Myosin cleft closure by double electron–electron resonance and dipolar EPR

Dipolar electron paramagnetic resonance (EPR) has been used to determine the conformation of the myosin actin-binding cleft in various states of actomyosin ATPase and binding to actin. Comparison of the crystal structures of isolated components and electron microscopy of the actin–myosin complex suggested that the cleft between the upper and lower 50 kDa subdomains closes on binding to actin. The changes in the myosin cleft might be coupled to the state of the nucleotide in the active site and provide communication between ATP hydrolysis and actin binding. In order to detect these changes in solution, we measured the distances between double cysteine mutants of the Dictyostelium discoidium myosin II MD by continuous wave EPR and pulsed EPR—double electron–electron resonance (DEER). We have engineered double cysteine mutants C416/C537 which are labelled with spin probes and measured the distances between the upper and lower domains in various nucleotide states and in the presence of actin. We found two major populations of distances at ∼15 Å (conventional EPR) and at 25 Å (DEER) and shifting in the populations as a function of nucleotide state and actin binding, which suggest that the cleft movement is coupled to nucleotides and actin binding and dissociation.