Kinetics and Thermodynamics of the Rate Limiting Conformational Change in the Myosin V Mechanochemical Cycle

Abstract

We have used FRET to examine the kinetics and thermodynamics of the structural changes associated with ADP release in myosin V, which is thought to be a strain sensitive step in many muscle and non-muscle myosins. We also use essential dynamics using FIRST/FRODA starting with three different myosin V X-ray crystal structures to examine the intrinsic flexibility and correlated motions. Our kinetic and steady-state FRET results demonstrate that the nucleotide binding pocket goes from a closed to an open conformation prior to the release of ADP while the actin binding cleft remains closed. Thermodynamic analysis of ADP binding to actomyosin V suggests the collision complex formation is driven by a large enthalpy change and a small change in entropy. The transition from the open to closed pocket actomyosin.ADP state is associated with a large unfavorable decrease in entropy, which suggests the closed pocket conformation is more rigid than the open pocket conformation. Although no crystal structure is available of the closed pocket myosin V.ADP state, our FRET analysis reveals that this conformation may be similar to the myosin V.ATP state. FIRST/FRODA analysis is consistent with these conclusions as the myosin V.ADP structure is more flexible than the Apo structure, while the myosin V.ATP structure is more rigid than myosin V.ADP. Principal component analysis demonstrates that opening and closing of the nucleotide binding pocket correlates with the motions of loop 1 and the transducer region in all three crystal structures. Interestingly, we find that the temperature dependence of the maximum actin-activated myosin V ATPase rate correlates with the pocket opening step, suggesting this is the rate limiting step in the ATPase cycle. Our results provide insight into the structural mechanism of strain-dependent ADP release in myosins.