Registration of the rod is not critical for the phosphorylation-dependent regulation of smooth muscle myosin.

Abstract

A recent report has suggested that the interaction between the head and the rod region of smooth muscle myosin at S2 is important for the phosphorylation-mediated regulation of myosin motor activity [Trybus, K. M., Freyzon, Y., Faust, L. Z., and Sweeney, H. L. (1997) Proc. Natl. Acad. Sci. U.S.A. 74, 48-52]. To investigate whether specific amino acid residues at S2 or whether the registration of the 7-residue/28-residue repeat appearing in the alpha-helical coiled-coil structure of the rod are critical for such an interaction, two smooth muscle myosin mutants were constructed in which the N-terminal sequences of S2 were deleted to various extents. One mutant contained a deletion of 71 residues at the position immediately C-terminal to the invariant proline (Pro849) linking the S1 domain directly to the downstream sequence of the rod, while in another mutant, 53 residues were deleted at a position 56 residues downstream of Pro849. Despite these alterations which change the registration of both the 28-residue repeat and the 7-residue repeat found in myosin rod sequence, both myosin mutants showed a stable double-headed structure by electron microscopic observation. Both the actin-activated ATPase activity and the actin translocating activity of the mutants were completely regulated by the phosphorylation of the regulatory light chain. The actin sliding velocity of the two mutant myosins was the same as the wild-type recombinant myosin. Furthermore, the head configuration critical for myosin filament formation (extended or folded) was unchanged in either mutant. These results indicate that neither the specific amino acid residues nor the registration of the amino acid repeat in S2 is critical for the head configuration. These results indicate that neither a specific amino acid sequence at the head-rod junction nor the rod sequence registration is critical for the regulation of smooth muscle myosin.