Locking Regulatory Myosin in the Off-state with Trifluoperazine
2000; Elsevier BV; Volume: 275; Issue: 7 Linguagem: Inglês
10.1074/jbc.275.7.4880
ISSN1083-351X
AutoresHitesh Patel, Sarkis S. Margossian, Peter D. Chantler,
Tópico(s)Neuroscience and Neural Engineering
ResumoScallop striated adductor muscle myosin is a regulatory myosin, its activity being controlled directly through calcium binding. Here, we show that millimolar concentrations of trifluoperazine were effective at removal of all regulatory light chains from scallop myosin or myofibrils. More important, 200 μm trifluoperazine, a concentration 10-fold less than that required for light-chain removal, resulted in the reversible elimination of actin-activated and intrinsic ATPase activities. Unlike desensitization induced by metal ion chelation, which leads to an elevation of activity in the absence of calcium concurrent with regulatory light-chain removal, trifluoperazine caused a decline in actin-activated MgATPase activity both in the presence and absence of calcium. Procedures were equally effective with respect to scallop myosin, myofibrils, subfragment-1, or desensitized myofibrils. Increased α-helicity could be induced in the isolated essential light chain through addition of 100–200 μm trifluoperazine. We propose that micromolar concentrations of trifluoperazine disrupt regulation by binding to a single high-affinity site located in the C-terminal domain of the essential light chain, which locks scallop myosin in a conformation resembling the off-state. At millimolar trifluoperazine concentrations, additional binding sites on both light chains would be filled, leading to regulatory light-chain displacement. Scallop striated adductor muscle myosin is a regulatory myosin, its activity being controlled directly through calcium binding. Here, we show that millimolar concentrations of trifluoperazine were effective at removal of all regulatory light chains from scallop myosin or myofibrils. More important, 200 μm trifluoperazine, a concentration 10-fold less than that required for light-chain removal, resulted in the reversible elimination of actin-activated and intrinsic ATPase activities. Unlike desensitization induced by metal ion chelation, which leads to an elevation of activity in the absence of calcium concurrent with regulatory light-chain removal, trifluoperazine caused a decline in actin-activated MgATPase activity both in the presence and absence of calcium. Procedures were equally effective with respect to scallop myosin, myofibrils, subfragment-1, or desensitized myofibrils. Increased α-helicity could be induced in the isolated essential light chain through addition of 100–200 μm trifluoperazine. We propose that micromolar concentrations of trifluoperazine disrupt regulation by binding to a single high-affinity site located in the C-terminal domain of the essential light chain, which locks scallop myosin in a conformation resembling the off-state. At millimolar trifluoperazine concentrations, additional binding sites on both light chains would be filled, leading to regulatory light-chain displacement. trifluoperazine essential light chain regulatory light chain myosin subfragment-1 4-morpholinepropanesulfonic acid Trifluoperazine (TFP),1a member of the phenothiazine class of drugs, is one of the strongest antagonists of calmodulin action known, capable of binding to calmodulin in the presence of calcium and preventing its stimulatory effects (1.Levin R.M. Weiss B. Mol. Pharmacol. 1976; 12: 581-589PubMed Google Scholar, 2.Prozialeck W.C. Weiss B. J. Pharmacol. Exp. Ther. 1982; 222: 509-516PubMed Google Scholar, 3.Roufogalis B.D. Minocherhomjee A.M. Al-Jobore A. Can. J. Biochem. Cell Biol. 1983; 61: 927-933Crossref PubMed Scopus (78) Google Scholar). The structure of the TFP·calmodulin complex in the presence of calcium has been determined at 2.45-Å resolution (4.Cook W.J. Walter L.J. Walter M.R. Biochemistry. 1994; 33: 15259-15265Crossref PubMed Scopus (124) Google Scholar, 5.Vandonselaar M. Hickie R.A. Quail J.W. Delbaere L.T.J. Nat. Struct. Biol. 1994; 1: 795-801Crossref PubMed Scopus (185) Google Scholar), where it was shown that TFP induces a profound conformational change in calmodulin, converting the elongated dumbbell to a compact globular structure, analogous to the form obtained through the binding of calmodulin to a target peptide (6.Meador W.E. Means A.R. Quiocho F.A. Science. 1992; 257: 1251-1255Crossref PubMed Scopus (939) Google Scholar, 7.Ikura M. Marius Clore G. Gronenborn A.M. Zhu G. Klee C.B. Bax A. Science. 1992; 256: 632-638Crossref PubMed Scopus (1176) Google Scholar). This effect was accomplished when a single TFP molecule bound to the predominant binding site, a hydrophobic pocket within the C-terminal domain (4.Cook W.J. Walter L.J. Walter M.R. Biochemistry. 1994; 33: 15259-15265Crossref PubMed Scopus (124) Google Scholar). Additional TFP-binding sites are apparent when the TFP concentration is raised (5.Vandonselaar M. Hickie R.A. Quail J.W. Delbaere L.T.J. Nat. Struct. Biol. 1994; 1: 795-801Crossref PubMed Scopus (185) Google Scholar). Recently, TFP-binding sites on the related calcium-binding protein troponin C have also been identified (8.Kleererkoper Q. Liu W. Choi D. Putkey J.A. J. Biol. Chem. 1998; 273: 8153-8160Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar).Myosin light chains belong to the same large family of calcium-binding proteins as calmodulin, to which they display a similar overall structure (9.Persechini A. Moncrief N.D. Kretsinger R.H. Trends Neurosci. 1989; 12: 462-467Abstract Full Text PDF PubMed Scopus (292) Google Scholar, 10.Houdusse A. Cohen C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10644-10647Crossref PubMed Scopus (99) Google Scholar). There are two types of light chain, termed regulatory (R-LC) and essential (E-LC); one member of each type binds to each of the two heads of conventional myosin II. In those regulatory myosins that are activated directly by calcium binding (for review, see Ref. 11.Szent-Gyorgyi A.G. Chantler P.D. Engel A.G. Franzini-Armstrong C. Myology. II. McGraw-Hill Book Co., New York1994: 506-528Google Scholar), the exact relationship of the heavy chain to the light chains, in the presence of calcium, is now known in detail, the structure of the regulatory domain of scallop myosin having first been established at 2.8-Å resolution (12.Xie X. Harrison D.H. Schlichting I. Sweet R.M. Kalabokis V.N. Szent-Gyorgyi A.G. Cohen C. Nature. 1994; 368: 306-312Crossref PubMed Scopus (265) Google Scholar) and then refined to 2.0-Å resolution (13.Houdusse A. Cohen C. Structure. 1996; 4: 21-32Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Solution studies (14.Huang W. Wilson G.J. Brown L.J. Lam H. Hambly B. Eur. J. Biochem. 1998; 257: 457-465Crossref PubMed Scopus (16) Google Scholar) indicate that TFP can bind to the isolated light chains: measurements of the circular dichroism indicated half-maximal binding of TFP with a dissociation constant in the range of 14–50 μm, the binding of which resulted in a significant change in secondary structure consistent with an increase in α-helical content, whereas EPR spectroscopy detected binding at sites of lower affinity with half-maximal effects yielding dissociation constants in the range of 370–800 μm.The removal of R-LCs from scallop myosin through chelation of metal ions is a well known phenomenon (15.Szent-Gyorgyi A.G. Szentkiralyi E.M. Kendrick-Jones J. J. Mol. Biol. 1973; 74: 179-203Crossref PubMed Scopus (274) Google Scholar, 16.Kendrick-Jones J. Szentkiralyi E.M. Szent-Gyorgyi A.G. J. Mol. Biol. 1976; 104: 747-775Crossref PubMed Scopus (229) Google Scholar), complete dissociation being achieved in a reversible manner at elevated temperatures (17.Chantler P.D. Szent-Gyorgyi A.G. J. Mol. Biol. 1980; 138: 473-492Crossref PubMed Scopus (129) Google Scholar, 18.Chantler P.D. J. Mol. Biol. 1985; 181: 557-560Crossref PubMed Scopus (6) Google Scholar). However, the same treatment has proven ineffective when applied to smooth muscle and other myosins (11.Szent-Gyorgyi A.G. Chantler P.D. Engel A.G. Franzini-Armstrong C. Myology. II. McGraw-Hill Book Co., New York1994: 506-528Google Scholar). Recently, TFP was shown to facilitate both R-LC exchange and R-LC dissociation from smooth muscle myosin (19.Trybus K.M. Waller G.S. Chatman T.A. J. Cell Biol. 1994; 124: 963-969Crossref PubMed Scopus (62) Google Scholar, 20.Yang Z. Sweeney H.L. J. Biol. Chem. 1995; 270: 24646-24649Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 21.Katoh T. Morita F. J. Biol. Chem. 1996; 271: 9992-9996Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). It was therefore of interest to see whether or not TFP would prove effective at R-LC removal from scallop myosin. Here, we demonstrate that TFP in the millimolar range can remove R-LCs from scallop myosin. However, unlike the ensuing loss of regulation that accompanies metal ion chelation, brought about through anelevation of actin-activated MgATPase in the absence of calcium (17.Chantler P.D. Szent-Gyorgyi A.G. J. Mol. Biol. 1980; 138: 473-492Crossref PubMed Scopus (129) Google Scholar), TFP binding results in a monotonous decline in actin-activated MgATPase both in the presence and absence of calcium. Furthermore, we demonstrate that the loss of regulation brought about by TFP occurs at concentrations an order of magnitude lower than those required to effect R-LC dissociation. These results are discussed in the light of current structural knowledge: we formulate a hypothesis suggesting that TFP may act by interfering with the conformational relay mechanism operating through the interface between the C-terminal lobe of E-LC and regions of the heavy chain, thereby fixing the "off-state" of scallop myosin.DISCUSSIONThe selective and total dissociation of R-LC from scallop myosin by TFP treatment (Fig. 1) is reminiscent of R-LC removal from scallop myosin as a consequence of 10 mm EDTA treatment at elevated temperatures (17.Chantler P.D. Szent-Gyorgyi A.G. J. Mol. Biol. 1980; 138: 473-492Crossref PubMed Scopus (129) Google Scholar). Furthermore, increasing concentrations of TFP gave rise to a decline in actin-activated MgATPase in the presence of calcium (Figs. 2 and 3), superficially also similar to the effect of R-LC loss through metal ion chelation (17.Chantler P.D. Szent-Gyorgyi A.G. J. Mol. Biol. 1980; 138: 473-492Crossref PubMed Scopus (129) Google Scholar). However, the effects of TFP and EDTA on scallop myosin are not the same and differ from each other in several ways, as described below.Of fundamental importance are our observations of the effect of TFP on actin-activated MgATPase in the absence of calcium: the inhibited state of intact scallop myosin. In the case of R-LC removal through divalent cation chelation, a biphasic rise in this ATPase activity as a function of R-LC loss was observed (17.Chantler P.D. Szent-Gyorgyi A.G. J. Mol. Biol. 1980; 138: 473-492Crossref PubMed Scopus (129) Google Scholar, 18.Chantler P.D. J. Mol. Biol. 1985; 181: 557-560Crossref PubMed Scopus (6) Google Scholar), providing insight into the cooperative nature of regulatory myosins. By contrast, TFP treatment of scallop myosin or myofibrils led to a monotonic decline in actin-activated MgATPase (−Ca2+) (Figs. 2 and 3). Furthermore, the sensitivity of actin-activated MgATPase activity to TFP took place over a concentration range an order of magnitude lower than that required to elicit R-LC removal (compare Figs. 1 cand 2). In both the presence and absence of calcium, 50% inactivation of actin-activated myosin MgATPase occurred at 100–150 μm TFP, and full inactivation was achieved at 300 μm TFP (Fig. 2), whereas R-LC removal began only at TFP concentrations >500 μm, with full dissociation being achieved at 3.0 mm TFP (Fig. 1). These effects were reversible at 200 μm TFP (Table I).TFP-induced removal of R-LC was specific, with no E-LC being lost during treatment (Fig. 1). When scallop R-LC was removed totally by EDTA treatment prior to TFP addition, E-LC removal remained refractory to TFP treatment, although some small losses (<5%) did occur (data not shown). Such losses are consistent with data suggesting that prior R-LC removal destabilizes E-LC attachment to the myosin heavy chain (21.Katoh T. Morita F. J. Biol. Chem. 1996; 271: 9992-9996Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 29.Ashiba G. Szent-Gyorgyi A.G. Biochemistry. 1985; 24: 6618-6623Crossref PubMed Scopus (18) Google Scholar). For smooth muscle myosin, a combined treatment of 5–10 mm TFP and 4.5 m ammonium chloride facilitates E-LC exchange and even complete removal of both R-LCs and E-LCs (21.Katoh T. Morita F. J. Biol. Chem. 1996; 271: 9992-9996Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar,30.Katoh T. Numata T. Konishi K. Furuya K. Yazawa M. Anal. Biochem. 1997; 253: 78-84Crossref PubMed Scopus (6) Google Scholar). E-LC exchange, in the absence of R-LC removal, has been shown to occur through the action of 1 mm TFP on permeabilized smooth muscle cells (31.Matthew J.D. Khromov A.S. Trybus K.M. Somlyo A.P. Somlyo A.V. J. Biol. Chem. 1998; 273: 31289-31296Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar).It is significant that the incremental addition of TFP to EDTA-desensitized myofibrils was just as effective at attenuating actin-activated MgATPase as the addition of TFP to intact myofibrils (Fig. 3). TFP also inactivated actin-activated S-1 MgATPase (Table II), analogous to its effect on myosin (Fig. 2) and myofibrils (Fig. 3). Whereas complete removal of R-LCs by EDTA treatment does not impair the K+EDTA-ATPase of scallop myosin (17.Chantler P.D. Szent-Gyorgyi A.G. J. Mol. Biol. 1980; 138: 473-492Crossref PubMed Scopus (129) Google Scholar), TFP treatment does cause a significant decline in this indicator of active-site function, with 75% calcium-specific binding remained at 150 μmTFP, a concentration where actin-activated MgATPase was >50% inactivated and where no loss of R-LC had occurred. Although this relationship between calcium binding and actin activation is expected for heavy meromyosin, myosin should exhibit nearly 100% activity at this level of calcium binding (26.Chantler P.D. Sellers J.R. Szent-Gyorgyi A.G. Biochemistry. 1981; 20: 210-216Crossref PubMed Scopus (63) Google Scholar); that it does not do so is indicative of an inhibitory effect by TFP above and beyond its effect on calcium binding. At 500 μm TFP, only 40–50% calcium-specific binding remained in the case of scallop myosin, whereas calcium-specific binding to Ca·Mg·S-1 remained unimpaired (Fig. 5). This result emphasizes the functional dislocation of regulation from activity in scallop Ca·Mg·S-1, a molecule in which regulatory capability is completely impaired (15.Szent-Gyorgyi A.G. Szentkiralyi E.M. Kendrick-Jones J. J. Mol. Biol. 1973; 74: 179-203Crossref PubMed Scopus (274) Google Scholar, 33.Kalabokis V.N. Szent-Gyorgyi A.G. Biochemistry. 1997; 36: 15834-15840Crossref PubMed Scopus (34) Google Scholar). Scallop Ca·Mg·S-1 is also known to be more refractory to EDTA-induced dissociation of R-LC as compared with the two-headed myosin structure (24.Stafford W.F. Szentkiralyi E.M. Szent-Gyorgyi A.G. Biochemistry. 1979; 18: 5273-5280Crossref PubMed Scopus (99) Google Scholar).From the above results, it is clear that the effects of TFP binding are manifest over two distinct concentration ranges, indicative of at least two different sets of binding sites. Whereas 300 μm TFP is required to eliminate actin activation and regulation (50% inactivation at 100–150 μm TFP), 3000 μmTFP is required to remove R-LCs (50% inactivation at 1300–1600 μm TFP). Spectroscopic studies on TFP binding to skeletal muscle myosin light chains in solution have demonstrated the presence of two types of TFP-binding site, differing in affinity by at least an order of magnitude. Measurements of the far-UV CD spectra indicated that TFP elicited increases in helical content with half-maximal effects at ∼20–50 μm TFP for LC1 and LC3 and ∼14 μm for LC2 (14.Huang W. Wilson G.J. Brown L.J. Lam H. Hambly B. Eur. J. Biochem. 1998; 257: 457-465Crossref PubMed Scopus (16) Google Scholar). By contrast, paramagnetic spin probes attached to the same light chains exhibited a change in rotational correlation time upon TFP addition, the effect being half-maximal at ∼370–809 μm TFP, an order of magnitude higher than the CD results (14.Huang W. Wilson G.J. Brown L.J. Lam H. Hambly B. Eur. J. Biochem. 1998; 257: 457-465Crossref PubMed Scopus (16) Google Scholar). Here, through far-UV CD analysis (Fig. 6), we have confirmed that micromolar concentrations of TFP have a direct effect in inducing an increase in α-helicity in isolated scallop E-LC; the effect is maximal by ∼100 μm. The higher affinities of the two sets of TFP-dependent transitions determined from spectroscopic data on isolated light chains as compared with the two ranges of TFP concentration required for the two different functional results obtained here on scallop myosin may reflect the fact that functional effects can be assessed on only the multichain structure.TFP binding to the hydrophobic pocket within the C-terminal lobe of E-LC would appear to be the most likely cause of ATPase inactivation. There is a monotonic decline in actin-activated MgATPase (±Ca2+) that is complete at 300 μm TFP, and prior removal of R-LC by EDTA still permits TFP inactivation of the desensitized myosin and exhibits the same concentration dependence (Fig. 3), suggesting that the heavy chain·E-LC complex can be induced to attain this extreme inhibited state. Also, TFP can induce, directly, a conformational change in isolated E-LC (Fig. 6). In the stoichiometric TFP·calmodulin complex, the hydrophobic tricyclic ring of TFP localizes to a hydrophobic pocket within the C-terminal lobe of calmodulin, entrapping TFP·calmodulin in the same conformation as that seen when calmodulin binds to a target peptide substrate (4.Cook W.J. Walter L.J. Walter M.R. Biochemistry. 1994; 33: 15259-15265Crossref PubMed Scopus (124) Google Scholar, 6.Meador W.E. Means A.R. Quiocho F.A. Science. 1992; 257: 1251-1255Crossref PubMed Scopus (939) Google Scholar,7.Ikura M. Marius Clore G. Gronenborn A.M. Zhu G. Klee C.B. Bax A. Science. 1992; 256: 632-638Crossref PubMed Scopus (1176) Google Scholar). Although the C-terminal lobes of scallop R-LC and E-LC exhibit semi-open conformations, in contrast to the C-terminal lobe of calmodulin, which displays a fully open conformation (10.Houdusse A. Cohen C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10644-10647Crossref PubMed Scopus (99) Google Scholar), the required hydrophobic pocket remains available, albeit less deeply placed (Fig.7, a and b). Of the 12 residues directly involved in TFP binding within the C-terminal lobe of calmodulin (4.Cook W.J. Walter L.J. Walter M.R. Biochemistry. 1994; 33: 15259-15265Crossref PubMed Scopus (124) Google Scholar), 7 are identical to sequences found within eitherArgopecten irradians (27.Goodwin E.B. Leinwand L.A. Szent-Gyorgyi A.G. J. Mol. Biol. 1990; 216: 85-99Crossref PubMed Scopus (25) Google Scholar) or Pecten maximus (34.Janes D. Patel H. Chantler P.D. J. Muscle Res. Cell Motil. 1999; 20: 80Google Scholar) E-LC, and another two represent conservative replacements; furthermore, the spatial configuration of these residues appears to be conserved (Fig. 7, a and b). This places TFP within the hydrophobic pocket located between the two domains of the C-terminal lobe of E-LC (Fig. 7 b).Currently, crystal structures of myosin heads from two regulatory myosins are available: the chicken smooth muscle myosin motor domain·E-LC complex, crystallized in the presence of MgADP·AlF4− or MgADP·BeFxto 3.5- and 3.6-Å resolution, respectively (35.Dominguez R. Freyzon Y. Trybus K.M. Cohen C. Cell. 1998; 94: 559-571Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar); and scallop (A. irradians) S-1, crystallized in the presence of MgADP to 2.5-Å resolution (36.Houdusse A. Kalabokis V.N. Himmel D. Szent-Gyorgyi A.G. Cohen C. Cell. 1999; 97: 459-470Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). These are very different structures (Fig. 7,c and d). With respect to the chicken smooth muscle myosin structure (35.Dominguez R. Freyzon Y. Trybus K.M. Cohen C. Cell. 1998; 94: 559-571Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar), the G-helix within the TFP-binding lobe of E-LC abuts the motor domain close to the 25/50-kDa loop (Ala198–Lys204) (Fig. 7 c), a region of the heavy chain implicated as a major determinant in the rate of ADP release (37.Spudich J.A. Nature. 1994; 372: 515-518Crossref PubMed Scopus (423) Google Scholar, 38.Sweeney H.L. Rosenfeld S.S. Brown F. Faust L. Smith J. Xing J. Stein L.A. Sellers J.R. J. Biol. Chem. 1998; 273: 6262-6270Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 39.Perreault-Micale C.L. Kalabokis V.N. Nyitray L. Szent-Gyorgyi A.G. J. Muscle Res. Cell Motil. 1996; 17: 543-553Crossref PubMed Scopus (52) Google Scholar, 40.Rovner A.S. Freyzon Y. Trybus K.M. J. Muscle Res. Cell Motil. 1997; 18: 103-110Crossref PubMed Scopus (114) Google Scholar, 41.Kurzawa-Goertz S.E. Perreault-Micale C.L. Trybus K.M. Szent-Gyorgyi A.G. Geeves M.A. Biochemistry. 1998; 37: 7517-7525Crossref PubMed Scopus (64) Google Scholar). Additionally, other surface loop structures (Met140–His147, Ser163–Asp170, and Phe256–Tyr261 in the chicken smooth muscle myosin structure (35.Dominguez R. Freyzon Y. Trybus K.M. Cohen C. Cell. 1998; 94: 559-571Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar)) are capable of interaction with this TFP-binding lobe of E-LC. By contrast, within the scallop MgADP·S-1 structure (36.Houdusse A. Kalabokis V.N. Himmel D. Szent-Gyorgyi A.G. Cohen C. Cell. 1999; 97: 459-470Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar), the TFP-binding lobe of E-LC abuts the motor domain close to the N terminus (especially Ala45–Lys60) (Fig.7 d) within the so-called SH3 domain, which has been suggested to limit the potential swing of the lever arm (35.Dominguez R. Freyzon Y. Trybus K.M. Cohen C. Cell. 1998; 94: 559-571Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar). This region of contact is close to the heavy-chain region (Glu21–Asn29) found in the nucleotide-free skeletal myosin structure (42.Rayment I. Rypniewski W.R. Schmidt-Base K. Smith R. Tomchick D.R. Benning M.M. Winkelmann D.A. Wesenberg G. Holden H.M. Science. 1993; 261: 50-58Crossref PubMed Scopus (1859) Google Scholar), where it abuts the F-helix within the C-terminal domain of E-LC (as described (35.Dominguez R. Freyzon Y. Trybus K.M. Cohen C. Cell. 1998; 94: 559-571Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar)). Although crystallized in the presence of MgADP, the point is well made (36.Houdusse A. Kalabokis V.N. Himmel D. Szent-Gyorgyi A.G. Cohen C. Cell. 1999; 97: 459-470Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar) that scallop MgADP·S-1 may be considered to be in an "ATP-like" or pre-power stroke state; consequently, this structure is classified as being in the weak binding state. Here, too, the F-helix of E-LC makes contact with heavy-chain residues, located within an α-helix close to the N terminus (Fig. 7 d) (36.Houdusse A. Kalabokis V.N. Himmel D. Szent-Gyorgyi A.G. Cohen C. Cell. 1999; 97: 459-470Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). If the conformational off-state locked by TFP corresponds to either of the above structures (Fig. 7,c and d), it can be seen that TFP will bind at a key location along alternative sequential conformational relays that carry information from the triggering site within the N-terminal domain of E-LC, via the C-terminal lobe of E-LC, to the ATP-binding pocket. We speculate that the interface between the 25/50-kDa loop and E-LC is most likely to participate in the off-state (as seen in Fig.7 c) because the 25/50-kDa loop is known to be a major determinant of the rate of ADP release (37.Spudich J.A. Nature. 1994; 372: 515-518Crossref PubMed Scopus (423) Google Scholar, 38.Sweeney H.L. Rosenfeld S.S. Brown F. Faust L. Smith J. Xing J. Stein L.A. Sellers J.R. J. Biol. Chem. 1998; 273: 6262-6270Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 39.Perreault-Micale C.L. Kalabokis V.N. Nyitray L. Szent-Gyorgyi A.G. J. Muscle Res. Cell Motil. 1996; 17: 543-553Crossref PubMed Scopus (52) Google Scholar, 40.Rovner A.S. Freyzon Y. Trybus K.M. J. Muscle Res. Cell Motil. 1997; 18: 103-110Crossref PubMed Scopus (114) Google Scholar, 41.Kurzawa-Goertz S.E. Perreault-Micale C.L. Trybus K.M. Szent-Gyorgyi A.G. Geeves M.A. Biochemistry. 1998; 37: 7517-7525Crossref PubMed Scopus (64) Google Scholar), and the rate-limiting state in the absence of calcium is likely to be a weak binding ADP·Pi intermediate (45.Marston S.B. Lehman W. Nature. 1974; 252: 38-39Crossref PubMed Scopus (9) Google Scholar, 46.Jackson A.P. Bagshaw C.R. Biochem. J. 1988; 251: 515-526Crossref PubMed Scopus (26) Google Scholar), not an ATP-like structure. Further work is needed to test this hypothesis through both structural and functional means. However, it is unlikely that either structure, alone, can account for all features of regulation, two heads being required (26.Chantler P.D. Sellers J.R. Szent-Gyorgyi A.G. Biochemistry. 1981; 20: 210-216Crossref PubMed Scopus (63) Google Scholar, 43.Kalabokis V.N. Vibert P. York M.L. Szent-Gyorgyi A.G. J. Biol. Chem. 1996; 271: 26779-26782Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 44.Trybus K.M. Freyzon Y. Faust L.Z. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 48-52Crossref PubMed Scopus (128) Google Scholar).Our results may also suggest that different mechanisms for the control of the off-state exist in regulatory myosins controlled either by direct calcium binding or through phosphorylation. Although the triggers are very different (calcium binds to scallop myosin E-LC (12.Xie X. Harrison D.H. Schlichting I. Sweet R.M. Kalabokis V.N. Szent-Gyorgyi A.G. Cohen C. Nature. 1994; 368: 306-312Crossref PubMed Scopus (265) Google Scholar,47.Fromherz S. Szent-Gyorgyi A.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7652-7656Crossref PubMed Scopus (40) Google Scholar) whereas phosphorylation occurs on smooth muscle myosin R-LC (48.Sellers J.R. Curr. Opin. Cell Biol. 1991; 3: 98-104Crossref PubMed Scopus (172) Google Scholar)), conventional wisdom has been that the conformational sequelae of the two triggering mechanisms rapidly converge toward a common pathway in these conserved structures to facilitate full activation at the active site. However, smooth muscle heavy meromyosin lacking E-LC (LC17), produced through overexpression of recombinant baculovirus, exhibited a 75% reduction in the rate of displacement of actin filaments, yet this movement remained phosphorylation-dependent (49.Trybus K.M. J. Biol. Chem. 1994; 269: 20819-20822Abstract Full Text PDF PubMed Google Scholar). Furthermore, smooth muscle myosin lacking LC17, produced through R-LC (LC20) readdition to light chain-depleted myosin, had reduced rates of superprecipitation and actin-activated MgATPase, yet these activities remained phosphorylation-dependent (21.Katoh T. Morita F. J. Biol. Chem. 1996; 271: 9992-9996Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). If, as our results suggest, E-LC is vital for maintenance of the off-state, it is unlikely that regulated activity could remain in the absence of E-LC. Consequently, in addition to distinct triggering mechanisms, these two forms of myosin-linked regulation may also exhibit different conformational relays between their respective triggering sites and the active site.In summary, we have shown that TFP has a dual effect on scallop myosin. Application of millimolar concentrations of TFP leads to dissociation of R-LC from scallop myosin. However, concentrations of TFP an order of magnitude lower than those required to displace R-LCs can eliminate the actin-activated MgATPase activity of scallop myosin, irrespective of the presence and absence of calcium. This occurs in a manner that is independent of the presence or absence of R-LC. Furthermore, TFP can directly affect the conformation of E-LC; comparison with calmodulin indicates that the putative binding site is conserved. The exact manner by which this inhibited state is maintained remains to be determined, but we hypothesize that this structure closely mimics the off-state of a regulatory myosin, which has, so far, eluded structural analysis. Trifluoperazine (TFP),1a member of the phenothiazine class of drugs, is one of the strongest antagonists of calmodulin action known, capable of binding to calmodulin in the presence of calcium and preventing its stimulatory effects (1.Levin R.M. Weiss B. Mol. Pharmacol. 1976; 12: 581-589PubMed Google Scholar, 2.Prozialeck W.C. Weiss B. J. Pharmacol. Exp. Ther. 1982; 222: 509-516PubMed Google Scholar, 3.Roufogalis B.D. Minocherhomjee A.M. Al-Jobore A. Can. J. Biochem. Cell Biol. 1983; 61: 927-933Crossref PubMed Scopus (78) Google Scholar). The structure of the TFP·calmodulin complex in the presence of calcium has been determined at 2.45-Å resolution (4.Cook W.J. Walter L.J. Walter M.R. Biochemistry. 1994; 33: 15259-15265Crossref PubMed Scopus (124) Google Scholar, 5.Vandonselaar M. Hickie R.A. Quail J.W. Delbaere L.T.J. Nat. Struct. Biol. 1994; 1: 795-80
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