A bent monomeric conformation of myosin from smooth muscle.

Abstract

Smooth muscle myosin filaments formed in 0.15 M KCI are depolymerized by MgATP to a lOS component, rather than to the 6S component typical of myosin monomer in high salt concentrations. This lOS species is also monomeric as determined by sedimentation equilibrium and calculated from the diffusion and sedimentation coefficients. The conformation of lOS myosin is, however, very different from that of 6S myosin, which has a flexible but extended rod. The Stokes radius and the viscosity of lOS myosin are less than those of 6S myosin, consistent with a structure in which the rod is bent. Electron microscopy of rotaryshadowed preparations confirmed that the light meromyosin region of the rod is bent back on subfragment 2, that region of the rod adjacent to the two globular heads. MgATP and dephosphorylation of the 20,000 molecular weight light chain increase the amount of 10S myosin present in 0.15 M KCI; addition of salt converts lOS myosin back to the typical 6S conformation. We conclude that smooth muscle myosin preferentially forms a bent or folded conformation instead ofthe extended shape usually associated with skeletal muscle myosin, provided that the salt concentration is kept sufficiently low. Here we show that 10S myosin is not a dimer, but a monomer in which the rod is folded back upon itself such that the light meromyosin (LMM) region interacts with its own subfragment-2 (S-2) region. Addition of salt reforms the 6S conformation in which the rod is extended. The salt concentration thus has a large effect on the conformation of monomeric smooth muscle myosin, with this unusual bent form occurring at physiological ionic strength. MATERIALS AND METHODSPreparation of Calf Aorta Myosin. Myosin was prepared as described by Megerman and Lowey (7). To minimize phosphorylation of myosin by light chain kinase, 1 mM EGTA was added to all buffers, and MgATP was added to the crude myosin just prior to gel filtration. In this way, myosin was isolated with <10% phosphorylation. Myosin concentration was determined from Al% = 4.8.Gel Electrophoresis. NaDodSO4/polyacrylamide gel electrophoresis was performed according to the method ofLaemmli (8), with a 5-20% gradient ofacrylamide and a 4% stacking gel. Crosslinked myosin was separated on 2.5% acrylamide/0.5% agarose slab gels in the Laemmli buffer system. Phosphorylated and dephosphorylated LC20s were separated on glycerol/ acrylamide gels. The running gel contained 40% (vol/vol) glycerol and 10% acrylamide; the stacking gel, 40% glycerol and 3.5% acrylamide. The running and gel buffers were 20 mM Tris/ glycine, pH 8.6 (9). Myosin samples were applied to the gel in 30 mM Tris/glycine, pH 8.6/8 M urea/0.2 mM EDTA/0.1% 2-mercaptoethanol.Crosslinldng. A 20 mg/ml solution ofdimethyl suberimidate was prepared immediately before use. Myosin at 1 mg/ml (10 mM KPi, pH 7.5/5 mM MgCl2/1 mM EGTA/1 mM MgATP/ 0.15 or 0.6 M KCl) was crosslinked by addition of dimethyl suberimidate at 6 mg/ml for 1 hr at room temperature. As determined by sedimentation velocity, crosslinked 10S ...

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

“…It is known, for example, that regulation is highly ionic strength-dependent (8), and it is increasing ionic strength to which HMM is subjected during rotary shadowing specimen preparation. These effects can be somewhat offset by using peptide crosslinkers, and in those few instances where a crosslinker has been used, a clearly more compact arrangement of the myosin heads can be detected, as predicted by our model (25,27).…”

“…Before this study, the only evidence for the structure of dephosphorylated smooth muscle HMM came from rotary shadowing electron microscopy of isolated molecules (25). Typically, these preparations reveal a conformation with the heads folded backwards toward the S2 domain (26).…”

Complete nucleotide sequence and deduced polypeptide sequence of a nonmuscle myosin heavy chain gene from Acanthamoeba: evidence of a hinge in the rodlike tail.

“…It will be interesting to see whether vertebrate smooth muscle and nonmuscle myosins, which adopt the 10S monomer conformation requiring sharp bends at two constant positions within the rod (11,43), have similar proline-containing hinges. For striated muscle myosins, a hinge region has been postulated to exist about one-third of the way down the length of the rod, based on sequence analysis, electron microscopy, protease sensitivity, and biophysical measurements (for review see reference 19).…”

Requirement of the 20-kDa light chain for the papain-resistant conformation of gizzard myosin

“…It has been reported that unphosphorylated gizzard myosin disassembly by ATP has a sedimentation coefficient of 10 S and a molecular mass of 500 kDa, the latter value being the same to that of 6-S myosin in 0.6 M KC1 [2,10]. The structural difference between the 6-S and 10-S forms of smooth muscle myosin has been clarified by electron microscopic studies as follows : while 6-S myosin in high-salt medium exhibits an extended tail, 10-S myosin in low-salt solution containing ATP shows a looped tail conformation with a crossing point between the S2 region of the myosin neck and the specific hinge at one-third from the C-terminus of the rod [2,11]. Accordingly, it is likely that ATP induces some structural changes in each filament component, that is, each myosin molecule.…”

“…Formations of actin thin filament and myosin thick filament in smooth muscle and non-muscle tissues seem to be prerequisites for actomyosin to generate mechanical force for the contraction. Recently, evidence has been accumulating that Ca2 + calmodulin-dependent myosin light chain kinase phosphorylates the regulatory light chain, resulting in the assembly of myosins from various sources, including gizzard [I], aorta [2], thymus [3, 41, platelet [3] and thyroid [5]. However, the detailed mechanism of myosin assembly by the phosphorylation of its light chain remains to be investigated.…”