GLYCINE-699 IS PIVOTAL FOR THE MOTOR-ACTIVITY OF SKELETAL-MUSCLE MYOSIN

Myosin couples ATP hydrolysis to the translocation of actin filaments to power many forms of cellular motility, A striking feature of the st ructure of the muscle myosin head domain is a 9-nm long ''lever arm'' that has been postulated to produce a 5-10-nm power stroke. This motio n must be coupled to conformational changes around the actin and nucle otide binding sites, The linkage of these sites to the lever arm has b een analyzed by site-directed mutagenesis of a conserved glycine resid ue (G699) found in a bend joining two helices containing the highly re active and mobile cysteine residues, SH1 and SH2, Alanine mutagenesis of this glycine (G699A) dramatically alters the motor activity of skel etal muscle myosin, inhibiting the velocity of actin filament movement by >100-fold. Analysis of the defect in the G699A mutant myosin is co nsistent with a marked slowing of the transition within the motor doma in from a strong binding to a weak binding interaction with actin. Thi s result is interpreted in terms of the role of this residue (G699) as a pivot point for motion of the lever arm. The recombinant myosin use d in these experiments has been produced in a unique expression system , A shuttle vector containing a regulated muscle-specific promoter has been developed for the stable expression of recombinant myosin in C2C 12 cells. The vector uses the promoter/enhancer region, the first two and the last five exons of an embryonic rat myosin gene, to regulate t he expression of an embryonic chicken muscle myosin cDNA. Stable cell lines transfected with this vector express the unique genetically engi neered myosin after differentiation into myotubes, The myosin assemble s into myofibrils, copurifies with the endogenous myosin, and contains a complement of muscle-specific myosin light chains. The functional a ctivity of the recombinant myosin is readily analyzed with an in vitro motility assay using a species-specific anti-S2 mAb to selectively as say the recombinant protein. This expression system has facilitated ma nipulation and analysis of the skeletal muscle myosin motor domain and is also amenable to a wide range of structure-function experiments ad dressing questions unique to the muscle-specific cytoarchitecture and myosin isoforms.