A point mutation in the SH1 helix alters elasticity and thermal stability of myosin II

Abstract

Movement generated by the myosin motor is generally thought to be driven by distortion of an elastic element within the myosin molecule and subsequent release of the resulting strain. However, the location of this elastic element in myosin remains unclear. The myosin motor domain consists of four major subdomains connected by flexible joints. The SH1 helix is the joint that connects the converter subdomain to the other domains, and is thought to play an important role in arrangements of the converter relative to the motor. To investigate the involvement of the SH1 helix in elastic distortion in myosin, we have introduced a point mutation into the SH1 helix of Dictyostelium myosin II (R689H), which in human nonmuscle myosin IIA causes nonsyndromic hereditary deafness, DFNA17. The mutation resulted in a significant impairment in motile activities, whereas actin-activated ATPase activity was only slightly affected. Single molecule mechanical measurements using optical trap showed that the step size was not shortened by the mutation, suggesting that the slower motility is caused by altered kinetics. The single molecule measurements demonstrated that the mutation significantly reduced cross-bridge stiffness. Motile activities produced by mixtures of wild-type and mutant myosins also suggested that the mutation affected the elasticity of myosin. These results suggest that the SH1 helix is involved in modulation of myosin elasticity, presumably by modulating the converter flexibility. Consistent with this, the mutation was also shown to reduce thermal stability and induce thermal aggregation of the protein, which might be implicated in the disease process. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.