Smooth Muscle Heavy Meromyosin Phosphorylated on One of Its Two Heads Supports Force and Motion

Walcott, Sam; Fagnant, Patricia M.; Trybus, Kathleen M.; Warshaw, David M.

doi:10.1074/jbc.m109.003293

Cited by 26 publications

(32 citation statements)

References 44 publications

(42 reference statements)

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“…Because the heads come from different molecules, it implies that RLC phosphorylation does not alter the characteristics of the binding interface between myosin MDs, but rather alters the myosin head structure in such a way as to affect the ability of the two heads of a single smHMM molecule to adopt the inactive conformation. We propose that the effect of phosphorylation on the inactive conformation is not equivalent for the two heads, and that phosphorylation of the “blocked head” will lead to activation, consistent with the partial activation that occurs when smHMM is phosphorylated on only one RLC 22 ; 23 .…”

Section: Introductionmentioning

confidence: 64%

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Phosphorylated Smooth Muscle Heavy Meromyosin Shows an Open Conformation Linked to Activation

Baumann¹,

Taylor²,

Huang³

et al. 2012

Journal of Molecular Biology

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Smooth muscle myosin and heavy meromyosin (smHMM) are activated by regulatory light chain (RLC) phosphorylation but the mechanism remains unclear. Dephosphorylated, inactive smHMM assumes a closed conformation with asymmetric intramolecular head-head interactions between motor domains. The “free head” can bind to actin, but the actin-binding interface of the “blocked head” is involved in interactions with the free head. We report here a 3-D structure for phosphorylated, active smHMM obtained using electron crystallography of 2-D arrays. Head-head interactions of phosphorylated smHMM resemble those found in the dephosphorylated state, but occur between different molecules, not within the same molecule. The light chain binding domain structure of phosphorylated smHMM differs markedly from that of the “blocked” head of dephosphorylated smHMM. We hypothesize that RLC phosphorylation opens the inhibited conformation primarily by its effect on the blocked head. Singly phosphorylated smHMM is not compatible with the closed conformation if the blocked head is phosphorylated. This concept has implications for the extent of myosin activation at low levels of phosphorylation in smooth muscle.

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Section: Introductionmentioning

confidence: 64%

“…Conversely, if the phosphorylated head was destined to become the blocked head, folding would be unstable. Asymmetry in the effect of phosphorylation may explain why smHMM phosphorylated on only one of its two heads appears to exist in an equilibrium between a fully active state and an inhibited conformation 22; 23; 47 .…”

Section: Discussionmentioning

confidence: 99%

Phosphorylated Smooth Muscle Heavy Meromyosin Shows an Open Conformation Linked to Activation

Baumann¹,

Taylor²,

Huang³

et al. 2012

Journal of Molecular Biology

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“…Muscle myosin II has a force-velocity relation, even at the level of a few molecules (21)(22)(23), of the form:…”

Section: Modelsmentioning

confidence: 99%

A mechanical model of actin stress fiber formation and substrate elasticity sensing in adherent cells

Walcott

Sun²

2010

Proc. Natl. Acad. Sci. U.S.A.

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210

161

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Tissue cells sense and respond to the stiffness of the surface on which they adhere. Precisely how cells sense surface stiffness remains an open question, though various biochemical pathways are critical for a proper stiffness response. Here, based on a simple mechanochemical model of biological friction, we propose a model for cell mechanosensation as opposed to previous more biochemically based models. Our model of adhesion complexes predicts that these cell-surface interactions provide a viscous drag that increases with the elastic modulus of the surface. The force-velocity relation of myosin II implies that myosin generates greater force when the adhesion complexes slide slowly. Then, using a simple cytoskeleton model, we show that an external force applied to the cytoskeleton causes actin filaments to aggregate and orient parallel to the direction of force application. The greater the external force, the faster this aggregation occurs. As the steady-state probability of forming these bundles reflects a balance between the time scale of bundle formation and destruction (because of actin turnover), more bundles are formed when the cytoskeleton time-scale is small (i.e., on stiff surfaces), in agreement with experiment. As these large bundles of actin, called stress fibers, appear preferentially on stiff surfaces, our mechanical model provides a mechanism for stress fiber formation and stiffness sensing in cells adhered to a compliant surface.biological friction | cell biomechanics | cytoskeleton | focal adhesions | mechanosensitivity A ll cells sense and respond to their environment. A prototypical example is chemical sensing mediated by cell-surface receptors, e.g., a neuron cell can sense small changes in external neural transmitter concentration and responds by opening ion channels and changing internal membrane polarization. A different kind of sensing has been receiving attention lately where some cells directly respond to mechanical properties of their environment, such as the stiffness of the surface to which they adhere. Understanding how these cells sense and respond to their mechanical environment and how they are able to translate mechanical cues into a chemical response are both topics of great interest in cell biomechanics.Here, we are motivated by how stem cells sense and respond to their local mechanical environment. Viability of mesenchymal stem cells depends on their adherence to a surface. Interestingly, the differentiation of these cells is dependent on substrate stiffness. For example, stem cells on soft surfaces become primarily brain cells; on intermediate surfaces they become muscle cells and on stiff surfaces they become bone (1). Precisely how these cells sense surface stiffness, and how surface mechanics leads to the underlying biochemical changes that determine cell identity are still open questions (2). Several processes are thought to be involved. In particular, adhesion complexes (ACs), nonmuscle myosin II, and stress fibers are all thought to play a role. In fact, it seems likel...

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“…Unphosphorylated SMM is auto-inhibited by interactions between the two catalytic domains (1, 2) that are relieved by RLC phosphorylation (3,4). Although regulation requires both heads of myosin (5)(6)(7)(8), phosphorylation of one RLC is sufficient to activate both heads (9,10).…”

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confidence: 99%

Phosphorylation-induced structural changes in smooth muscle myosin regulatory light chain

Kast

Espinoza-Fonseca

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

111

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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. fluorescence resonance energy transfer | FRET | molecular dynamics | molecular dynamics simulation | time-resolved fluorescence S mooth muscle myosin (SMM) is a member of the myosin superfamily of motor proteins, which use chemical energy from ATP hydrolysis to perform mechanical work on actin. Motor properties of myosin are activated by Ca 2þ . In skeletal muscle, Ca 2þ moves actin-bound inhibitory proteins to allow active myosin-actin interaction. In smooth muscle, activation requires phosphorylation of regulatory light chain (RLC) (Fig. 1), which is distant from the myosin active site on the catalytic domain (Fig. 1). Unphosphorylated SMM is auto-inhibited by interactions between the two catalytic domains (1, 2) that are relieved by RLC phosphorylation (3, 4). Although regulation requires both heads of myosin (5-8), phosphorylation of one RLC is sufficient to activate both heads (9, 10).The structural mechanism by which phosphorylation of RLC at S19 activates SMM is a mystery, primarily because there is no highresolution structure of any myosin that contains the N-terminal 24 residues of RLC (Fig. 1). Site-directed spin labeling demonstrated that this N-terminal segment acts as a coherent domain in response to phosphorylation, so it is now referred to as the phosphorylation domain (PD) (11). Removal of PD abolishes regulation, but charge replacement or deletion of PD causes only partial activ...

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Smooth Muscle Heavy Meromyosin Phosphorylated on One of Its Two Heads Supports Force and Motion

Cited by 26 publications

References 44 publications

Phosphorylated Smooth Muscle Heavy Meromyosin Shows an Open Conformation Linked to Activation

Phosphorylated Smooth Muscle Heavy Meromyosin Shows an Open Conformation Linked to Activation

A mechanical model of actin stress fiber formation and substrate elasticity sensing in adherent cells

Phosphorylation-induced structural changes in smooth muscle myosin regulatory light chain

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