Novel Sensors of the Regulatory Switch on the Regulatory Light Chain of Smooth Muscle Myosin

Abstract

Here we used three different approaches to test this notion, which are reactivity of cysteine thiols, pyrene and acrylodan spectral analysis, and pyrene fluorescence quenching. All methods detected significant differences between the unphosphorylated and phosphorylated regulatory light chain N termini in heavy meromyosin, a double-headed subfragment with an intact regulatory switch. These differences were not observed for subfragment-1, a single-headed, unregulated subfragment. In the presence of either ATP or ADP, phosphorylation increased the solvent exposure and decreased the polarity of the environment about position 23 of the regulatory light chain of heavy meromyosin. These phosphorylation-induced structural changes were not as evident in the absence of nucleotides. Nucleotide binding to unphosphorylated heavy meromyosin caused a decrease in exposure and an increase in polarity of the N terminus, whereas the effects of nucleotide on phosphorylated heavy meromyosin were the opposite. We showed a direct correlation between the kinetics of nucleotide binding/turnover and the conformational change reported by acrylodan at position 23 of the regulatory light chain. Acrylodan-A23C also reports the heads up (extended) to flexed (folded) transition in unphosphorylated heavy meromyosin. This is the first demonstration of direct coupling of nucleotide binding to conformational changes in the N terminus of the regulatory light chain.Smooth muscle myosin (SMM) 1 and nonmuscle myosin are hexameric motor ATPases of ϳ500 kDa mass composed of pairs of HC, ELC, and RLC. A globular motor domain (N-terminal HC) and a light chain binding domain (HC ϩ ELC ϩ RLC) form a head (S1), and the two heads are dimerized by the HC C-terminal halves, which form a coiled-coil. The motor domain contains the catalytic site and the actin binding site (1). Only the head domain and its subfragments have been crystallized to date so there are no atomic resolution structures of a doubleheaded construct.The actin-activated ATPase activity and motor properties of SMM and nonmuscle myosin are regulated by phosphorylation of Ser-19 of the RLC, which is greater than 10 nm from the catalytic site (1-4). Phosphorylation enhances the ATPase activity by more than 1000-fold at saturating actin concentrations (5, 6). Domain requirements for regulation have been elucidated through studies of various proteolytic and expressed subfragments of SMM and nonmuscle myosin. HMM, which lacks the C-terminal two-thirds of the tail, is double-headed and regulated (5-7), but expressed HMM constructs with truncated tails too short to form stable double-headed structures are unregulated (8 -10) as is S1 (7,11,12) and single-headed myosin (13,14). Furthermore, a nonmuscle HMM construct without one of the motor domains but with both regulatory domains is not regulated (15). Therefore, two full heads connected together with enough coiled-coil to allow for dimerization are required for regulation.Removal of the RLC abolishes regulation (16), but the ELC can be removed with re...

Regulatory and Catalytic Domain Dynamics of Smooth Muscle Myosin Filaments

“…Studies with calcium-regulated scallop HMM show similar trends induced by calcium binding (12). The two RLC can be cross-linked in the unphosphorylated, but not phosphorylated smooth HMM (13), and phosphorylation causes a large structural rearrangement of the RLC Nterminus (14) to a more exposed and nonpolar environment (15) consistent with recent EPR studies (16). Cryo-EM image reconstructions of nucleotide-bound smooth HMM ordered on a lipid monolayer have visualized contacts between the two CDs that are abolished upon phosphorylation (17,18).…”

“…But it is clear that nucleotide at the active site biases the structure more toward an OFF state. Mazhari et al (15) showed that the environment of the RLC N-terminus (phosphorylated or not) in the absence of nucleotides was intermediate between the nucleotide bound unphosphorylated and phosphorylated states in HMM. The fact that we see little effect of phosphorylation on the dynamics in the absence of nucleotides is consistent with the general trend of the Mazhari et al data (15).…”

“…Mazhari et al (15) showed that the environment of the RLC N-terminus (phosphorylated or not) in the absence of nucleotides was intermediate between the nucleotide bound unphosphorylated and phosphorylated states in HMM. The fact that we see little effect of phosphorylation on the dynamics in the absence of nucleotides is consistent with the general trend of the Mazhari et al data (15). Therefore, the Taylor model (17,18) may represent a state that is more biased toward the OFF structure than the state that we are measuring in the absence of nucleotides.…”

“…Nucleotides have little effect upon the distribution of the relative head-to-head positions of both phosphorylated and unphosphorylated SMM at low ionic strength as determined by atomic force microscopy (11). Large structural differences between the conformation of the RLC N-terminal domain are observed in the absence of nucleotides (15,16), although nucleotide binding can enhance the structural differences (15). Two measures that are consistent with partial release of cross-bridges from the vertebrate skMM filament surface by phosphorylation, the phosphorylation-induced decrease in the chemical cross-linking rate between the S2 and the filament surface and the increase in the proteolytic susceptibility of the HMM-LMM junction, are observed without nucleotides (26).…”

Broad disorder and the allosteric mechanism of myosin II regulation by phosphorylation

“…27) versus HMM (used here), and (iii) a large structural transition in the N-terminal domain from disordered to ordered helix was found in HMM (without ATP), but not in S1 (27). Yet another study has shown that fluorophores on the RLC N-terminal domain sense changes in solvent exposure upon ATP binding and phosphorylation but only in regulated constructs HMM but not S1 (29). Therefore it is likely that backbone conformational heterogeneity in the N-terminal domain contributes to the heterogeneity in the RLC-RLC distances, consistent with the variation of the distance distribution widths measured here.…”