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.     

Calcium modulates conformational changes in F-actin induced by smooth muscle heavy meromyosin

The effect of Ca²⁺ on conformational changes in rhodamine-phalloidin-labeled F-actin induced by binding of smooth muscle heavy meromyosin (HMM) with either phosphorylated or dephosphorylated regulatory light chains (LC₂₀) was studied by polarized fluorimetry. LC₂₀ phosphorylation caused alterations in the F-actin structure typical of the force-producing (strong-binding) state, while dephosphorylation of the chains led to alterations typical of the formation of non-force-producing (weak-binding) state of the actomyosin complex. The presence of Ca²⁺ enhanced the effect of LC₂₀ phosphorylation and weakened the effect of LC₂₀ dephosphorylation. These data suggest that Ca²⁺ modulates actin-myosin interaction in smooth muscle by promoting formation of the strong-binding state.     

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.     

Caldesmon inhibits the cooperative turning-on of the smooth muscle heavy meromyosin by tropomyosin-actin

The 38-kDa chymotryptic fragment of caldesmon, which possesses the actin/calmodulin binding domain, was purified and utilized to study the mechanism for the inhibition of acto-myosin ATPase by caldesmon. The intact caldesmon inhibited the acto-HMM ATPase although it caused an increase in the binding of HMM to actin, presumably due to the interaction between the S-2 region of HMM and the caldesmon located on the actin filament. The 38-kDa fragment, which lacks the S-2 binding domain, inhibited both the acto-HMM ATPase and the HMM binding to actin. The ATPase and the HMM binding to actin decreased in parallel on increasing the 38-kDa fragment bound to actin. In the presence of tropomyosin, the ATPase activity fell more rapidly than did the HMM binding to actin. Binding of intact caldesmon or 38-kDa fragment to actin inhibited the cooperative turning-on of tropomyosin-actin by NEM.S-1, which forms rigor complexes in the presence of ATP. The absence of cooperative turning-on of the acto-HMM ATPase by rigor complexes in the presence of 38-kDa fragment was associated with an inhibition of the binding of HMM to tropomyosin-actin. Addition of NEM.S-1 to tropomyosin-actin-caldesmon caused a gradual decrease in the caldesmon-induced binding of HMM to actin. The calmodulin restored the caldesmon-induced binding of HMM to tropomyosin-actin, but it had only a slight effect on the acto-HMM ATPase. These data suggest that the cooperative turning-on of the smooth muscle tropomyosin-actin by rigor bonds is modulated by the interaction of caldesmon, tropomyosin, and calmodulin on the thin filament.     

Evidence for the association between two myosin heads in rigor acto-smooth muscle heavy meromyosin

The rigor complexes that formed between rabbit skeletal muscle F-actin and chicken gizzard heavy meromyosin (HMM), in which the heavy chains had been cleaved with trypsin into 24K, 50K, and 68K fragments, were examined by using the zero-length chemical cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). Two cross-linked products of approximate Mr 115K and 60K were generated. These products were not obtained by EDC treatment of HMM in the absence of F-actin. The HMM fragments that participated in cross-linking were identified by fluorescent labeling and amino acid composition studies. The 115K peptide was determined to be a covalently cross-linked complex that formed between actin and the COOH-terminal 68K fragment of the HMM heavy chain. Our results are in agreement with a previous study which proposed that the site of cross-linking between HMM and F-actin resides within the COOH-terminal 22K fragment of the myosin subfragment 1 heavy chain [Marianne-Pépin, T., Mornet, D., Bertrand, R., Labbé, J.-P., & Kassab, R. (1985) Biochemistry 24, 3024-3029]. The 60K peptide, however, was not a product of cross-linking between HMM and F-actin. On the basis of its amino acid composition, we concluded that this 60K peptide was a cross-linked dimer of the NH2-terminal 24K fragments of the HMM heavy chain. The cross-linking of acto-gizzard HMM significantly increased the Mg-ATPase activity of gizzard HMM without any observable phosphorylation of the regulatory (20K) light chains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lys-65 and Glu-168 are the residues for carbodiimide-catalyzed cross-linking between the two heads of rigor smooth muscle heavy meromyosin

We have previously demonstrated that the two heads of chicken gizzard heavy meromyosin (HMM) in a rigor complex with rabbit skeletal F-actin could be cross-linked by the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Here, we report the location of the cross-linked sites in the amino acid sequence of the HMM heavy chain. One of the cross-linked residues was identified as Glu-168 by sequencing the CN1.CN6 cross-linked peptide containing residues 1-77 (CN1) and 164-203 (CN6). This site is located close to the ATP-binding site of HMM. Since the other site was further into the amino acid sequence of CN1, another cross-linked peptide corresponding to residues 53-66 and 145-182 was isolated from the 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide-treated acto-tryptic gizzard HMM digested further by other proteolytic enzymes. The amino acid sequence of this peptide and its cyanogen bromide fragment indicated that the cross-linking occurred between Glu-168 and Lys-65. Our results suggests that these two amino acid side chains are in contact with each other in the acto-gizzard HMM rigor complex and participate in the electrostatic interaction between the two HMM heads bound to F-actin. Based on the head-to-head contact, we propose a three-dimensional model for the attachment of gizzard HMM heads to F-actin.     

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 mechanism for the inhibition of actin-activated ATPase of smooth muscle heavy meromyosin by calponin.

Calponin, an actin-binding protein, inhibited the acto-heavy meromyosin (HMM) MgATPase and lowered the binding of HMM to actin. The amount of calponin bound to actin or tropomyosin-actin was the same when the ATPase was inhibited 80-90%. While the KATPase was diminished only less than 2-fold in the presence of calponin, the Vmax was decreased 6-fold and 2-fold with actin and tropomyosin-actin, respectively. A comparison of the kinetic constants for the ATP hydrolysis obtained in the presence of actin-calponin and tropomyosin-actin-calponin revealed that the tropomyosin augmented the Vmax 5-fold from the inhibited level, but there was no effect on the KATPase.     

The light chain binding domain of expressed smooth muscle heavy meromyosin acts as a mechanical lever

Structural data led to the proposal that the molecular motor myosin moves actin by a swinging of the light chain binding domain, or "neck." To test the hypothesis that the neck functions as a mechanical lever, smooth muscle heavy meromyosin (HMM) mutants were expressed with shorter or longer necks by either deleting or adding light chain binding sites. The mutant HMMs were characterized kinetically and mechanically, with emphasis on measurements of unitary displacements and forces in the laser trap assay. Two shorter necked constructs had smaller unitary step sizes and moved actin more slowly than WT HMM in the motility assay. A longer necked construct that contained an additional essential light chain binding site exhibited a 1.4-fold increase in the unitary step size compared with its control. Kinetic changes were also observed with several of the constructs. The mutant lacking a neck produced force at a somewhat reduced level, while the force exerted by the giraffe construct was higher than control. The single molecule displacement and force data support the hypothesis that the neck functions as a rigid lever, with the fulcrum for movement and force located at a point within the motor domain.     

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.     

Two smooth muscle myosin heavy chains differ in their light meromyosin fragment

Smooth muscle myosin heavy chains [SM1, approximately 205 kilodaltons (kDa), and SM2, approximately 200 kDa] were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Peptide maps of the two heavy chains showed unique patterns. Limited proteolytic cleavage of purified swine stomach myosin was performed by using a variety of proteases to produce the major myosin fragments which were resolved on SDS gels. A single band was obtained for heavy meromyosin in the soluble fraction following chymotrypsin digestion. However, a variable number of bands were observed for light meromyosin fragments in the insoluble fraction after chymotrypsin digestion. Peptide mapping indicated that the two bands observed after short digestion times with chymotrypsin had relative mobility and solubility properties consistent with approximately 100- and 95-kDa light meromyosin (LMM) fragments. These results indicate that the region of difference between SM1 and SM2 lies in the LMM fragment.     

The effects of caldesmon on smooth muscle heavy actomeromyosin ATPase activity and binding of heavy meromyosin to actin

Caldesmon was purified to homogeneity from both chicken gizzard and bovine aortic smooth muscles. Caldesmon purified from bovine aorta was slightly larger than caldesmon purified from chicken gizzards (Mr = 140,000) when the two were compared electrophoretically. Caldesmon bound tightly to actin saturating at a molar ratio of 1 caldesmon monomer per 6.6 actin monomers. Ca2+-calmodulin appeared to reduce the affinity of caldesmon for actin. Caldesmon was also a potent inhibitor of heavy actomeromyosin ATPase activity producing a maximal effect at a ratio of 1 caldesmon monomer per 7-10 actin monomers. This effect was also antagonized by Ca2+-calmodulin. While caldesmon inhibited heavy actomeromyosin ATPase activity, it greatly enhanced binding of both unphosphorylated and phosphorylated heavy meromyosin to actin in the presence of MgATP, reducing the Kd for binding by a factor of 40 for each form of heavy meromyosin. Although we did identify a Ca2+-calmodulin-stimulated "caldesmon kinase" activity in caldesmon preparations purified under nondenaturing conditions, we observed no effect of phosphorylation (2 mol of PO4/mol of caldesmon) on the capacity to inhibit heavy actomeromyosin ATPase activity. Our results suggest that caldesmon could serve some role in smooth muscle function by enhancing cross-bridge affinity while inhibiting actomyosin ATPase activity.     

Reversible phosphorylation of smooth muscle myosin, heavy meromyosin, and platelet myosin

Smooth muscle myosin was purified from turkey gizzards with the 20,000-dalton light chains in the unphosphorylated state. The actin-activated MgATPase activity was 4 nmol/min/mg at 25 degrees C. When the myosin was phosphorylated to 2 mol of Pi/mol of myosin using purified myosin light chain kinase, calmodulin, and ATP, the actin-activated MgATPase activity rose to 51 nmol/min/mg. Complete dephosphorylation of the same myosin by a purified phosphatase lowered the activity to 5 nmol/min/mg, and complete rephosphorylation of the myosin following inhibition of the phosphatase raised it again to 46 nmol/min/mg. Human platelet myosin could be substituted for turkey gizzard myosin, with similar results. A chymotryptic fragment of smooth muscle myosin which retains the phosphorylated site on the 20,000-dalton light chain of myosin was prepared. Using the same scheme for reversible phosphorylation, this smooth muscle heavy meromyosin was found to show the same positive correlation between phosphorylation of the myosin light chain and the actin-activated MgATPase activity. The results with smooth muscle heavy meromyosin show that the effect of phosphorylation on the actin-activated MgATPase activity can be separated from the effects of phosphorylation on myosin filament assembly.     

Phosphorylation of smooth muscle heavy meromyosin by calcium-activated, phospholipid-dependent protein kinase. The effect on actin-activated MgATPase activity

Smooth muscle heavy meromyosin (HMM) can serve as a substrate for the Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) as well as for the Ca2+/calmodulin-dependent kinase, myosin light chain kinase. When turkey gizzard HMM is incubated with protein kinase C, 1.7-2.2 mol of phosphate are incorporated per mol of HMM, all of it into the 20,000-Da light chain of HMM. Two-dimensional peptide mapping following tryptic hydrolysis revealed that protein kinase C phosphorylated a different site on the 20,000-Da HMM light chain than did myosin light chain kinase. Moreover, sequential phosphorylation of HMM by myosin light chain kinase and protein kinase C resulted in the incorporation of 4 mol of phosphate/mol of HMM, i.e. 2 mol of phosphate into each 20,000-Da light chain. When unphosphorylated HMM was phosphorylated by myosin light chain kinase, its actin-activated MgATPase activity increased from 4 nmol to 156 nmol of phosphate released/mg of HMM/min. Subsequent phosphorylation of this phosphorylated HMM by protein kinase C decreased the actin-activated MgATPase activity of HMM to 75 nmol of phosphate released/mg of HMM/min.     

Structures of smooth muscle myosin and heavy meromyosin in the folded, shutdown state

Remodelling the contractile apparatus within smooth muscle cells allows effective contractile activity over a wide range of cell lengths. Thick filaments may be redistributed via depolymerisation into inactive myosin monomers that have been detected in vitro, in which the long tail has a folded conformation. Using negative stain electron microscopy of individual folded myosin molecules from turkey gizzard smooth muscle, we show that they are more compact than previously described, with heads and the three segments of the folded tail closely packed. Heavy meromyosin (HMM), which lacks two-thirds of the tail, closely resembles the equivalent parts of whole myosin. Image processing reveals a characteristic head region morphology for both HMM and myosin, with features identifiable by comparison with less compact molecules. The two heads associate asymmetrically: the tip of one motor domain touches the base of the other, resembling the blocked and free heads of this HMM when it forms 2D crystals on lipid monolayers. The tail of HMM lies between the heads, contacting the blocked motor domain, unlike in the 2D crystal. The tail of whole myosin is bent sharply and consistently close to residues 1175 and 1535. The first bend position correlates with a skip in the coiled coil sequence, the second does not. Tail segments 2 and 3 associate only with the blocked head, such that the second bend is near the C-lobe of the blocked head regulatory light chain. Quantitative analysis of tail flexibility shows that the single coiled coil of HMM has an apparent Young's modulus of about 0.5 GPa. The folded tail of the whole myosin is less flexible, indicating interactions between the segments. The folded tail does not modify the compact head arrangement but stabilises it, indicating a structural mechanism for the very low ATPase activity of the folded molecule.     

Effect of phosphorylation on the binding of smooth muscle heavy meromyosin X ADP to actin

Relaxation of both smooth and skeletal muscles appears to be caused primarily by inhibition of the step associated with Pi release in the actomyosin ATPase cycle, rather than by a block in the binding of the myosin X ATP and myosin X ADP X Pi complexes to actin. In skeletal muscle, troponin-tropomyosin not only causes marked inhibition of Pi release, but it also markedly inhibits the binding of myosin subfragment-1 X ADP to actin, raising the possibility that the two phenomena are coupled in some way. In the present study we determined whether phosphorylation of smooth muscle heavy meromyosin (HMM) also affects both the binding of HMM X ADP to actin and the Pi release step. This was done by having phosphorylated and unphosphorylated HMM X ADP compete for sites on F-actin. At mu = 30 mM, phosphorylation increased the affinity of the HMM molecule for actin about 12-fold and at mu = 170 mM, there was less than a 3-fold increase in the affinity of HMM. If phosphorylation affects the binding of each head of HMM to the same extent, then phosphorylation caused about a 4- and 2-fold increase in the affinity of each head of HMM for actin at mu = 30 and 170 mM, respectively. In contrast, at both ionic strengths, phosphorylation caused more than 100-fold actin activation of the ATPase activity of smooth muscle HMM. Therefore, the marked activation of Pi release in the acto X HMM ATPase cycle upon phosphorylation of HMM is not accompanied by a comparable increase in the affinity of HMM X ADP for actin. We have also found that phosphorylation increases by only 4-fold the rate of Pi release from HMM alone. These results suggest that in smooth muscle, phosphorylation accelerates the step associated with the release of Pi both in the forward and the reverse direction without correspondingly affecting the binding of myosin X ADP to actin.     

Phosphorylation-dependent structural changes in the regulatory light chain domain of smooth muscle heavy meromyosin.

Smooth muscle heavy meromyosin, a double-headed proteolytic fragment of myosin lacking the COOH-terminal two-thirds of the tail, has been shown previously to be regulated by phosphorylation. To examine phosphorylation-dependent structural changes near the head-tail junction, we prepared five well regulated heavy meromyosins containing single-cysteine mutants of the human smooth muscle regulatory light chain labeled with the photocross-linking reagent, benzophenone-iodoacetamide. For those mutants that generated cross-links, only one type of cross-linked species was observed, a regulatory light chain dimer. Irradiated mutants fell into two classes. First, for Q15C, A23C, and wild type (Cys-108), a regulatory light chain dimer was formed for dephosphorylated but not thiophosphorylated heavy meromyosin. These data provide direct chemical evidence that in the dephosphorylated state, Gln-15, Ala-23, and Cys-108 on one head are positioned near (within 8.9 A) the regulatory light chain of the partner head and that thiophosphorylation abolishes proximity. This behavior was also observed for the Q15C mutant on a truncated heavy meromyosin lacking both catalytic domains. For the actin-heavy meromyosin complex, cross-links were formed in both de- and thiophosphorylated states. S59C and T134C mutants were in a second mutant class, where regulatory light chain dimers were not detected in dephosphorylated or thiophosphorylated heavy meromyosin, suggesting positions outside the region of interaction of the regulatory light chains.     

Protein kinase C modulates in vitro phosphorylation of the smooth muscle heavy meromyosin by myosin light chain kinase

Protein kinase C phosphorylates different sites on the 20,000-Da light chain of smooth muscle heavy meromyosin (HMM) than did myosin light chain kinase (Nishikawa, M., Hidaka, H., and Adelstein, R. S. (1983) J. Biol. Chem. 258, 14069-14072). Although protein kinase C incorporates 1 mol of phosphate into 1 mol of 20,000-Da light chain when either HMM or the whole myosin molecule is used as a substrate, it catalyzes the incorporation of up to 3 mol of phosphate/mol of 20,000-Da light chain when the isolated light chains are used as a substrate. Threonine is the major phosphoamino acid resulting from phosphorylation of HMM by protein kinase C. Prephosphorylation of HMM by protein kinase C decreases the rate of phosphorylation of HMM by myosin light chain kinase due to a 9-fold increase of the Km for prephosphorylated HMM compared to that of unphosphorylated HMM. Prephosphorylation of HMM by myosin light chain kinase also results in a decrease of the rate of phosphorylation by protein kinase C due to a 2-fold increase of the Km for HMM. Both prephosphorylations have little or no effect on the maximum rate of phosphorylation. The sequential phosphorylation of HMM by myosin light chain kinase and protein kinase C results in a decrease in actin-activated MgATPase activity due to a 7-fold increase of the Km for actin over that observed with phosphorylated HMM by myosin light chain kinase but has little effect on the maximum rate of the actin-activated MgATPase activity. The decrease of the actin-activated MgATPase activity correlates well with the extent of the additional phosphorylation of HMM by protein kinase C following initial phosphorylation by myosin light chain kinase.     

The binding of smooth muscle heavy meromyosin to actin in the presence of ATP. Effect of phosphorylation

Phosphorylation of the 20,000-dalton light chains of smooth muscle heavy meromyosin (HMM) from turkey gizzards results in a large increase in the actin-activated MgATPase activity over that observed with unphosphorylated HMM. In an attempt to define which step in the kinetic cycle is affected by phosphorylation, we have measured the binding of both unphosphorylated and phosphorylated HMM to actin in the presence of ATP using sedimentation. There was only a 4-fold difference in the actin binding constants of unphosphorylated HMM (5.35 x 10(3) M-1) and fully phosphorylated HMM (2.35 x 10(4) M-1). In contrast, the maximum rate of the actin-activated MgATPase activity (Vmax) of phosphorylated HMM was 25 times greater than that for unphosphorylated HMM. These data rule out a mechanism whereby the unphosphorylated light chain of myosin regulates actin-myosin interaction by directly or indirectly blocking the binding of HMM to actin. This implies that some step in the kinetic cycle other than the binding of HMM to actin must be regulated. We have also measured the rate constant for ATP hydrolysis (the initial phosphate burst) under the same conditions and found that this step was very fast compared to the steady state ATPase rate and was unaffected by phosphorylation. This suggests that the step which is regulated by phosphorylation is either phosphate release or a step preceding phosphate release but following ATP hydrolysis.