Chimeric Substitutions of the Actin-binding Loop Activate Dephosphorylated but Not Phosphorylated Smooth Muscle Heavy Meromyosin

Abstract

Regulatory light chain (RLC) phosphorylation is necessary to activate smooth muscle myosin, unlike constitutively active striated muscle myosins. Here we show that an actin-binding surface loop located at the 50/20-kDa junction contributes to this fundamental difference between myosins. Substitution of the native actin-binding loop of smooth muscle heavy meromyosin (HMM) with that from either skeletal or ␤-cardiac myosin caused the chimeric HMMs to become unregulated like the myosin from which the loop was derived. Dephosphorylated chimeric HMMs gained the ability to move actin in a motility assay and had 50 -70% of the actinactivated ATPase activity of phosphorylated wild-type HMM. Direct binding measurements showed that the affinity of HMM for actin in the presence of MgATP was unaffected by loop substitution; thus the rate of a step other than binding is increased. Phosphorylation of the chimeras did not lead to a higher V max than obtained for wild-type HMM. In the absence of actin, a foreign loop did not affect nucleotide trapping. Native regulated molecules have thus evolved a loop sequence which prevents rapid product release by actin when the RLC is dephosphorylated, thereby allowing activity to be controlled by RLC phosphorylation.Myosins differ from each other in how fast they can move actin, the rates of their actin-activated ATPases, and whether or not these activities are regulated. It has been recently suggested that a divergent surface loop at the actin-binding interface "tunes" the rate of phosphate release and thus sets the maximum velocity (V max ) for ATPase activity. A second loop, at the 25/50-kDa interface near the ATP-binding site, was proposed to control the rate of ADP release and thus be responsible for determining the velocity at which different myosins move actin (Spudich, 1994). The actin-activated ATPase activity of a chimeric Dictyostelium myosin containing the actin-binding loop from skeletal muscle myosin was 5-fold higher than wildtype Dictyostelium myosin, providing experimental evidence in support of the first part of this hypothesis (Uyeda et al., 1994). This study did not show if such a generalization would hold true for other myosin motors nor was the question of heavy chain sequences involved in regulation of activity addressed.The molecular step that is controlled by light chain phosphorylation in smooth muscle myosin is phosphate release from the active site (Sellers, 1985). It has been recently proposed that myosin is a "back door" enzyme, whereby the cleaved phosphate leaves via a cleft in the 50-kDa domain, instead of through the nucleotide-binding pocket from which it entered. Binding of actin is suggested to promote movement of the highly conserved P-loop near the active site so that phosphate can leave (Yount et al., 1995). The interaction of actin with myosin probably involves several steps; the first is thought to be a weak interaction between the N terminus of actin and the 50/20-kDa junction of myosin, followed by stronger interactions involving hydrophobic res...

Three-dimensional image reconstruction of dephosphorylated smooth muscle heavy meromyosin reveals asymmetry in the interaction between myosin heads and placement of subfragment 2

“…Chimeric substitutions of the actin-binding loop (HC residues 626-653) with the corresponding loop from skeletal muscle myosin produced a molecule enzymatically active in the dephosphorylated state (32). In our structure, the part of this insert that is visible in the ''blocked'' head (residues 626-634) is located immediately behind the surface that interacts with the ''free'' head.…”

Phosphorylation of a Single Head of Smooth Muscle Myosin Activates the Whole Molecule

“…In constructs where the rod region was truncated to 7-heptads or less of coiled-coil, but dimerization was enforced with a leucine zipper, dephosphorylated HMM molecules displayed a range of activities which were a sizeable percentage of the activity of doubly phosphorylated controls (10). Chimeric smooth muscle HMM constructs in which Loop 2 was replaced by skeletal myosin sequence moved actin filaments in the motility assay and had at least 50% of wild-type, phosphorylated ATPase activity, all in the absence of phosphorylation (35). These findings reinforce the idea that formation of the inhibited, intramolecular complex described above requires specific head/head and head/rod interactions, which can be disrupted in mutant or truncated constructs.…”

Kinetic Differences in Cardiac Myosins with Identical Loop 1 Sequences

“…The sequences of the loops have been shown to vary significantly between and within classes of myosins (3), and these variations are thought to account for differences in the motile properties of these motor proteins (4). Studies using chimeric Dictyostelium myosin (5) and chimeric smooth muscle myosin (6) have shown that differences in loop 2 alter the steady state ATPase activity, although it is still not known whether loop 2 is a determinant of V max , K m , or both. These studies also show that the ATPase activities (in solution) and motor velocities of myosins are not proportional, a finding that is consistent with previous suggestions that actin-activated ATPase and motility have different rate-limiting steps (7) and might therefore be controlled by distinct molecular mechanisms.…”

“…Results from recombinant Dictyostelium myosins suggested that V max is determined by the sequence of loop 2 (5). Subsequent studies on recombinant smooth muscle myosins showed that K m (not V max ) is determined by loop 2 (6) and that V max is influenced by alterations in loop 1 (10,11). In view of these earlier results, the sequences of loop 1 in the myosins used in the present study were also assessed.…”