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

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 phosphorylat ion-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 whet her the registration of the 7-residue/28-residue repeat appearing in the ct -helical coiled-coil structure of the rod are critical for such an interact ion, two smooth muscle myosin mutants were constructed in which the N-termi nal sequences of S2 were deleted to various extents. One mutant contained a deletion of 71 residues at the position immediately C-terminal to the inva riant proline (Pro849) linking the S1 domain directly to the downstream seq uence of the rod, while in another mutant, 53 residues were deleted at a po sition 56 residues downstream of Pro849. Despite these alterations which ch ange the registration of both the 28-residue repeat and the 7-residue repea t found in myosin rod sequence, both myosin mutants showed a stable double- headed structure by electron microscopic observation. Both the actin-activa ted ATPase activity and the actin translocating activity of the mutants wer e 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 residue s nor the registration of the amino acid repeat in S2 is critical for the h ead configuration. These results indicate that neither a specific amino aci d sequence at the head-rod junction nor the rod sequence registration is cr itical for the regulation of smooth muscle myosin.