A long, weakly charged actin-binding loop is required for phosphorylation-dependent regulation of smooth muscle myosin.

Chimeric substitution of the weak actin-binding loop (ABL) from chicken skeletal muscle myosin for that of gizzard smooth muscle heavy meromyosin (HMM) causes activation of the dephosphorylated mutant (SABL HMM; Rovner, A. S., Freyzon, Y., and Trybus, K. M. (1995) J. Biol. Chem. 270, 30260-30263). The present study determined whether this loss of regulation is due to the greater positive charge density (5 versus 3 clustered lysine residues) or lesser length (14 versus 26 residues) of the mutant ABL. Charge augmentation had little effect on regulation of expressed mutants, but elimination of the 12 N-terminal amino acids from the wild-type ABL significantly increased actin-activated ATPase activity of the dephosphorylated relative to the phosphorylated molecule while conferring the ability to move actin filaments in vitro on the former. Addition of the same 12 residues to the SABL mutant increased the ratio of phosphorylated to dephosphorylated ATPase activity while imparting wild type-like regulation to motility. However, full actin activation of dephosphorylated ATPase activity required both the shorter length and greater positive charge density found in the SABL loop. These results demonstrate that, compared with skeletal, both the greater length and lesser positive charge density of the smooth muscle myosin ABL are required for proper phosphorylation-mediated regulation of the molecule.