During the recovery
stroke, the
myosin motor is primed for the next power
stroke by a 60 degree rotation of its lever arm. This reversible motion is coupled to the activation of the
ATPase function of
myosin through conformational changes along the relay helix, which runs from the Switch-2 loop near the
ATP to the converter domain carrying the lever arm. Via a hydrogen bond between the side-chain of Asn475 on the relay helix and the Gly457/Ser456
peptide group on the Switch-2, the rotation of the converter domain is coupled to the formation of a hydrogen bond between Gly457 and gamma-
phosphate that is essential for
ATP hydrolysis. Here, molecular dynamics simulations of Dictyostelium discoideum
myosin II in the two end conformations of the recovery
stroke with different
nucleotide states (
ATP,
ADP x
Pi, ADP) reveal that the side-chain of Asn475 breaks away from Switch-2 upon
ATP hydrolysis to make a hydrogen bond with Tyr573. This sensing of the
nucleotide state is achieved by a small displacement of the cleaved gamma-
phosphate towards Gly457 which in turn pushes Asn475 away. The sensing plays a dual role by (i) preventing the wasteful reversal of the recovery
stroke while the
nucleotide is in the
ADP x Pi state, and (ii) decoupling the relay helix from Switch-2, thus allowing the power
stroke to start upon initial binding to actin while Gly457 of Switch-2 keeps interacting with the Pi (known to be released only later after tight actin binding). A catalytically important
salt bridge between Arg238 (on Switch-1) and Glu459 (on Switch-2), which covers the hydrolysis site, is seen to form rapidly when
ATP is added to the pre-recovery
stroke conformer and remains stable after the recovery
stroke, indicating that it has a role in shaping the
ATP binding site by induced fit.