Myosin II Trapped In A Weak Actin-binding State Through A Chemical Crosslink Across The Actin-Binding Cleft

We have trapped Dictyostelium myosin II in a weak actin-binding conformation by chemically crosslinking two engineered Cys across the actin-binding cleft using a bifunctional spin label (BSL). With sites in the lower and upper 50 kDa domains, the crosslink restricts the conformation of the actin-binding cleft. The crosslinking reaction was monitored by electron paramagnetic resonance (EPR) based on the spin label immobilization that occurs upon reaction of both Cys. The EPR spectrum of crosslinked myosin is sensitive to structural changes induced by both nucleotide- and actin-binding. Functional assays demonstrate that crosslinking partially impairs actin binding and actin-activation but has negligible effects on basal ATPase activity. We propose that crosslinked myosin is trapped in a weak actin-binding structure in which phosphate release is inhibited by the presence of actin. This conformation presumably binds actin weakly but cannot transition to the “closed” cleft structure that is populated with strong actin-binding (Klein et al, 2008, PNAS 105:12867-72). The weak actin-binding structure has proven difficult to characterize because of its transient nature; BSL-crosslinked myosin provides a stable model system for analysis of structural dynamics. We are using EPR to analyze the orientation of BSL-crosslinked myosin attached to actin in skinned muscle fibers, and we are using nucleotide probes to investigate the coupling between the actin-binding cleft and the nucleotide-binding pocket. This work is complementary to a study in which BSL was used to crosslink SH1 (C707) and SH2 (C697) in the force-generating domain of myosin, producing a stable complex that bound weakly to actin with slow orientational disorder (Thompson et al., 2008, Biophys. J., in press). This work was supported by grants from NIH (AR32961, AR07612) and the Minnesota Supercomputing Institute.