Search

We have performed molecular dynamics simulations of the phosphorylated (at S-19) and the unphosphorylated 25-residue N-terminal phosphorylation domain of the regulatory light chain (RLC) of smooth muscle myosin to provide insight into the structural basis of regulation. This domain does not appear in any crystal structure, so these simulations were combined with site-directed spin labeling to define its structure and dynamics. Simulations were carried out in explicit water at 310 K, starting with an ideal alpha-helix. In the absence of phosphorylation, large portions of the domain (residues S-2 to K-11 and R-16 through Y-21) were metastable throughout the simulation, undergoing rapid transitions among alpha-helix, pi-helix, and turn, whereas residues K-12 to Q-15 remained highly disordered, displaying a turn motif from 1 to 22.5 ns and a random coil pattern from 22.5 to 50 ns. Phosphorylation increased alpha-helical order dramatically in residues K-11 to A-17 but caused relatively little change in the immediate vicinity of the phosphorylation site (S-19). Phosphorylation also increased the overall dynamic stability, as evidenced by smaller temporal fluctuations in the root mean-square deviation. These results on the isolated phosphorylation domain, predicting a disorder-to-order transition induced by phosphorylation, are remarkably consistent with published experimental data involving site-directed spin labeling of the intact RLC bound to the two-headed heavy meromyosin. The simulations provide new insight into structural details not revealed by experiment, allowing us to propose a refined model for the mechanism by which phosphorylation affects the N-terminal domain of the RLC of smooth muscle myosin.

The use of site-directed spectroscopic probes in elucidating the molecular mechanism of force generation in muscle is reviewed, focusing on recent technical and conceptual advances. High-resolution crystal structures of actin and myosin suggest mechanistic models for force generation, but spectroscopic probe experiments under functional conditions are required to test and refine these models. An array of complementary spectroscopic techniques has been developed that can detect disorder and dynamics directly, resolve multiple conformational states, and can be applied to complex protein assemblies, taking advantage of the beautiful symmetry of the myofibrillar lattice. Recently developed instrumentation has the capacity to resolve simultaneously both biochemical and structural kinetics of myosin and/or actin. These new insights support a model in which structural and biochemical transitions are not tightly coupled, and disorder-to-order transitions play central roles in both force generation and its regulation.

We have performed complementary time-resolved fluorescence resonance energy transfer (TR-FRET) experiments and molecular dynamics (MD) simulations to elucidate structural changes in the phosphorylation domain (PD) of smooth muscle regulatory light chain (RLC) bound to myosin. PD is absent in crystal structures, leaving uncertainty about the mechanism of regulation. Donor-acceptor pairs of probes were attached to three site-directed di-Cys mutants of RLC, each having one Cys at position 129 in the C-terminal lobe and the other at position 2, 3, or 7 in the N-terminal PD. Labeled RLC was reconstituted onto myosin subfragment 1 (S1). TR-FRET resolved two simultaneously populated structural states of RLC, closed and open, in both unphosphorylated and phosphorylated biochemical states. All three FRET pairs show that phosphorylation shifts the equilibrium toward the open state, increasing its mol fraction by ∼20%. MD simulations agree with experiments in remarkable detail, confirming the coexistence of two structural states, with phosphorylation shifting the system toward the more dynamic open structural state. This agreement between experiment and simulation validates the additional structural details provided by MD simulations: In the closed state, PD is bent onto the surface of the C-terminal lobe, stabilized by interdomain salt bridges. In the open state, PD is more helical and straight, resides farther from the C-terminal lobe, and is stabilized by an intradomain salt bridge. The result is a vivid atomic-resolution visualization of the first step in the molecular mechanism by which phosphorylation activates smooth muscle.

Spectroscopy of myosin and actin has entered a golden age. High-resolution crystal structures of isolated actin and myosin have been used to construct detailed models for the dynamic actomyosin interactions that move muscle. Improved protein mutagenesis and expression technologies have facilitated site-directed labeling with fluorescent and spin probes. Spectroscopic instrumentation has achieved impressive advances in sensitivity and resolution. Here we highlight the contributions of site-directed spectroscopic probes to understanding the structural dynamics of myosin II and its actin complexes in solution and muscle fibers. We emphasize studies that probe directly the movements of structural elements within the myosin catalytic and light-chain domains, and changes in the dynamics of both actin and myosin due to their alternating strong and weak interactions in the ATPase cycle. A moving picture emerges in which single biochemical states produce multiple structural states, and transitions between states of order and dynamic disorder power the actomyosin engine.

We have engineered site-directed probe pairs in the regulatory light chain (RLC) for exchange into smooth muscle heavy meromyosin (HMM) and subfragment-1 (S1), in order to examine the phosphorylation-induced changes in RLC structural states using time-resolved FRET (TR-FRET). Phosphorylation of the RLC is required for activation of contraction in smooth muscle and modulates force in striated muscle. Force development in smooth muscle is triggered by phosphorylation of the RLC's N-terminal phosphorylation domain (PD) at Ser19, alleviating inhibitory interactions between the two heads. Since crystal structures of the RLC lack the PD, the mechanism by which RLC phosphorylation allosterically triggers disruption of HMM auto-inhibition remains unresolved. Our site-directed FRET probe pairs resolved intramolecular atomic distance measurements between the RLC core and the dynamic PD. Smooth muscle RLC assumes two structural states in both states of activation: phosphorylation shifts the equilibrium of these two structural states from primarily occupying the compact closed state to favoring the extended open state of the PD, relative to the RLC core. Compared to single-headed S1 (Kast et al., PNAS., 2010), the open state RLC conformation of unphosphorylated HMM is more compact, presumably due to head-head or RLC-RLC interactions. All-atom MD simulations corroborate our TR-FRET studies, and reveal specific salt-bridges that stabilize each structural state. The hypotheses generated from our MD simulations are being tested experimentally by TR-FRET via site-directed mutagenesis to manipulate charge. These studies offer the first atomic-resolution insight into the structural dynamics of RLC phosphorylation, and a similar approach should be applicable to striated muscle. Spectroscopy was performed in the Biophysical Spectroscopy Center at the University of Minnesota. This work was funded by NIH grants to DDT (R01 AR32961, P30 AR0507220, T32 AR007612).

We have performed molecular dynamics simulations of the phosphorylation domain (PD) of the regulatory light chain (RLC) of smooth muscle myosin, to gain insight into the thermodynamic principles governing the phosphorylation-induced disorder-to-order transition. Simulations were performed in explicit water under near-physiological conditions, starting with an ideal alpha-helix. In the absence of phosphorylation, the helical periodicity of the peptide was disrupted at residues T9-K11, while phosphorylation significantly favored the helical periodicity, in agreement with experimental data. Using the MM/PBSA approach, we calculated a relative free energy of -7.1 kcal/mol for the disorder-to-order transition. A large enthalpic decrease was compensated by a large loss of conformational entropy, despite the small helical increase (no more than three residues) upon phosphorylation. Phosphorylation decreased the conformational dynamics of K and R side chains, especially R16, which forms a salt bridge with pS19. Mutation of R16 to A or E prevented this phosphorylation-dependent ordering. We propose that phosphorylation balances the enthalpy-entropy compensation of the disorder-to-order transition of RLC via short and long-range electrostatic interactions with positively charged residues of the phosphorylation domain. We suggest that this balance is necessary to induce a disorder-to-order conformational change through a subtle energy switching.

We have used site-directed time-resolved FRET to determine light-chain domain transitions in expressed Dictyostelium full-length myosin S1. Myosin S1 is proposed to undergo large rotations of the lever-arm in response to the nucleotide state. Previous fluorescence experiments (Shih et al., 2000) have shown that S1 adopts two populations of pre-stroke states and the equilibrium between these states is driven by nucleotide. To observe these population changes in detail, and to observe the effect of actin, we have used time-resolved fluorescence resonance energy transfer (TR-FRET) to measure the distribution of distances between the labeled catalytic domain (A250C) and a labeled RLC (M129C). In the present work, we show that the lever arm adopts multiple orientations in solution. In the absence of nucleotide, the light-chain domain probe is at a mean distance of 8.4 nm from the catalytic domain probe. This distance extends to >9.0 nm when flS1 is bound to actin. The addition of ATP gives three roughly equal populations of distances at 3.6, 4.3, and >9.0 nm. ADP.Vi stabilizes the shorter of these populations, indicating that ADP.Pi induces a conformational change of the light-chain domain. This work was supported by grants from NIH (AR32961, AR07612). We thank Igor Negrashov for excellent technical assistance.

Using site-directed fluorescence labeling, transient time resolved FRET ([TR]2FRET) and time resolved fluorescence anisotropy, we have measured actin- and nucleotide-induced structural changes within the actin binding cleft of Dictyostelium myosin II. In a recent report using time-resolved pulsed EPR spectroscopy, distances were measured between paramagnetic probes attached to the upper and lower 50 kDa subdomains of Dictyostelium myosin II (Klein et al., PNAS, 105:12867-72). These results support the hypothesis that the actin-binding cleft closes partially upon actin binding, but also suggested that both open and closed conformations are simultaneously present, with nucleotides and actin controlling the open-closed equilibrium. Due to technical constraints, those EPR distance measurements were limited to frozen samples. In the present study, time-resolved fluorescence is used to probe the cleft in solution under more physiologically relevant conditions, including the transient phase of the ATPase reaction. Fluorescent probes were attached to engineered Cys residues in the upper and lower 50 kDa subdomains and used to measure the distance across the cleft. Single probes attached to either subdomain were used in combination with fluorescent nucleotides to monitor the coupling between the actin-binding cleft and the active site. Here we use a combination of transient time resolved FRET and transient time resolved fluorescence anisotropy to probe the equilibrium between open and closed cleft conformations. In the key experiments, a complete nanosecond time-resolved fluorescence decay was measured, defining the detailed distance distribution between probes, every 0.1 ms following rapid mixing (stopped flow), thus yielding high-resolution structural information on the sub-millisecond time scale. The results provide new insights into the coupling between the actin-binding cleft, the active site, and actin binding.

We have combined time-resolved fluorescence resonance energy transfer (TR-FRET) and molecular dynamics (MD) simulations to elucidate structural changes in the phosphorylation domain (PD) of chicken gizzard smooth muscle regulatory light chain (RLC) bound to smooth muscle myosin. The PD is absent in crystal structures, leaving uncertainty about the mechanism of regulation. Donor-acceptor pairs of probes were attached to three site-directed di-Cys mutants, each having one Cys at position 129 in the C-terminal lobe and the other at position 2, 3, or 7 in the N-terminal PD. Labeled RLCs, with and without phosphorylation at S19, were reconstituted into myosin S1. Time-resolved FRET identified two structural states of the RLC, closed and open, which are present in both unphosphorylated and phosphorylated biochemical states. Phosphorylation shifts the equilibrium toward the open state by 20-30%. Molecular dynamics simulations, guided by these and previous spectroscopic studies, confirm the existence of the open and closed structural states and produce atomic resolution models of these states. In the closed state, the PD interacts with the surface of the C-terminal lobe. In the open state, the PD is more helical and straight, resides farther from the C-terminal lobe, and is more mobile. Phosphorylation stabilizes the open state by forming a specific salt bridge between R16 and phosphorylated S19. This conformational shift is consistent with a mechanism of regulation that catalyzes large structural changes within myosin at a low energetic cost. This work was supported by grants from NIH (AR32961, AR07612) and the Minnesota Supercomputing Institute. We thank Igor Negrashov for excellent technical assistance.