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Modeling smooth muscle myosin's two heads: long-lived enzymatic roles and phosphorylation-dependent equilibria.

ABSTRACT: Smooth muscle myosin has two heads, each capable of interacting with actin to generate force and/or motion as it hydrolyzes ATP. These heads are inhibited when their associated regulatory light chain is unphosphorylated (0P), becoming active and hydrolyzing ATP maximally when phosphorylated (2P). Interestingly, with only one of the two regulatory light chains phosphorylated (1P), smooth muscle myosin is active but its ATPase rate is <2P. To explain published 1P single ATP turnover and steady-state ATPase activities, we propose a kinetic model in which 1P myosin exists in an equilibrium between being fully active (2P) and inhibited (0P). Based on the single ATP turnover data, we also propose that each 2P head adopts a hydrolytic role distinct from its partner at any point in time, i.e., one head strongly binds actin and hydrolyzes ATP at its actin-activated rate while the other weakly binds actin. Surprisingly, the heads switch roles slowly (<0.1 s(-1)), suggesting that their activities are not independent. The phosphorylation-dependent equilibrium between active and inhibited states and the hydrolytic role that each head adopts during its interaction with actin may have implications for understanding regulation and mechanical performance of other members of the myosin family of molecular motors.

SUBMITTER: Walcott S

PROVIDER: S-EPMC2920627 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Modeling smooth muscle myosin's two heads: long-lived enzymatic roles and phosphorylation-dependent equilibria.

Walcott Sam S Warshaw David M DM

Biophysical journal 20100801 4

Smooth muscle myosin has two heads, each capable of interacting with actin to generate force and/or motion as it hydrolyzes ATP. These heads are inhibited when their associated regulatory light chain is unphosphorylated (0P), becoming active and hydrolyzing ATP maximally when phosphorylated (2P). Interestingly, with only one of the two regulatory light chains phosphorylated (1P), smooth muscle myosin is active but its ATPase rate is <2P. To explain published 1P single ATP turnover and steady-sta ...[more]

PMID: 20712996

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Project description:Caldesmon is a major calmodulin- and actin-binding protein of smooth muscle which interacts with calmodulin in a Ca2+-dependent manner or with actin in a Ca2+-independent manner. Isolated caldesmon is capable of inhibiting the actin-activated Mg2+-ATPase of smooth-muscle myosin, suggesting a possible physiological role for caldesmon in regulating the contractile state of smooth-muscle. Caldesmon can be phosphorylated in vitro by a co-purifying Ca2+/calmodulin-dependent protein kinase and dephosphorylated by a protein phosphatase, both of which are present in smooth muscle. We investigated further the phosphorylation of caldesmon and the effects which phosphorylation has on the functional properties of the protein. The kinetics of caldesmon phosphorylation were similar whether the caldesmon substrate was free or bound to actin, actin/tropomyosin or thin filaments. Caldesmon containing endogenous kinase activity was rapidly phosphorylated (to approx. 1 mol of Pi/mol of caldesmon in 5 min) when reconstituted with actin, myosin, tropomyosin, calmodulin and myosin light-chain kinase in the presence of Ca2+ and MgATP2-. Under conditions in which unphosphorylated caldesmon showed substantial inhibition of the actin-activated myosin Mg2+-ATPase, no inhibition was observed with phosphorylated caldesmon. This was the case whether caldesmon was phosphorylated before addition to the actomyosin Mg2+-ATPase system, or phosphorylation was allowed to take place during the ATPase reaction. Binding studies revealed maximal binding of 1 mol of unphosphorylated caldesmon/9.5 mol of actin and 1 mol of phosphorylated caldesmon/11.7 mol of actin. All the bound phosphorylated caldesmon could be released by Ca2+/calmodulin, with half-maximal release at 0.11 microM-Ca2+, whereas only 62% of the bound unphosphorylated caldesmon could be removed, with half-maximal release at 0.16 microM-Ca2+. However, under conditions in which inhibition of actomyosin Mg2+-ATPase activity by non-phosphorylated but not by phosphorylated caldesmon was observed, both forms of caldesmon would remain bound to the thin filament. These observations suggest a possible mechanism whereby caldesmon phosphorylation may prevent its inhibitory action on the actomyosin Mg2+-ATPase.

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Modeling smooth muscle myosin's two heads: long-lived enzymatic roles and phosphorylation-dependent equilibria.

Publications

Modeling smooth muscle myosin's two heads: long-lived enzymatic roles and phosphorylation-dependent equilibria.

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