Requirement of the two‐headed structure for the phosphorylation dependent regulation of smooth muscle myosin

Matsuura, Motoi; Ikebe, Mitsuo

doi:10.1016/0014-5793(95)00326-5

Cited by 31 publications

(38 citation statements)

References 30 publications

(26 reference statements)

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“…Unphosphorylated WT and mutant smooth muscle HMM showed no ATPase activity activation in the presence of 190 M actin. Phosphorylated WT HMM showed Ͼ30-fold maximal actin activation in agreement with previous results (42,43). We did not directly measure the extent of RLC phosphorylation.…”

Section: Methodssupporting

confidence: 93%

“…5 and from measurements using the unphosphorylated HMM isoforms are summarized in Table III. Table III shows that phosphorylation increases V max for the WT HMM by ϳ36-fold, in agreement with published results (42,43). In the phosphorylated species, V max is identical for the WT and G362A species but increases ϳ2-fold in the SmR370E mutant.…”

Section: Effect Of C-loopsupporting

confidence: 92%

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The Myosin Cardiac Loop Participates Functionally in the Actomyosin Interaction

Ajtai

Garamszegi

Watanabe

et al. 2004

Journal of Biological Chemistry

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The motor protein myosin in association with actin transduces chemical free energy in ATP into work in the form of actin translation against an opposing force. Mediating the actomyosin interaction in myosin is an actin binding site distributed among several peptides on the myosin surface including surface loops contributing to affinity and actin regulation of myosin ATPase. A structured surface loop on ␤-cardiac myosin, the cardiac or C-loop, was recently demonstrated to affect myosin ATPase and was indirectly implicated in the actomyosin interaction. The C-loop is a conserved feature of all myosin isoforms with crystal structures, suggesting that it is an essential part of the core energy transduction machinery. It is shown here that proteolytic digestion of the C-loop in ␤-cardiac myosin eliminates actin-activated myosin ATPase and reduces actomyosin affinity in rigor more than 100-fold. Studies of C-loop function in smooth muscle myosin were also undertaken using sitedirected mutagenesis. Mutagenesis of a single charged residue in the C-loop of smooth muscle myosin alters actomyosin affinity and doubles myosin in vitro motility and actin-activated ATPase velocities, thereby involving a charged region of the loop in the actomyosin interaction. It appears likely that the C-loop is an essential electrostatic binding site for actin involved in modulation of actomyosin affinity and regulation of actomyosin ATPase velocity.The motor protein myosin is an ATPase and an actin-binding protein transducing ATP free energy into work. In muscle, myosin, actin, and ATP constitute the unitary work producer. The cyclical interaction of these components, a sequence of states each characterized as a static relation between the proteins and an intermediate in the degradation of ATP, is a contraction cycle. In a contraction cycle, myosin hydrolyzes ATP in its active site and forms a weak association with actin at a separate actin binding site. Release of phosphate from the active site initiates strong binding to actin and a large conformation change in myosin that produces work in the form of actin translation against a load. Several solved crystal structures represent intermediates in ATP hydrolysis and define myosin conformation changes in the absence of actin (1-6). Presently, we lack a detailed understanding of actomyosin structure and in particular how the elements making up the actomyosin interface contribute to mutual affinity, actin regulation of phosphate release, and the ability of myosin-bound nucleotide to modulate actomyosin affinity.Myosin consists of a globular head domain containing the enzymatic portion of the molecule and a tail involved in filament assembly. The globular head separated from its tail portion is called subfragment 1 (S1). 1 S1 interacts with filamentous actin (F-actin) consisting of polymerized actin monomers. An atomic model for F-actin built from monomeric actin crystal structures (7-9) satisfies x-ray diffraction data restraints (10) and is consistent with electron microscopic imagery (11). In...

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Section: Methodssupporting

confidence: 93%

Section: Effect Of C-loopsupporting

confidence: 92%

The Myosin Cardiac Loop Participates Functionally in the Actomyosin Interaction

Ajtai

Garamszegi

Watanabe

et al. 2004

Journal of Biological Chemistry

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“…An ApaI site was created 2319 bp from the initiation codon in chicken skeletal MHC by using a transformer site-directed mutagenesis kit (CLONTECH). Another ApaI site was introduced 2337 bp in a truncated chicken gizzard MHC cDNA containing an introduced stop codon at base pair 3331 (23,24). These unique ApaI sites were used to generate a cDNA encoding a 127-kDa skeletal͞smooth chimeric MHC (Met 1 -Gly 773 are skeletal and Gly 773 -Ser 1104 are smooth) (Fig.…”

Section: Methodsmentioning

confidence: 99%

The motor domain and the regulatory domain of myosin solely dictate enzymatic activity and phosphorylation-dependent regulation, respectively

Sata

Stafford

Mabuchi

et al. 1997

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

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While the structures of skeletal and smooth muscle myosins are homologous, they differ functionally from each other in several respects, i.e., motor activities and regulation. To investigate the molecular basis for these differences, we have produced a skeletal͞smooth chimeric myosin molecule and analyzed the motor activities and regulation of this myosin. The produced chimeric myosin is composed of the globular motor domain of skeletal muscle myosin (Met 1 -Gly 773 ) and the C-terminal long ␣-helix domain of myosin subfragment 1 as well as myosin subfragment 2 (Gly 773 -Ser 1104 ) and light chains of smooth muscle myosin. Both the actin-activated ATPase activity and the actin-translocating activity of the chimeric myosin were completely regulated by light chain phosphorylation. On the other hand, the maximum actin-activated ATPase activity of the chimeric myosin was the same as skeletal myosin and thus much higher than smooth myosin. These results show that the C-terminal light chainassociated domain of myosin head solely confers regulation by light chain phosphorylation, whereas the motor domain determines the rate of ATP hydrolysis. This is the first report, to our knowledge, that directly determines the function of the two structurally separated domains in myosin head.

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“…see Refs. [5][6][7]. The structural basis of the inhibitory mechanism has been difficult to decipher partly because the high resolution crystal structure of the smooth muscle myosin S1 fragment does not include the regulatory light chain (8); the scallop muscle protein structure contains both essential and regulatory light chains, but the protein is regulated by calcium binding rather than by phosphorylation (9,10); and the residue (Ser-13) equivalent to the phosphorylation site in smooth myosin RLC is not seen in the structure of striated muscle myosin (11).…”

mentioning

confidence: 99%

Cryo-atomic Force Microscopy of Unphosphorylated and Thiophosphorylated Single Smooth Muscle Myosin Molecules

Sheng

Gao

Khromov

et al. 2003

Journal of Biological Chemistry

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The purpose of this study was to determine whether steric blockage of one head by the second head of native two-headed myosin was responsible for the inactivity of nonphosphorylated two-headed myosin compared with the high activity of single-headed myosin, as suggested on the basis of electron microscopy of two-dimensional crystals of heavy meromyosin (Wendt, T., Taylor indicates that thiophosphorylation of the regulatory light chain increases the separation of the two heads of a single myosin molecule, but the thermodynamic probability of steric hindrance by strong binding between the two heads was not determined. We now report this probability determined by cryo-AFM of single whole myosin molecules shown to have normal low ATPase activity (0.007 s ؊1 ). We found that the thermodynamic probability of the relative head positions of nonphosphorylated myosin was approximately equal between separated heads as compared with closely apposed heads (energy difference of 0.24 kT (where k is a Boltzman constant and T is the absolute temperature)), and thiophosphorylation increased the number of molecules having separated heads (energy advantage of ؊1.2 kT (where k is a Boltzman constant and I is the absolute temperature)). Our results do not support the suggestion that strong binding of one head to the other stabilizes the blocked conformation against thermal fluctuations resulting in steric blockage that can account for the low activity of nonphosphorylated two-headed myosin.One of the most fundamental, but not yet understood, questions about smooth muscle and nonmuscle myosin II is how phosphorylation of the regulatory light chain (RLC), 1 located C-terminally in the myosin head, activates the N-terminal motor domain at a nearly 15-nm distance; or conversely, how dephosphorylated RLCs maintain myosin in the "off state." Myosin II is composed of two heavy chains each having at its N terminus a catalytic (motor) and a regulatory domain bound with essential light chain (LC 17 ) and RLC. Whereas the hexamer containing unphosphorylated RLCs is inactive and requires RLC phosphorylation for ATPase activity, a single head (S1) (4) or a construct of a single head with the heavy chain tail and two associated light chains is active without RLC phosphorylation (e.g. see Refs. 5-7). The structural basis of the inhibitory mechanism has been difficult to decipher partly because the high resolution crystal structure of the smooth muscle myosin S1 fragment does not include the regulatory light chain (8); the scallop muscle protein structure contains both essential and regulatory light chains, but the protein is regulated by calcium binding rather than by phosphorylation (9, 10); and the residue (Ser-13) equivalent to the phosphorylation site in smooth myosin RLC is not seen in the structure of striated muscle myosin (11). Therefore, before atomic structures of both phosphorylated and dephosphorylated RLC bound to twoheaded smooth muscle myosin are obtained, information at less than atomic resolution may lead to critical insights about...

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Requirement of the two‐headed structure for the phosphorylation dependent regulation of smooth muscle myosin

Cited by 31 publications

References 30 publications

The Myosin Cardiac Loop Participates Functionally in the Actomyosin Interaction

The Myosin Cardiac Loop Participates Functionally in the Actomyosin Interaction

The motor domain and the regulatory domain of myosin solely dictate enzymatic activity and phosphorylation-dependent regulation, respectively

Cryo-atomic Force Microscopy of Unphosphorylated and Thiophosphorylated Single Smooth Muscle Myosin Molecules

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