Structural Model of the Regulatory Domain of Smooth Muscle Heavy Meromyosin

Wahlstrom, Jan L.; Randall, M. Allen; Lawson, J. David; Lyons, Derek E.; Siems, William F.; Crouch, Greg J.; Barr, Regina; Facemyer, Kevin C.; Cremo, Christine R.

doi:10.1074/jbc.m206963200

Cited by 32 publications

(49 citation statements)

References 40 publications

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“…6). The two heads of dephosphorylated myosin have been shown to be photo-crosslinked through the RLCs (within 8.9 Å of each other) (28), and thiophosphorylation abolishes this cross-linking (29) consistent with the greater separation of the two thiophosphorylated heads in solution (present study and Ref. 3).…”

Section: Figsupporting

confidence: 68%

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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Section: Figsupporting

confidence: 68%

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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“…Fig. 1A shows our homology model of the regulatory domain (23), and Fig. 1B shows the RLC sequence with substituted cysteines.…”

Section: Resultsmentioning

confidence: 99%

“…Because our preparations of HMM and S1 have residual phosphatase activity, phosphorylation was in 20 mM MOPS (pH 7.0), 10 g/ml calmodulin, 30 g/ml myosin light chain kinase, 3 mM CaCl 2 , 3 mM MgCl 2 , 120 mM NaF, 4 M microcystin, and 2 mM ATP on ice with slow stirring for 3 h. 1 mM EGTA was maintained in all solutions thereafter where phosphorylation was stable for days. All (thio)phosphorylations were confirmed by PAGE electrophoresis (23). All UP samples were treated exactly as the thio(phosphorylated), except no ATP was added.…”

Section: Methodsmentioning

confidence: 99%

“…Unfortunately, none of the crystal structures contain coordinates for the RLC N terminus (residues 1-24), presumably due to its lack of ordered secondary structure in single-head constructs. Our photocross-linking studies (22,23) provide structural information about the RLC N terminus in UP-HMM. Our data (23) suggest that phosphorylation of Ser-19 causes a large movement of the N terminus from an extended to a folded conformation.…”

mentioning

confidence: 99%

“…Our photocross-linking studies (22,23) provide structural information about the RLC N terminus in UP-HMM. Our data (23) suggest that phosphorylation of Ser-19 causes a large movement of the N terminus from an extended to a folded conformation. The UP N terminus may stabilize the RLC-RLC or RLC-S2 interaction that allosterically down-regulates the ATPase activity.…”

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confidence: 99%

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Novel Sensors of the Regulatory Switch on the Regulatory Light Chain of Smooth Muscle Myosin

Mazhari

Selser

Cremo

2004

Journal of Biological Chemistry

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Here we used three different approaches to test this notion, which are reactivity of cysteine thiols, pyrene and acrylodan spectral analysis, and pyrene fluorescence quenching. All methods detected significant differences between the unphosphorylated and phosphorylated regulatory light chain N termini in heavy meromyosin, a double-headed subfragment with an intact regulatory switch. These differences were not observed for subfragment-1, a single-headed, unregulated subfragment. In the presence of either ATP or ADP, phosphorylation increased the solvent exposure and decreased the polarity of the environment about position 23 of the regulatory light chain of heavy meromyosin. These phosphorylation-induced structural changes were not as evident in the absence of nucleotides. Nucleotide binding to unphosphorylated heavy meromyosin caused a decrease in exposure and an increase in polarity of the N terminus, whereas the effects of nucleotide on phosphorylated heavy meromyosin were the opposite. We showed a direct correlation between the kinetics of nucleotide binding/turnover and the conformational change reported by acrylodan at position 23 of the regulatory light chain. Acrylodan-A23C also reports the heads up (extended) to flexed (folded) transition in unphosphorylated heavy meromyosin. This is the first demonstration of direct coupling of nucleotide binding to conformational changes in the N terminus of the regulatory light chain.Smooth muscle myosin (SMM) 1 and nonmuscle myosin are hexameric motor ATPases of ϳ500 kDa mass composed of pairs of HC, ELC, and RLC. A globular motor domain (N-terminal HC) and a light chain binding domain (HC ϩ ELC ϩ RLC) form a head (S1), and the two heads are dimerized by the HC C-terminal halves, which form a coiled-coil. The motor domain contains the catalytic site and the actin binding site (1). Only the head domain and its subfragments have been crystallized to date so there are no atomic resolution structures of a doubleheaded construct.The actin-activated ATPase activity and motor properties of SMM and nonmuscle myosin are regulated by phosphorylation of Ser-19 of the RLC, which is greater than 10 nm from the catalytic site (1-4). Phosphorylation enhances the ATPase activity by more than 1000-fold at saturating actin concentrations (5, 6). Domain requirements for regulation have been elucidated through studies of various proteolytic and expressed subfragments of SMM and nonmuscle myosin. HMM, which lacks the C-terminal two-thirds of the tail, is double-headed and regulated (5-7), but expressed HMM constructs with truncated tails too short to form stable double-headed structures are unregulated (8 -10) as is S1 (7,11,12) and single-headed myosin (13,14). Furthermore, a nonmuscle HMM construct without one of the motor domains but with both regulatory domains is not regulated (15). Therefore, two full heads connected together with enough coiled-coil to allow for dimerization are required for regulation.Removal of the RLC abolishes regulation (16), but the ELC can be removed with re...

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ESR Spectroscopy in Membrane Biophysics

Hemminga¹,

Berliner²

2007

Biological Magnetic Resonance

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Structural Model of the Regulatory Domain of Smooth Muscle Heavy Meromyosin

Cited by 32 publications

References 40 publications

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

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

Novel Sensors of the Regulatory Switch on the Regulatory Light Chain of Smooth Muscle Myosin

ESR Spectroscopy in Membrane Biophysics

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