Kinetics of smooth muscle heavy meromyosin with one thiophosphorylated head

Actin-activated MgATPase of smooth muscle heavy meromyosin is activated by thiophosphorylation of two regulatory light chains, one on each head domain. To understand cooperativity between heads, we examined the kinetics of heavy meromyosin (HMM) with one thiophosphorylated head. Proteolytic gizzard heavy meromyosin regulatory light chains were partially exchanged with recombinant thiophosphorylated His-tagged light chains, and HMM with one thiophosphorylated head was isolated by nickel-affinity chromatography. In vitro motility was observed. By steady-state kinetic analysis, one-head thiophosphorylated heavy meromyosin had a similar K(m) value for actin but a V(max) value of approximately 50% of the fully thiophosphorylated molecule. However, single turnover analysis, which is not sensitive to small amounts of active heads, showed that one-head thiophosphorylated heavy meromyosin was 46-120 times more active than unphosphorylated HMM but only 7-19% as active as the fully thiophosphorylated molecule. Discrepancy between the single turnover and steady-state values could be explained by a small fraction of rigor heads. These rigor heads would have a large effect on the steady-state kinetics of one-head thiophosphorylated HMM. In summary, thiophosphorylation of one head leads to a molecule with unique intermediate kinetics suggesting that thiophosphorylation of one head cooperatively alters the kinetics of the partner head and vice versa.     

Phosphorylated smooth muscle heavy meromyosin shows an open conformation linked to activation

Smooth muscle myosin and smooth muscle heavy meromyosin (smHMM) are activated by regulatory light chain 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 three-dimensional structure for phosphorylated, active smHMM obtained using electron crystallography of two-dimensional 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 regulatory light chain 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.     

Structural model of the regulatory domain of smooth muscle heavy meromyosin

The goal of this study was to provide structural information about the regulatory domains of double-headed smooth muscle heavy meromyosin, including the N terminus of the regulatory light chain, in both the phosphorylated and unphosphorylated states. We extended our previous photo-cross-linking studies (Wu, X., Clack, B. A., Zhi, G., Stull, J. T., and Cremo, C. R. (1999) J. Biol. Chem. 274, 20328-20335) to determine regions of the regulatory light chain that are cross-linked by a cross-linker attached to Cys(108) on the partner regulatory light chain. For this purpose, we have synthesized two new biotinylated sulfhydryl reactive photo-cross-linking reagents, benzophenone, 4-(N-iodoacetamido)-4'-(N-biotinylamido) and benzophenone, 4-(N-maleimido)-4'-(N-biotinylamido). Cross-linked peptides were purified by avidin affinity chromatography and characterized by Edman sequencing and mass spectrometry. Labeled Cys(108) from one regulatory light chain cross-linked to (71)GMMSEAPGPIN(81), a loop in the N-terminal half of the regulatory light chain, and to (4)RAKAKTTKKRPQR(16), a region for which there is no atomic resolution data. Both cross-links were to the partner regulatory light chain and occurred in unphosphorylated but not phosphorylated heavy meromyosin. Using these data, data from our previous study, and atomic coordinates from various myosin isoforms, we have constructed a structural model of the regulatory domain in an unphosphorylated double-headed molecule that predicts the general location of the N terminus. The implications for the structural basis of the phosphorylation-mediated regulatory mechanism are discussed.     

The N-terminal lobes of both regulatory light chains interact with the tail domain in the 10 S-inhibited conformation of smooth muscle myosin

In the presence of ATP, unphosphorylated smooth muscle myosin can form a catalytically inactive monomer that sediments at 10 Svedbergs (10 S). The tail of 10 S bends into thirds and interacts with the regulatory domain. ADP-P(i) is "trapped" at the active site, and consequently the ATPase activity is extremely low. We are interested in the structural basis for maintenance of this off state. Our prior photocross-linking work with 10 S showed that tail residues 1554-1583 are proximal to position 108 in the C-terminal lobe of one of the two regulatory light chains ( Olney, J. J., Sellers, J. R., and Cremo, C. R. (1996) J. Biol. Chem. 271, 20375-20384 ). These data suggested that the tail interacts with only one of the two regulatory light chains. Here we present data, using a photocross-linker on position 59 on the N-terminal lobe of the regulatory light chain (RLC), demonstrating that both regulatory light chains of a single molecule can cross-link to the light meromyosin portion of the tail. Mass spectrometric data show four specific cross-linked regions spanning residues 1428-1571 in the light meromyosin portion of the tail, consistent with cross-linking two RLC to one light meromyosin. In addition, we find that position 59 can cross-link internally to residues 42-45 within the same RLC subunit. The internal cross-link only forms in 10 S and not in unphosphorylated heavy meromyosin (lacking the light meromyosin), suggesting a structural rearrangement within the RLC attributed to the interaction of the tail with the head.     

Regulatory light chain influences alterations of myosin head induced by actin.

The effect of magnesium-for-calcium exchange and phosphorylation of regulatory light chain (LC2) on structural organization of rabbit skeletal myosin head was studied by limited tryptic digestion. In the presence of actin, exchange of magnesium bound to LC2 by calcium in dephosphorylated myosin accelerates the digestion of myosin and heavy meromyosin heavy chain and increases the accumulation of a 50 kDa fragment. This effect is significantly diminished in the case of phosphorylated myosin. Thus, both phosphorylation and cation exchange influences the effect of actin binding on the structural organization of myosin head.     

A new method to specifically label thiophosphorylatable proteins with extrinsic probes. Labeling of serine-19 of the regulatory light chain of smooth muscle myosin.

We present a new method to specifically and stably label proteins by attaching extrinsic probes to amino acids that are thiophosphorylated by protein kinases and ATP gamma S. The method was demonstrated for labeling of a thiophosphorylatable serine of the isolated regulatory light chain of smooth muscle myosin. We stoichiometrically blocked the single thiol (Cys-108) either by forming a reversible intermolecular disulfide bond or by reacting with iodoacetic acid. The protein was stoichiometrically thiophosphorylated at Ser-19 by myosin light chain kinase and ATP gamma S. The nucleophilic sulfur of the protein phosphorothioate was coupled at pH 7.9 and 25 degrees C to the fluorescent haloacetate [3H]-5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1- sulfonic acid ([3H]IAEDANS) by displacement of the iodide. Typical labeling efficiencies were 70-100%. The labeling was specific for the thiophosphorylated Ser-19, as determined from the sequences of two labeled peptides isolated from a tryptic digest of the labeled protein. [3H]IAEDANS attached to the thiophosphorylated Ser-19 was stable at pH 3-10 at 25 degrees C, and to boiling in high concentrations of reductant. The labeled light chains were efficiently exchanged for unlabeled regulatory light chains of the whole myosin molecule. The resulting labeled myosin had normal ATPase activities in the absence of actin, indicating that the modification of Ser-19 and the exchange of the labeled light chain into myosin did not significantly disrupt the protein. The labeled myosin partially retained the elevated actin-activated Mg(2+)-ATPase activity which is characteristic of thiophosphorylated myosin. This indicates that labeling of the thiophosphate group with [3H]IAEDANS did not completely disrupt the functional properties of the thiophosphorylated protein in the presence of actin.     

Refined model of the 10S conformation of smooth muscle myosin by cryo-electron microscopy 3D image reconstruction

The actin-activated ATPase activity of smooth muscle myosin and heavy meromyosin (smHMM) is regulated by phosphorylation of the regulatory light chain (RLC). Complete regulation requires two intact myosin heads because single-headed myosin subfragments are always active. 2D crystalline arrays of the 10S form of intact myosin, which has a dephosphorylated RLC, were produced on a positively charged lipid monolayer and imaged in 3D at 2.0 nm resolution by cryo-electron microscopy of frozen, hydrated specimens. An atomic model of smooth muscle myosin was constructed from the X-ray structures of the smooth muscle myosin motor domain and essential light chain and a homology model of the RLC was produced based on the skeletal muscle S1 structure. The initial model of the 10S myosin, based on the previous reconstruction of smHMM, was subjected to real space refinement to obtain a quantitative fit to the density. The smHMM was likewise refined and both refined models reveal the same asymmetric interaction between the upper 50 kDa domain of the "blocked" head and parts of the catalytic, converter domains and the essential light chain of the "free" head observed previously. This observation suggests that this interaction is not simply due to crystallographic packing but is enforced by elements of the myosin heads. The 10S reconstruction shows additional alpha-helical coiled-coil not seen in the earlier smHMM reconstruction, but the location of one segment of S2 is the same in both.     

Decoration of actin filaments with skeletal muscle heavy meromyosin containing either phosphorylated or dephosphorylated regulatory light chains

Heavy meromyosin containing almost intact regulatory light chains (LC2) was obtained from monomeric phosphorylated and dephosphorylated rabbit fast skeletal muscle myosin by brief chymotryptic digestion in the presence of CaCl2. Actin filaments, complexed with heavy meromyosin, display two different forms of arrowhead, depending on the form of LC2.     

Both heads of tissue-derived smooth muscle heavy meromyosin bind to actin in the presence of ADP

The effect of ADP and phosphorylation upon the actin binding properties of heavy meromyosin was investigated using three fluorescence methods that monitor the number of heavy meromyosin heads that bind to pyrene-actin: (i) amplitudes of ATP-induced dissociation, (ii) amplitudes of ADP-induced dissociation of the pyrene-actin-heavy meromyosin complex, and (iii) amplitudes of the association of heavy meromyosin with pyrene-actin. Both heads bound to pyrene-actin, irrespective of regulatory light chain phosphorylation or the presence of ADP. This behavior was found for native regulated heavy meromyosin prepared by proteolytic digestion of chicken gizzard myosin with between 5 and 95% heavy chain cleavage at the actin-binding loop, showing that two-head binding is a property of heavy meromyosin with uncleaved heavy chains. These data are in contrast to a previous study using an uncleaved expressed preparation (Berger, C. E., Fagnant, P. M., Heizmann, S., Trybus, K. M., and Geeves, M. A. (2001) J. Biol. Chem. 276, 23240-23245), which showed that one head of the unphosphorylated heavy meromyosin-ADP complex bound to actin and that the partner head either did not bind or bound weakly. Possible explanations for the differences between the two studies are discussed. We have shown that unphosphorylated heavy meromyosin appears to adopt a special state in the presence of ADP based upon analysis of actin-heavy meromyosin association rate constants. Data were consistent with one head binding rapidly and the second head binding more slowly in the presence of ADP. Both heads bound to actin at the same rate for all other states.     

Monoclonal antibodies detect and stabilize conformational states of smooth muscle myosin

Antibodies with epitopes near the heavy meromyosin/light meromyosin junction distinguish the folded from the extended conformational states of smooth muscle myosin. Antibody 10S.1 has 100-fold higher avidity for folded than for extended myosin, while antibody S2.2 binds preferentially to the extended state. The properties of these antibodies provide direct evidence that the conformation of the rod is different in the folded than the extended monomeric state, and suggest that this perturbation may extend into the subfragment 2 region of the rod. Two antihead antibodies with epitopes on the heavy chain map at or near the head/rod junction. Magnesium greatly enhances the binding of these antibodies to myosin, showing that the conformation of the heavy chain in the neck region changes upon divalent cation binding to the regulatory light chain. Myosin assembly is also altered by antibody binding. Antibodies that bind to the central region of the rod block disassembly of filaments upon MgATP addition. Antibodies with epitopes near the COOH terminus of the rod, in contrast, promote filament depolymerization, suggesting that this region of the tail is important for assembly. The monoclonal antibodies described here are therefore useful both for detecting and altering conformational states of smooth muscle myosin.     

Regulation by molluscan myosins

Molluscan myosins are regulated molecules that control muscle contraction by the selective binding of calcium. The essential and the regulatory light chains are regulatory subunits. Scallop myosin is the favorite material for studying the interactions of the light chains with the myosin heavy chain since the regulatory light chains can be reversibly removed from it and its essential light chains can be exchanged. Mutational and structural studies show that the essential light chain binds calcium provided that the Ca-binding loop is stabilized by specific interactions with the regulatory light chain and the heavy chain. The regulatory light chains are inhibitory subunits. Regulation requires the presence of both myosin heads and an intact headrod junction. Heavy meromyosin is regulated and shows cooperative features of activation while subfragment-1 is non-cooperative. The myosin heavy chains of the functionally different phasic striated and the smooth catch muscle myosins are products of a single gene, the isoforms arise from alternative splicing. The differences between residues of the isoforms are clustered at surface loop-1 of the heavy chain and account for the different ATPase activity of the two muscle types. Catch muscles contain two regulatory light chain isoforms, one phosphorylatable by gizzard myosin light chain kinase. Phosphorylation of the light chain does not alter ATPase activity. We could not find evidence that light chain phosphorylation is responsible for the catch state.     

Proximity relationships between sites on myosin and actin. Resonance energy transfer determination of the following distances, using a hybrid myosin: those between Cys-55 on the Mercenaria regulatory light chain, SH-1 on the Aequipecten myosin heavy chain, and Cys-374 of actin

Resonance energy transfer measurements have been made on hybrid myosins in order to map distances between sites on the regulatory light chain, heavy chain, and actin as well as to assess potential conformational changes of functional importance. Using scallop (Aequipecten) myosin hybrid molecules possessing clam (Mercenaria) regulatory light chains, we have been able to map the distance between Cys-55 on the regulatory light chain and the fast-reacting thiol on the myosin heavy chain (SH-1). This distance is shown to be approximately 6.4 nm, and it is not altered by the presence or absence of Ca2+, MgATP, or actin. Experiments performed at low ionc strength on heavy meromyosin (HMM) derived from these hybrid myosins gave results similar to those performed on the soluble parent myosin preparations. The distances between Cys-374 on actin and each of the above sites were also measured. Mercenaria regulatory light-chain Cys-55, within the hybrid myosin molecule, was found to be greater than 8.0 nm away from actin Cys-374. Scallop heavy-chain SH-1 is shown to be approximately 4.5 nm away from actin Cys-374, in broad agreement with earlier measurements made by others in nonregulatory myosins. The significance of our results is discussed with respect to putative conformational changes within the region of the heavy chain connecting SH-1 to the N-terminal region of the light chain.     

Novel sensors of the regulatory switch on the regulatory light chain of smooth muscle Myosin

Smooth muscle myosin can be switched on by phosphorylation of Ser-19 of the regulatory light chain. Our previous photocross-linking results suggested that an element of the structural mechanism for the regulatory switch was a phosphorylation-induced motion of the regulatory light chain N terminus (Wahlstrom, J. L., Randall, M. A., Jr., Lawson, J. D., Lyons, D. E., Siems, W. F., Crouch, G. J., Barr, R., Facemyer, K. C., and Cremo, C. R. (2003) J. Biol. Chem. 278, 5123-5131). 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.     

Calcium ions modulate regulation of smooth muscle contraction mediated by phosphorylation of myosin regulatory light chains

The effect of calcium ions on conformational changes of F-actin initiated by decoration of thin filaments with phosphorylated and dephosphorylated heavy meromyosin from smooth muscles was studied by fluorescence polarization spectroscopy. It is shown that heavy meromyosin with phosphorylated regulatory light chains (pHMM) promotes structural changes of F-actin which are typical for the "strong" binding of actin to the myosin heads. Heavy meromyosin with dephosphorylated regulatory light chains (dpHMM) causes conformational changes of F-actin which are typical for the "weak" binding of actin to the myosin heads. The presence of calcium enhances the pHMM effect and attenuates the dpHMM effect. We propose that a Ca2+-dependent mechanism exists in smooth muscles which modulates the regulation of actin--myosin interaction occurring via phosphorylation of myosin regulatory light chains.     

The interaction between the regulatory light chain domains on two heads is critical for regulation of smooth muscle myosin

Recent findings have suggested that the interaction between the two heads is critical for phosphorylation-dependent regulation of smooth muscle myosin. We hypothesized that the interaction between the two regulatory light chains on two heads of myosin dictates the regulation of myosin motor function. To evaluate this notion, we engineered and characterized smooth muscle heavy meromyosin (HMM), which is composed of one entire HMM heavy chain and one motor domain truncated heavy chain containing the S2 rod and regulatory light chain (RLC) binding site, as well as the bound RLC (SMDHMM). SMDHMM was inactive for both actin-translocating activity and actin-activated ATPase activity in the dephosphorylated state, demonstrating that the interaction between the two RLC domains on the two heads and/or a motor domain and a RLC domain in a distinct head is sufficient for the inhibition of smooth muscle myosin motor activity. When phosphorylated, SMDHMM was activated for both actin-translocating activity and actin-activated ATPase activity; however, these activities were lower than those of double-headed HMM, implying partial release of inhibition by phosphorylation in SMDHMM and/or cooperativity between the two heads of smooth muscle myosin. The present results indicate that the RLC domain is critical for phosphorylation-dependent regulation of smooth muscle myosin motor activity. On the other hand, similar to double-headed HMM, SMDHMM showed both "folded" and "extended" conformations, and the ratio of those conformations is dependent on ionic strength, suggesting that the RLC domain is sufficient to regulate the conformational transition in myosin.     

Two functional heads are required for full activation of smooth muscle myosin

The motor activity of smooth muscle myosin II is regulated by the regulatory light chain phosphorylation, but it is not understood how phosphorylation activates motor activity. To address this question, we produced asymmetric heavy meromyosin (HMM), which is composed of a wild-type (WT) heavy chain and a mutant heavy chain having no motor activity (i.e. S236T or G457A). The actin-activated ATPase activities (Vmax) of asymmetric HMMs were only 21.8 and 8.4% of the wild-type HMM for S236A/WT HMM and G456A/WT HMM, respectively. If the two heads of HMM are independent for their ATPase activities, asymmetric HMM should show 50% of the activity of wild-type HMM; however, the activity of asymmetric HMM was much lower than the expected value. The results suggest that the activity of the wild-type head is attenuated by the presence of inactive head. Consistently, the actin-gliding velocity of the asymmetric HMM (i.e. S236T/WT or G457A/WT) was less than one-fifth of the wild-type HMM. The present study supports an idea that the two heads of smooth muscle myosin II interact with each other and the presence of two active heads is required for full activation.     

Visualization of head-head interactions in the inhibited state of smooth muscle myosin

The structural basis for the phosphoryla- tion-dependent regulation of smooth muscle myosin ATPase activity was investigated by forming two- dimensional (2-D) crystalline arrays of expressed unphosphorylated and thiophosphorylated smooth muscle heavy meromyosin (HMM) on positively charged lipid monolayers. A comparison of averaged 2-D projections of both forms at 2.3-nm resolution reveals distinct structural differences. In the active, thiophosphorylated form, the two heads of HMM interact intermolecularly with adjacent molecules. In the unphosphorylated or inhibited state, intramolecular interactions position the actin-binding interface of one head onto the converter domain of the second head, thus providing a mechanism whereby the activity of both heads could be inhibited.     

Effect of skeletal muscle myosin light chain 2 on the Ca2+-sensitive interaction of myosin and heavy meromyosin with regulated actin

A low-speed centrifugation assay has been used to examine the binding of myosin filaments to F-action and to regulated actin in the presence of MgATP. While the cross-linking of F-actin by myosin was Ca2+ insensitive, much less regulated actin was cross-linked by myosin in the absence of Ca2+ than in its presence. Removal of the 19000-dalton, phosphorylatable light chain from myosin resulted in the loss of this Ca2+ sensitivity. Readdition of this light chain partially restored the Ca2+-sensitive cross-linking of regulated actin by myosin. Urea gel electrophoresis has been used to distinguish that fraction of heavy meromyosin which contains intact phosphorylatable light chain from that which contains a 17000-dalton fragment of this light chain. In the absence of Ca2+, heavy meromyosin which contained digested light chain bound to regulated actin in MgATP about 10-fold more tightly than did heavy meromyosin which contained intact light chain. The regulated actin-activated ATPases of heavy meromyosin also showed that cleavage of this light chain causes a substantial increase in the affinity of heavy meromyosin for regulated actin in the absence of Ca2+. Thus, the binding of both myosin and heavy meromyosin to regulated actin is Ca2+ sensitive, and this sensitivity is dependent on the phosphorylatable light chain.     

The disappearance of the dependence of actin-myosin interaction on the phosphorylation of myosin light chains in the "freezing" of the structure of heavy meromyosin by a bifunctional reagent

Using glycerinated muscle fibers, free of myosin, tropomyosin and troponin, a study was made of the structural state of F-actin modified by N-(iodoacetyl)-N'-(1-naphthyl-5-sulfo)-ethylendiamine (1.5-IAEDANS) and by rhodaminyl--phalloin at decoration of thin filaments with a proteolytic fragment of myosin--heavy meromyosin containing phosphorylated and dephosphorylated myosin light chains. The heavy meromyosin used has three SH-groups of heavy chain SH1, SH2 and SH chi modified by bifunctional reagent N,N'-n-phenylmaleimide (SH1-SH2, SH2-SH chi). At decoration of thin filaments with heavy meromyosin, some changes in polarized fluorescence of rhodaminyl--phalloin and 1.5-IAEDANS independent of phosphorylation of myosin light chains were found. Fluorescence anisotropy of the fiber was found to depend primarily on the character of heavy chain of SH-group modification. The ability of heavy chains to change their conformations is supposed to play an important role in the mechanism of myosin system modulation of muscle contraction.     

Asymmetric photocross-linking of singly phosphorylated smooth muscle heavy meromyosin

Although activities of smooth muscle myosin are regulated by phosphorylation, the molecular mechanisms of regulation have not been fully established. Phosphorylation of both heads of myosin is known to activate ATPase and motor activities, but the effects of phosphorylation of only one of the heads have not been established. Such information on singly phosphorylated myosin can serve to elucidate the molecular mechanism of the phosphorylation-dependent regulation. To understand the structural properties of the singly phosphorylated state, we prepared singly phosphorylated heavy meromyosin (HMM) containing a photoreactive benzophenone-labeled RLC and examined its photocross-linking reactivity. The two heads in the singly phosphorylated HMM showed different reactivities. The dephosphorylated RLC in the singly phosphorylated HMM was cross-linked to a heavy chain, like that in the dephosphorylated HMM, whereas the phosphorylated RLC did not react, like that in the fully phosphorylated HMM. These results indicate that the two heads of the singly phosphorylated HMM have an asymmetric structure, suggesting that phosphorylation of one head can to some extent activate smooth muscle HMM.