Heads Of Smooth Muscle Myosin Research Articles

Cooperation between the Two Heads of Smooth Muscle Myosin Is Essential for Full Activation of the Motor Function by Phosphorylation

The motor function of smooth muscle myosin (SmM) is regulated by phosphorylation of the regulatory light chain (RLC) bound to the neck region of the SmM heavy chain. It is generally accepted that unphosphorylated RLC induces interactions between the two heads and between the head and the tail, thus inhibiting the motor activity of SmM, whereas phosphorylation of RLC interrupts those interactions, thus reversing the inhibition and restoring the motor activity to the maximal value. One assumption of this model is that single-headed SmM is fully active regardless of phosphorylation. To re-evaluate this model, we produced a number of SmM constructs with coiled coils of various lengths and examined their structure and regulation. With these constructs we identified the segment in the coiled-coil key for the formation of a stable double-headed structure. In agreement with the current model, we found that the actin-activated ATPase activity of unphosphorylated SmM increased with shortening of the coiled-coil. However, contrary to the current model, we found that the actin-activated ATPase activity of phosphorylated SmM decreased with shortening coiled-coil and only the stable double-headed SmM was fully activated by phosphorylation. These results indicate that single-headed SmM is neither fully active nor fully inhibited. Based on our findings, we propose that cooperation between the two heads is essential, not only for the inhibition of unphosphorylated SmM, but also for the activation of phosphorylated SmM.

Stable Double-Headed Structure is Essential not only for the Inhibited State, but also for the Fully Activated State of Smooth Muscle Myosin

It is generally accepted that dephosphorylation of regulatory light chain (RLC) induces the interactions between the two heads of smooth muscle myosin (Sm) and between the head and the tail, thus inhibiting the motor activity, and phosphorylation of RLC interrupts above interactions, thus reversing the inhibition and stimulating the motor activity to maximal value. Thus it is predicted that Sm subfragment-1 (S1) containing only one head without the tail is fully active. However, no solid evident so far supports this prediction, although early studies showed that S1 produced by limited protease treatment is partially active. Here we produced a number of Sm truncations with various length of the tail. The stability of double-headed structure of Sm is dependent on the length of the coiled-coil tail. The Sm truncations with coiled-coil longer than 214 amino acids form stable double-headed structure (stable HMM), those with coiled-coil shorter than 179 amino acids form unstable double headed structure (unstable HMM), and that without coiled-coil, i.e. S1, is completely single-headed. Phosphorylation of RLC regulates the motor activity of stable HMM completely, regulates that of unstable HMM partially, and does not regulate that of S1. Unexpectedly, the actin-activated ATPase activity of S1, either unphosphorylated or phosphorylated, is higher than that of unphosphorylated stable HMM but less than 10% of that of phosphorylated stable HMM. The actin-activated ATPase activities of unphosphorylated Sm truncations increase with the shortening of the coiled-coil tail, and that of phosphorylated Sm truncations decrease with the shortening of the coiled-coil tail. These results indicate that the stable double-headed structure is critical not only for the inhibited state of unphosphorylated Sm, but also for the fully activated state of phosphorylated Sm.

Two Functional Heads Are Required for Full Activation of Smooth Muscle Myosin

The motor activity of smooth muscle myosin II is regulated by the regulatory light chain phosphorylation, but it is not understood how phosphorylation activates motor activity. To address this question, we produced asymmetric heavy meromyosin (HMM), which is composed of a wild-type (WT) heavy chain and a mutant heavy chain having no motor activity (i.e. S236T or G457A). The actin-activated ATPase activities (Vmax) of asymmetric HMMs were only 21.8 and 8.4% of the wild-type HMM for S236A/WT HMM and G456A/WT HMM, respectively. If the two heads of HMM are independent for their ATPase activities, asymmetric HMM should show 50% of the activity of wild-type HMM; however, the activity of asymmetric HMM was much lower than the expected value. The results suggest that the activity of the wild-type head is attenuated by the presence of inactive head. Consistently, the actin-gliding velocity of the asymmetric HMM (i.e. S236T/WT or G457A/WT) was less than one-fifth of the wild-type HMM. The present study supports an idea that the two heads of smooth muscle myosin II interact with each other and the presence of two active heads is required for full activation.

The Two Heads of Smooth Muscle Myosin Are Enzymatically Independent but Mechanically Interactive

The interaction between the two heads of myosin II during motion and force production is poorly understood. To examine this issue, we developed an expression and purification strategy to isolate homogeneous populations of heterodimeric smooth muscle heavy meromyosins containing heads with different properties. As an extreme example, we characterized a heterodimer containing one native head and one head locked in a "weak binding" state by a point mutation in switch 2 (E470A). The in vitro actin filament motility of this heterodimer was the same as the homodimeric control with two cycling heads, suggesting that only one head of a pair actively interacts with actin to generate maximal velocity. A second naturally occurring heterodimer contained two cycling heads with 2-fold different activity, due to the presence or absence of a 7-amino acid insert near the active site. Enzymatically this (+/-) insert heterodimer was indistinguishable from a (50:50) mixture of the two homodimers, but its motility averaged 17% less than that of the mixture. These data suggest that one head of a heterodimer can disproportionately affect the mechanics of double-headed myosin, a finding relevant to our understanding of heterozygous mutant myosins found in disease states like familial hypertrophic cardiomyopathy.

The interaction between the regulatory light chain domains on two heads is critical for regulation of smooth muscle myosin.

Recent findings have suggested that the interaction between the two heads is critical for phosphorylation-dependent regulation of smooth muscle myosin. We hypothesized that the interaction between the two regulatory light chains on two heads of myosin dictates the regulation of myosin motor function. To evaluate this notion, we engineered and characterized smooth muscle heavy meromyosin (HMM), which is composed of one entire HMM heavy chain and one motor domain truncated heavy chain containing the S2 rod and regulatory light chain (RLC) binding site, as well as the bound RLC (SMDHMM). SMDHMM was inactive for both actin-translocating activity and actin-activated ATPase activity in the dephosphorylated state, demonstrating that the interaction between the two RLC domains on the two heads and/or a motor domain and a RLC domain in a distinct head is sufficient for the inhibition of smooth muscle myosin motor activity. When phosphorylated, SMDHMM was activated for both actin-translocating activity and actin-activated ATPase activity; however, these activities were lower than those of double-headed HMM, implying partial release of inhibition by phosphorylation in SMDHMM and/or cooperativity between the two heads of smooth muscle myosin. The present results indicate that the RLC domain is critical for phosphorylation-dependent regulation of smooth muscle myosin motor activity. On the other hand, similar to double-headed HMM, SMDHMM showed both "folded" and "extended" conformations, and the ratio of those conformations is dependent on ionic strength, suggesting that the RLC domain is sufficient to regulate the conformational transition in myosin.

Two Heads Are Required for Phosphorylation-dependent Regulation of Smooth Muscle Myosin

Recent structural evidence (Rayment, I., Holden, H. M., Whittaker, M., Yohn, C. B., Lorenz, M., Holmes, K. C., and Milligan, R. A. (1993) Science 261, 58-65) suggests that the two heads of skeletal muscle myosin interact when the protein is bound to filamentous actin. Direct chemical cross-linking experiments show that the two heads of smooth muscle myosin interact in the presence of filamentous actin and the absence of ATP (Onishi, H., Maita, T., Matsuda, G., and Fujiwara, K. (1992) Biochemistry 31, 1201-1210). Head-head interactions may be important in the mechanism of phosphorylation-dependent regulation of smooth muscle myosin. To explore the structural elements essential for phosphorylation-dependent regulation, we purified a proteolytic fragment of chicken gizzard myosin containing only one head attached to an intact tail. This molecule contained a partially digested regulatory light chain, which was replaced with exogenously added intact light chain in either the thiophosphorylated or the unphosphorylated state. Control experiments showed that this replacement was nearly quantitative and did not alter the actin-activated ATPase of this myosin. Electron micrographs confirmed that the single-headed preparation contained an intact form of single-headed myosin. The unphosphorylated single-headed myosin hydrolyzed ATP rapidly and moved actin filaments in an in vitro motility assay. Phosphorylation had minimal effects upon these properties. Therefore, we conclude that phosphorylation-dependent regulation in this myosin requires two heads. These findings may have important implications in studies of other regulated motor proteins that contain two motor domains.

Interaction between two myosin heads in acto-smooth muscle heavy meromyosin rigor complex.

Muscle contraction occurs as a result of the cyclic interaction between actin and myosin, coupled with the hydrolysis of ATP by the myosin heads. A myosin molecule consists of two globular heads and a rod-shaped tail. It is thought that each myosin head functions as a contractile machinery unit, because each head shows ATPase activity and binds to F-actin. A still unsolved question is whether or not the two heads of a myosin molecule interact with each other during their cyclic interaction with F-actin. Here we report that when chicken gizzard heavy meromyosin (HMM) in its rigor complex with F-actin is reacted with the zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide (EDC), the two heads of the HMM molecule are cross-linked. This result suggests that the two heads of smooth muscle myosin are in contact with each other when myosin is attached to F-actin. It is thus possible that the two myosin heads interact with each other when cross-bridges are formed.

Reaction of two heads of gizzard myosin with ATP.

The reaction intermediates formed by the two heads of smooth muscle myosin were studied. The amount of myosin-phosphate-ADP complex, MPADP, formed was measured from the Pi-burst size over a wide range of ATP concentrations. At low concentrations of ATP, the Pi-burst size was 0.5 mol/mol myosin head, and the apparent Kd value was about 0.15 microM. However, at high ATP concentrations, the Pi burst size increased from 0.5 to 0.75 mol/mol myosin head with an observed Kd value of 15 microM. The binding of nucleotides to gizzard myosin during the ATPase reaction was directly measured by a centrifugation method. Myosin bound 0.5 mol of nucleotides (ATP and ADP) with high affinity (Kd congruent to 1 microM) and 0.35 mol of nucleotides with low affinity (Kd = 24 microM) for ATP. These results indicate that gizzard myosin has two kinds of nucleotide binding sites, one of which forms MPADP with high affinity for ATP while the other forms MPADP and MATP with low affinity for ATP. We studied the correlation between the formation of MPADP and the dissociation of actomyosin. The amount of Pi-burst size was not affected by the existence of F-actin, and when 0.5 mol of ATP per mol of myosin head was added to actomyosin (1 mg/ml F-actin, 5 microM myosin at 0 degrees C) most (93%) of the added ATP was hydrolyzed in the Pi-burst phase. All gizzard actomyosin dissociated when 1 mol of ATP per mol myosin head was added to actomyosin.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorylation of smooth muscle myosin: evidence for cooperativity between the myosin heads.

The relationship between the actin-activated adenosinetriphosphatase activity of smooth muscle myosin and the extent of myosin light chain phosphorylation is nonlinear. It is suggested that the phosphorylation of the two heads of smooth muscle myosin is an ordered process and that the two heads are influenced by cooperative interactions.