” position. In these free dynamics simulations, the applied force was released. To prevent the a7-helix from returning back to the “up” position in the simulations starting from 3.7 and 16 ns snapshots, we constrained the Ca atoms of the a7-helix in addition to the original constraint residues. Firstly, 30 ns free dynamics simulations were performed followed by 20 ns free dynamics simulations with the point charges of the two oxygen atoms of D239 carboxyl group reduced by 0.5e each. As shown in Fig. 3 with the RMSD time courses of the MIDAS ion between the simulated structure and its closed or open positions, in all three simulations, the MIDAS ions fluctuated around their closed position without any tendency to move towards the open position before the point charges were reduced. By comparison, after the point charges of the D239 carboxyl oxygen were reduced, in the simulations starting from 3.7 ns and 16 ns, the metal ion showed strong tendencies to move inward towards the open position, with the RMSD to the closed position reduced and that to the open position increased. For the simulation starting from 0 ns, the movement was also possible, but the duration was short. The simulated structure fluctuated around the closed position for the majority of simulation times. These simulations confirm that the position of the metal ion is related to the position of the a7-helix, consistent with the generally accepted contention that the position of the metal ion determines the ligand binding affinity of the aA domain. These results support the hypothesis that the closed, intermediate and open conformations of LFA-1 aA domain represent stable states and that sequential transitions from the closed to intermediate and from intermediate to open conformations can be induced by pulling the a7-helix. As primary force-bearing molecules governing cell-cell and cellmatrix adhesions, integrins are tightly regulated biochemically and mechanically.