Michael S. Carter

Biochemistry of smooth muscle myosin light chain kinase

The smooth muscle isoform of myosin light chain kinase (MLCK) is a Ca(2+)-calmodulin-activated kinase that is found in many tissues. It is particularly important for regulating smooth muscle contraction by phosphorylation of myosin. This review summarizes selected aspects of recent biochemical work on MLCK that pertains to its function in smooth muscle. In general, the focus of the review is on new findings, unresolved issues, and areas with the potential for high physiological significance that need further study. The review includes a concise summary of the structure, substrates, and enzyme activity, followed by a discussion of the factors that may limit the effective activity of MLCK in the muscle. The interactions of each of the many domains of MLCK with the proteins of the contractile apparatus, and the multi-domain interactions of MLCK that may control its behaviors in the cell are summarized. Finally, new in vitro approaches to studying the mechanism of phosphorylation of myosin are introduced.

Velocities of unloaded muscle filaments are not limited by drag forces imposed by myosin cross-bridges

Significance In vitro experiments that measure relative sliding velocities between actin and myosin filaments give insight into how the muscle motor protein myosin works in muscle. We show that when the physiological filamentous myosin moves along actin filaments, the velocities cannot be explained by the prevailing theory. Instead, we propose a paradigm in which myosin heads undergo unloaded “powerstrokes” rather than work in mechanical opposition to each other during contraction. This model is consistent with unloaded shortening velocities measured in muscle. It is not known which kinetic step in the acto-myosin ATPase cycle limits contraction speed in unloaded muscles (V0). Huxley’s 1957 model [Huxley AF (1957) Prog Biophys Biophys Chem 7:255–318] predicts that V0 is limited by the rate that myosin detaches from actin. However, this does not explain why, as observed by Bárány [Bárány M (1967) J Gen Physiol 50(6, Suppl):197–218], V0 is linearly correlated with the maximal actin-activated ATPase rate (vmax), which is limited by the rate that myosin attaches strongly to actin. We have observed smooth muscle myosin filaments of different length and head number (N) moving over surface-attached F-actin in vitro. Fitting filament velocities (V) vs. N to a detachment-limited model using the myosin step size d = 8 nm gave an ADP release rate 8.5-fold faster and ton (myosin’s attached time) and r (duty ratio) ∼10-fold lower than previously reported. In contrast, these data were accurately fit to an attachment-limited model, V = N·v·d, over the range of N found in all muscle types. At nonphysiologically high N, V = L/ton rather than d/ton, where L is related to the length of myosin’s subfragment 2. The attachment-limited model also fit well to the [ATP] dependence of V for myosin-rod cofilaments at three fixed N. Previously published V0 vs. vmax values for 24 different muscles were accurately fit to the attachment-limited model using widely accepted values for r and N, giving d = 11.1 nm. Therefore, in contrast with Huxley’s model, we conclude that V0 is limited by the actin–myosin attachment rate.

Effects of actin-myosin kinetics on the calcium sensitivity of regulated thin filaments

Activation of thin filaments in striated muscle occurs when tropomyosin exposes myosin binding sites on actin either through calcium-troponin (Ca-Tn) binding or by actin-myosin (A-M) strong binding. However, the extent to which these binding events contributes to thin filament activation remains unclear. Here we propose a simple analytical model in which strong A-M binding and Ca-Tn binding independently activates the rate of A-M weak-to-strong binding. The model predicts how the level of activation varies with pCa as well as A-M attachment, N·katt, and detachment, kdet, kinetics. To test the model, we use an in vitro motility assay to measure the myosin-based sliding velocities of thin filaments at different pCa, N·katt, and kdet values. We observe that the combined effects of varying pCa, N·katt, and kdet are accurately fit by the analytical model. The model and supporting data imply that changes in attachment and detachment kinetics predictably affect the calcium sensitivity of striated muscle mechanics, providing a novel A-M kinetic-based interpretation for perturbations (e.g. disease-related mutations) that alter calcium sensitivity.

The myosin duty ratio tunes the calcium sensitivity and cooperative activation of the thin filament.

In striated muscle, calcium binding to the thin filament (TF) regulatory complex activates actin-myosin ATPase activity, and actin-myosin kinetics in turn regulates TF activation. However, a quantitative description of the effects of actin-myosin kinetics on the calcium sensitivity (pCa50) and cooperativity (nH) of TF activation is lacking. With the assumption that TF structural transitions and TF-myosin binding transitions are inextricably coupled, we advanced the principles established by Kad et al. [Kad, N., et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 16990-16995] and Sich et al. [Sich, N. M., et al. (2011) J. Biol. Chem. 285, 39150-39159] to develop a simple model of TF regulation, which predicts that pCa50 varies linearly with duty ratio and that nH is maximal near physiological duty ratios. Using in vitro motility to determine the calcium sensitivity of TF sliding velocities, we measured pCa50 and nH at different myosin densities and in the presence of ATPase inhibitors. The observed effects of myosin density and actin-myosin duty ratio on pCa50 and nH are consistent with our model predictions. In striated muscle, pCa50 must match cytosolic calcium concentrations and a maximal nH optimizes calcium responsiveness. Our results indicate that pCa50 and nH can be predictably tuned through TF-myosin ATPase kinetics and that drugs and disease states that alter ATPase kinetics can, through their effects on calcium sensitivity, alter the efficiency of muscle contraction.

Sucrose increases the activation energy barrier for actin-myosin strong binding.

To determine the mechanism by which sucrose slows in vitro actin sliding velocities, V, we used stopped flow kinetics and a single molecule binding assay, SiMBA. We observed that in the absence of ATP, sucrose (880mM) slowed the rate of actin-myosin (A-M) strong binding by 71±8% with a smaller inhibitory effect observed on spontaneous rigor dissociation (21±3%). Similarly, in the presence of ATP, sucrose slowed strong binding associated with Pi release by 85±9% with a smaller inhibitory effect on ATP-induced A-M dissociation, kT (39±2%). Sucrose had no noticeable effect on any other step in the ATPase reaction. In SiMBA, sucrose had a relatively small effect on the diffusion coefficient for actin fragments (25±2%), and with stopped flow we showed that sucrose increased the activation energy barrier for A-M strong binding by 37±3%, indicating that sucrose inhibits the rate of A-M strong binding by slowing bond formation more than diffusional searching. The inhibitory effects of sucrose on the rate of A-M rigor binding (71%) are comparable in magnitude to sucroses effects on both V (79±33% decrease) and maximal actin-activated ATPase, kcat, (81±16% decrease), indicating that the rate of A-M strong bond formation significantly influences both kcat and V.

Kinetics of myosin light chain kinase activation of smooth muscle myosin in an in vitro model system.

During activation of smooth muscle contraction, one myosin light chain kinase (MLCK) molecule rapidly phosphorylates many smooth muscle myosin (SMM) molecules, suggesting that muscle activation rates are influenced by the kinetics of MLCK-SMM interactions. To determine the rate-limiting step underlying activation of SMM by MLCK, we measured the kinetics of calcium-calmodulin (Ca²⁺CaM)-MLCK-mediated SMM phosphorylation and the corresponding initiation of SMM-based F-actin motility in an in vitro system with SMM attached to a coverslip surface. Fitting the time course of SMM phosphorylation to a kinetic model gave an initial phosphorylation rate, kp(o), of ~1.17 heads s⁻¹ MLCK⁻¹. Also, we measured the dwell time of single streptavidin-coated quantum dot-labeled MLCK molecules interacting with surface-attached SMM and phosphorylated SMM using total internal reflection fluorescence microscopy. From these data, the dissociation rate constant from phosphorylated SMM was 0.80 s⁻¹, which was similar to the kp(o) mentioned above and with rates measured in solution. This dissociation rate was essentially independent of the phosphorylation state of SMM. From calculations using our measured dissociation rates and Kd values, and estimates of SMM and MLCK concentrations in muscle, we predict that the dissociation of MLCK from phosphorylated SMM is rate-limiting and that the rate of the phosphorylation step is faster than this dissociation rate. Also, association with SMM (11-46 s⁻¹) would be much faster than with pSMM (<0.1-0.2 s⁻¹). This suggests that the probability of MLCK interacting with unphosphorylated versus phosphorylated SMM is 55-460 times greater. This would avoid sequestering MLCK to unproductive interactions with previously phosphorylated SMM, potentially leading to faster rates of phosphorylation in muscle.

Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle

Smooth muscle myosin (SMM) light chain kinase (MLCK; UniProt accession no. Q15746-7) is a Ca-calmodulin (CaM)–activated kinase (Hong et al., 2011) that is required for smooth muscle contraction (Itoh et al., 1989; He et al., 2008; Zhang et al., 2010) and regulation of tone in most of the hollow organs of the body (Kamm and Stull, 2011). It phosphorylates the regulatory light chains (RLCs) of SMM (a myosin II), causing a switch from low to high actin–activated myosin ATPase activity. Similar kinases modulate the activities of other myosin IIs in skeletal and cardiac muscles, and also in nonmuscle cells (Kamm and Stull, 2001). The predominant form of MLCK in smooth muscle (Fig. 1 A) is a long multi-domain protein (Mabuchi et al., 2010) containing an N-terminal actin-binding domain (ABD; three DFRXXL motifs; Gallagher and Stull, 1997) within the first 75 residues, IgG and Fn domains, a kinase domain, and a C-terminal myosin-binding domain

Dynamics of Regulated Thin Filament Motility

The cooperative activation of regulated thin filaments (RTFs) is important for efficient calcium handling in striated muscle. Although myosin binding to an RTF has been proposed as a mechanism for cooperative activation, we have recently shown that myosin binding constitutively rather than cooperatively activates RTFs; in other words the binding of a single myosin head fully activates the regulatory unit of an RTF. The model predicts a well-defined relationship between the calcium concentration at half-maximal activation (pCa50) and all factor that influences actin-myosin attachment, katt, or detachment, kdet, kinetics. To test this model, we use an in vitro motility assay to determine the effects of altering katt and kdet on the calcium dependence of RTF sliding velocities, V. The model accurately describes our observed changes in pCa50 when katt and kdet are modulated with changes in [ATP] and [ADP], upon addition of inhibitors, and when myosin densities are varied. The model further predicts that partially activated V result from the RTF switching between fully active and inactive states rather than the RTF maintaining a partial level of activation. To test this hypothesis, we are using an in vitro motility assay to measure the dynamics of speckled fluorescent actin filaments with high spatial accuracy and temporal resolution. Our model and data suggest that calcium sensitivity of striated muscle is altered by changes in actin-myosin attachment and detachment kinetics.