Three-Dimensional Reconstruction of Tarantula Myosin Filaments Suggests How Phosphorylation May Regulate Myosin Activity

Álamo, Lorenzo; Wriggers, Willy; Pinto, Antonio; Bartoli, Fulvia; Salazar, Leiria; Zhao, Fa-Qing; Craig, Roger; Padrón, Raúl

doi:10.1016/j.jmb.2008.10.013

Cited by 139 publications

(341 citation statements)

References 85 publications

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“…One is very similar to the model of Wendt et al (9), at least with regard to the RLC-RLC distances. This suggests that the structure observed by Wendt et al is stable in the absence of a charged bilayer (9) or in the absence of potentially stabilizing interactions with the thick filament backbone (6). The second population, modeled by rigid body rotation, does not have interacting heads and is characterized by broader distance distributions as might be expected from noninteracting heads.…”

Section: Resultsmentioning

confidence: 80%

“…It is a difficult problem because regulation by phosphorylation requires the presence of both head domains. Cryogenic EM studies of double-headed constructs (6)(7)(8)(9) have incorporated data from crystallographic atomic resolution structures, which are limited to single head domains, to produce several pseudoatomic models. These static structures of the inhibited state all reveal that one head binds to the other through the MDs.…”

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confidence: 99%

“…The model suggests that the binding of the blocked head to actin is inhibited as well as the ATPase activity of the free head because its converter domain cannot rotate to release Pi from the active site. Interactions of portions of the heads with the proximal region of the tail (called S2) may also be important (6,10). Here the heads point "down," essentially lying on top of S2 (7).…”

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confidence: 99%

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Broad disorder and the allosteric mechanism of myosin II regulation by phosphorylation

Vileno

Chamoun

Liang

et al. 2011

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

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Double electron electron resonance EPR methods was used to measure the effects of the allosteric modulators, phosphorylation, and ATP, on the distances and distance distributions between the two regulatory light chain of myosin (RLC). Three different states of smooth muscle myosin (SMM) were studied: monomers, the shorttailed subfragment heavy meromyosin, and SMM filaments. We reconstituted myosin with nine single cysteine spin-labeled RLC. For all mutants we found a broad distribution of distances that could not be explained by spin-label rotamer diversity. For SMM and heavy meromyosin, several sites showed two heterogeneous populations in the unphosphorylated samples, whereas only one was observed after phosphorylation. The data were consistent with the presence of two coexisting heterogeneous populations of structures in the unphosphorylated samples. The two populations were attributed to an on and off state by comparing data from unphosphorylated and phosphorylated samples. Models of these two states were generated using a rigid body docking approach derived from EM [Wendt T, Taylor D, Trybus KM, Taylor K (2001) Proc Natl Acad Sci USA 98:4361-4366] (PNAS, 2001, 98:4361-4366), but our data revealed a new feature of the offstate, which is heterogeneity in the orientation of the two RLC. Our average off-state structure was very similar to the Wendt model reveal a new feature of the off state, which is heterogeneity in the orientations of the two RLC. As found previously in the EM study, our on-state structure was completely different from the off-state structure. The heads are splayed out and there is even more heterogeneity in the orientations of the two RLC.computational modeling | structural biology M yosin II is a motor protein that can transduce chemical energy from ATP hydrolysis into mechanical work. This process involves cyclic interactions between actin in thin and myosin in thick filaments causing relative filament sliding. Smooth muscle myosin (SMM), which contains two heads, each with a motor domain (MD) and regulatory domain (RD), connected by a long coiled-coil tail domain is an example of a large multisubunit protein that is allosterically regulated by phosphorylation (for review, see ref. 1). Phosphorylation of a single serine on each regulatory light chain (RLC), which are about 100 Å distant from the active sites, is sufficient to transform the protein from a state in which the ATPase activity is weakly actin-activated to one in which it is strongly actin-activated by controlling the rate of Pi release (2). Because phosphorylation modulates the rate of ADP release (3) and also alters the attitude of the lever arm domain with respect to the MD (4), it is likely that phosphorylation modulates strain between the two heads (5).The structural basis for the allosteric regulation has been a topic of intense study. It is a difficult problem because regulation by phosphorylation requires the presence of both head domains. Cryogenic EM studies of double-headed constructs (6-9) have incorporated data fr...

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Section: Resultsmentioning

confidence: 80%

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confidence: 99%

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confidence: 99%

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Broad disorder and the allosteric mechanism of myosin II regulation by phosphorylation

Vileno

Chamoun

Liang

et al. 2011

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

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“…Many aspects of the above results can be integrated and explained in terms of the concept of a regulatory transition in the conformation of the myosin heads in the thick filaments of heart muscle analogous to that in smooth muscle myosin (20,28) and the thick filaments of skeletal muscle (21,40). By analogy with those other muscle types and with direct support from EM reconstructions of isolated thick filaments from cardiac muscle (26,27), we may consider an OFF state of the cardiac thick filament (Fig.…”

Section: Length-dependent Activation and Crlc Phosphorylation May Usementioning

confidence: 90%

Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments

Kampourakis

Sun

Irving

2016

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

123

141

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Contraction of heart muscle is triggered by calcium binding to the actin-containing thin filaments but modulated by structural changes in the myosin-containing thick filaments. We used phosphorylation of the myosin regulatory light chain (cRLC) by the cardiac isoform of its specific kinase to elucidate mechanisms of thick filamentmediated contractile regulation in demembranated trabeculae from the rat right ventricle. cRLC phosphorylation enhanced active force and its calcium sensitivity and altered thick filament structure as reported by bifunctional rhodamine probes on the cRLC: the myosin head domains became more perpendicular to the filament axis. The effects of cRLC phosphorylation on thick filament structure and its calcium sensitivity were mimicked by increasing sarcomere length or by deleting the N terminus of the cRLC. Changes in thick filament structure were highly cooperative with respect to either calcium concentration or extent of cRLC phosphorylation. Probes on unphosphorylated myosin heads reported similar structural changes when neighboring heads were phosphorylated, directly demonstrating signaling between myosin heads. Moreover probes on troponin showed that calcium sensitization by cRLC phosphorylation is mediated by the thin filament, revealing a signaling pathway between thick and thin filaments that is still present when active force is blocked by Blebbistatin. These results show that coordinated and cooperative structural changes in the thick and thin filaments are fundamental to the physiological regulation of contractility in the heart. This integrated dual-filament concept of contractile regulation may aid understanding of functional effects of mutations in the protein components of both filaments associated with heart disease.cardiac muscle regulation | myosin regulatory light chain | phosphorylation

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“…Regulatory sites within the LCD control the conformation of the hinges to promote intramolecular interactions between the two heads and the S2 region in the off state (dephosphorylated or Ca 2+ -free state) (8)(9)(10)(11). Phosphorylated or Ca 2+ -bound LCD activates the motor because both heads become free and disordered and can interact with actin to produce force (12).…”

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confidence: 99%

Essential “ankle” in the myosin lever arm

Pylypenko

Houdusse

2010

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

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Three-Dimensional Reconstruction of Tarantula Myosin Filaments Suggests How Phosphorylation May Regulate Myosin Activity

Cited by 139 publications

References 85 publications

Broad disorder and the allosteric mechanism of myosin II regulation by phosphorylation

Broad disorder and the allosteric mechanism of myosin II regulation by phosphorylation

Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments

Essential “ankle” in the myosin lever arm

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