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• W2052806957 abstract "One of the putative actin-binding sites ofDictyostelium myosin II is the β-strand-turn-β-strand structure (Ile398-Leu-Ala-Gly-Arg-Asp403-Leu-Val405), the “myopathy loop,” which is located at the distal end of the upper 50-kDa subdomain and next to the conserved arginine (Arg397), whose mutation in human cardiac myosin results in familial hypertrophic cardiomyopathy. The myopathy loop contains the TEDS residue (Asp403), which is a target of the heavy-chain kinase in myosin I. Moreover, the loop contains a cluster of hydrophobic residues (Ile398, Leu399, Leu404, and Val405), whose side chains are fully exposed to the solvent. In our study, the myopathy loop was deleted from Dictyostelium myosin II to investigate its functional roles. The mutation abolished hydrophobic interactions of actin and myosin in the strong binding state during the ATPase cycle. Association of the mutant myosin and actin was maintained only through ionic interactions under these conditions. Without strong hydrophobic interactions, the mutant myosin still exhibited motor functions, although at low levels. It is likely that the observed defects resulted mainly from a loss of the cluster of hydrophobic residues, since replacement of Asp403 or Arg402 with alanine generated a mutant with less severe or no defects compared with those of the deletion mutant. One of the putative actin-binding sites ofDictyostelium myosin II is the β-strand-turn-β-strand structure (Ile398-Leu-Ala-Gly-Arg-Asp403-Leu-Val405), the “myopathy loop,” which is located at the distal end of the upper 50-kDa subdomain and next to the conserved arginine (Arg397), whose mutation in human cardiac myosin results in familial hypertrophic cardiomyopathy. The myopathy loop contains the TEDS residue (Asp403), which is a target of the heavy-chain kinase in myosin I. Moreover, the loop contains a cluster of hydrophobic residues (Ile398, Leu399, Leu404, and Val405), whose side chains are fully exposed to the solvent. In our study, the myopathy loop was deleted from Dictyostelium myosin II to investigate its functional roles. The mutation abolished hydrophobic interactions of actin and myosin in the strong binding state during the ATPase cycle. Association of the mutant myosin and actin was maintained only through ionic interactions under these conditions. Without strong hydrophobic interactions, the mutant myosin still exhibited motor functions, although at low levels. It is likely that the observed defects resulted mainly from a loss of the cluster of hydrophobic residues, since replacement of Asp403 or Arg402 with alanine generated a mutant with less severe or no defects compared with those of the deletion mutant. subfragment 1 4-morpholinepropanesulfonic acid In the absence of ATP, actin and myosin form a stable “rigor” complex, which is held together mainly by strong hydrophobic interactions. A three-dimensional reconstruction of electron microscopic images of the rigor complex of actin and myosin subfragment 1 (S1)1 revealed that S1 is in contact with actin at several sites (1Milligan R.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 21-26Crossref PubMed Scopus (156) Google Scholar, 2Schroder R.R. Manstein D.J. Jahn W. Holden H. Rayment I. Holmes K.C. Spudich J.A. Nature. 1993; 364: 171-174Crossref PubMed Scopus (266) Google Scholar). One of the putative actin-binding sites of S1 is the β-strand-turn-β-strand structure (Ile398-Leu-Ala-Gly-Arg-Asp403-Leu-Val405, in the case of Dictyostelium myosin II) located at the distal end of the upper 50-kDa subdomain (see Fig. 1) (3Rayment I. Rypniewski W.R. Schmidt B.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, 4Fisher A.J. Smith C.A. Thoden J.B. Smith R. Sutoh K. Holden H.M. Rayment I. Biochemistry. 1995; 34: 8960-8972Crossref PubMed Scopus (634) Google Scholar). Involvement of this β-turn-β structure in the actin-myosin interaction has been implied by two findings. First, mutation of Arg403 (equivalent to Arg397 inDictyostelium myosin II) to glutamine in human cardiac myosin is the cause of familial hypertrophic cardiomyopathy (5Geisterfer L.A. Kass S. Tanigawa G. Vosberg H.P. McKenna W. Seidman C.E. Seidman J.G. Cell. 1990; 62: 999-1006Abstract Full Text PDF PubMed Scopus (1030) Google Scholar) (thus, this β-turn-β structure is designated as the myopathy loop). In fact, R403Q mutation in cardiac myosin (6Sweeney H.L. Straceski A.J. Leinwand L.A. Tikunov B.A. Faust L. J. Biol. Chem. 1994; 269: 1603-1605Abstract Full Text PDF PubMed Google Scholar) and R397Q mutation inDictyostelium myosin II (7Fujita H. Sugiura S. Momomura S. Omata M. Sugi H. Sutoh K. J. Clin. Invest. 1997; 99: 1010-1015Crossref PubMed Scopus (61) Google Scholar) generated mutants with lower affinity to actin, suggesting the involvement of this conserved arginine residue in the actin-myosin interaction. Second, a threonine or serine residue of myosin I, whose location corresponds to a residue at the tip of the myopathy loop (Asp403 inDictyostelium myosin II), is phosphorylated by a myosin heavy-chain kinase (8Lee S.F. Cote G.P. J. Biol. Chem. 1995; 270: 11776-11782Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). This phosphorylation is required for the full activation of the actin-activated ATPase activity of myosin I. In the case of myosin II, however, the residue is glutamate or aspartate as inDictyostelium myosin II (TEDS rule (9Bement W.M. Mooseker M.S. Cell Motil. Cytoskeleton. 1995; 31: 87-92Crossref PubMed Scopus (147) Google Scholar), T or S in myosin I, and E or D in myosin II). In addition to this TEDS residue, the myopathy loop contains a cluster of hydrophobic residues (Ile398, Leu399, Leu404, and Val405) whose bulky side chains are fully exposed to the solvent (see Fig. 1), as if ready to interact with hydrophobic residues on actin.The second putative actin-binding site is located at the tip of the α-helix-loop-α-helix structure at the distal end of the lower 50-kDa subdomain, and it contains conserved hydrophobic residues (Fig. 1). Some of the hydrophobic side chains are fully exposed to the solvent and are likely to be in contact with hydrophobic residues located between subdomain 1 and subdomain 3 of actin (2Schroder R.R. Manstein D.J. Jahn W. Holden H. Rayment I. Holmes K.C. Spudich J.A. Nature. 1993; 364: 171-174Crossref PubMed Scopus (266) Google Scholar). These two hydrophobic actin-binding sites may be cooperatively involved in the rigor binding of actin and myosin in the absence of ATP as well as in their rigor-like binding in the presence of ADP, i.e. in the strong binding during the ATP hydrolysis cycle.The third putative site is the 50kDa-20kDa junction, a flexible loop easily recognized in almost all members of the myosin family (10Mooseker M.S. Cheney R.E. Annu. Rev. Cell Dev. Biol. 1995; 11: 633-675Crossref PubMed Scopus (386) Google Scholar). This loop, rich in basic residues and glycine residues, seems to be in contact with acidic residues of actin to form ionic bonds (1Milligan R.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 21-26Crossref PubMed Scopus (156) Google Scholar, 2Schroder R.R. Manstein D.J. Jahn W. Holden H. Rayment I. Holmes K.C. Spudich J.A. Nature. 1993; 364: 171-174Crossref PubMed Scopus (266) Google Scholar, 11Sutoh K. Ando M. Sutoh K. Toyoshima Y.Y. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7711-7714Crossref PubMed Scopus (109) Google Scholar,12Johara M. Toyoshima Y.Y. Ishijima A. Kojima H. Yanagida T. Sutoh K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2127-2131Crossref PubMed Scopus (70) Google Scholar). These ionic bonds are a dominant factor in maintaining the actin-myosin association in the weak-binding state during ATP hydrolysis.Dictyostelium discoideum cells have only one copy of the heavy-chain gene of myosin II (13Warrick H.M. De L.A. Leinwand L.A. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9433-9437Crossref PubMed Scopus (113) Google Scholar). Disruption of the gene generatesDictyostelium myosin-null cells with specific phenotypic defects (14De L.A. Spudich J.A. Science. 1987; 236: 1086-1091Crossref PubMed Scopus (752) Google Scholar, 15Manstein D.J. Titus M.A. De L.A. Spudich J.A. EMBO J. 1989; 8: 923-932Crossref PubMed Scopus (229) Google Scholar). The introduction of a mutant heavy-chain gene into these myosin-null cells generates transformants expressing the mutant myosin in place of the wild type. Using this Dictyosteliumexpression system developed by Spudich and co-workers (16O'Halloran T.J. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8110-8114Crossref PubMed Scopus (18) Google Scholar, 17Kubalek E.W. Uyeda T.Q. Spudich J.A. Mol. Biol. Cell. 1992; 3: 1455-1462Crossref PubMed Scopus (34) Google Scholar, 18Egelhoff T.T. Lee R.J. Spudich J.A. Cell. 1993; 75: 363-371Abstract Full Text PDF PubMed Scopus (241) Google Scholar, 19Uyeda T.Q. Spudich J.A. Science. 1993; 262: 1867-1870Crossref PubMed Scopus (103) Google Scholar, 20Ruppel K.M. Uyeda T.Q. Spudich J.A. J. Biol. Chem. 1994; 269: 18773-18780Abstract Full Text PDF PubMed Google Scholar, 21Uyeda T.Q. Ruppel K.M. Spudich J.A. Nature. 1994; 368: 567-569Crossref PubMed Scopus (188) Google Scholar), we investigated how the myopathy loop of Dictyostelium myosin II is involved in the actin-myosin association and the motor functions of myosin.DISCUSSIONIt has been proposed that the α-loop-α structure at the distal end of the lower 50-kDa subdomain interacts with the hydrophobic pocket formed by subdomain 1 and subdomain 3 of actin (2Schroder R.R. Manstein D.J. Jahn W. Holden H. Rayment I. Holmes K.C. Spudich J.A. Nature. 1993; 364: 171-174Crossref PubMed Scopus (266) Google Scholar). It has also been proposed that the β-turn-β structure at the distal end of the upper 50-kDa subdomain, the myopathy loop, is in contact with the upper surface of the ATP binding cleft of actin (2Schroder R.R. Manstein D.J. Jahn W. Holden H. Rayment I. Holmes K.C. Spudich J.A. Nature. 1993; 364: 171-174Crossref PubMed Scopus (266) Google Scholar). Thus, in the rigor binding, two distal ends of the upper and lower 50-kDa domains are likely to associate with actin through hydrophobic interactions and hold subdomain 1 of actin like two fingers holding a ball. Since deletion of one of the hydrophobic sites, i.e. the hydrophobic residues in the myopathy loop, abolished the rigor binding as shown here, cooperation between these two sites seems to be essential for the tight, hydrophobic association of actin and myosin. Consistent with this notion is the fact that replacement of one of the conserved hydrophobic residues in the α-loop-α structure (Phe535 in Dictyostelium myosin II) with alanine dramatically reduced the affinity of actin and myosin. 2N. Sasaki and K. Sutoh, unpublished result. It must be mentioned here that deletion of the loop abolished not only the rigor binding but also the rigor-like binding of actin and S1 in the presence of ADP, whereas replacement of Arg402 or Asp403 with alanine resulted in no or only slight destabilization of the complex under these conditions. The results show that the hydrophobic residues in this loop contribute to the hydrophobic interface to actin in the absence of nucleotide as well as in the presence of ADP,i.e. in the strong-binding state during ATPase cycle.Deletion of the loop did not completely abolish the motor functions of myosin. The basal MgATPase activity of Δ myosin was activated to some extent upon the addition of actin. Moreover, the mutant could support the slow sliding of actin filaments and exert a low level of force on them. The continuous, one-directional sliding of actin filaments driven by Δ myosin was disrupted upon increasing the concentration of KCl in the assay solvent from 25 to 75 mm. These results suggest that the hydrophobic actin-myosin binding in the strong-binding state is crucial for effective energy transduction for sliding and force generation but not essential for these motor functions. As far as weak ionic interactions between Δ myosin and actin being maintained in a low ionic strength solvent, Δ myosin could support the sliding of actin filaments and exert force on them, although at low levels. In view of the fact that the actin-Δ myosin interaction was highly dependent on the ionic strength of the solvent, this weak ionic association of Δ myosin and actin must have been disrupted in the living cells, consistent with the observation that Δ myosin was not functional in Dictyostelium myosin-null cells even though it retained a low level of in vitro motor functions.Removal of a negative charge from the TEDS residue by D403A mutation, which might mimic the dephosphorylation reaction, generated less severe defects than Δ mutation. Consistent with the result, the mutant myosin could completely complement the defects of myosin-null cells. Thus, the negative charge of the TEDS residue is not essential forin vivo and in vitro motor functions, although it is important for the full activation of ATPase activity of myosin and for efficient sliding of actin filaments. Removal of the positive charge in Arg402 rarely affected motor functions. It seems that the loss of the hydrophobic side chains was the main cause of defects observed for Δ myosin and Δ S1. Loss of the negative charge at Asp403 only partially contributed to the defects.In crystal structures of the motor domain of Dictyosteliummyosin II (4Fisher A.J. Smith C.A. Thoden J.B. Smith R. Sutoh K. Holden H.M. Rayment I. Biochemistry. 1995; 34: 8960-8972Crossref PubMed Scopus (634) Google Scholar, 31Smith C.A. Rayment I. Biochemistry. 1996; 35: 5404-5417Crossref PubMed Scopus (512) Google Scholar), the side chains of the hydrophobic residues in the myopathy loop are fully exposed to the solvent (Fig. 1). Chicken S1 has a similar cluster of exposed hydrophobic side chains at the corresponding location (3Rayment I. Rypniewski W.R. Schmidt B.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). Furthermore, almost all classes of myosins have a similar cluster of hydrophobic residues at a similar location along their sequences, although those amino acid residues are not conserved (10Mooseker M.S. Cheney R.E. Annu. Rev. Cell Dev. Biol. 1995; 11: 633-675Crossref PubMed Scopus (386) Google Scholar). This cluster of hydrophobic residues must be crucial for effective actin-activation of ATPase activity, sliding of actin filaments, and force generation. In the absence of ATP, actin and myosin form a stable “rigor” complex, which is held together mainly by strong hydrophobic interactions. A three-dimensional reconstruction of electron microscopic images of the rigor complex of actin and myosin subfragment 1 (S1)1 revealed that S1 is in contact with actin at several sites (1Milligan R.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 21-26Crossref PubMed Scopus (156) Google Scholar, 2Schroder R.R. Manstein D.J. Jahn W. Holden H. Rayment I. Holmes K.C. Spudich J.A. Nature. 1993; 364: 171-174Crossref PubMed Scopus (266) Google Scholar). One of the putative actin-binding sites of S1 is the β-strand-turn-β-strand structure (Ile398-Leu-Ala-Gly-Arg-Asp403-Leu-Val405, in the case of Dictyostelium myosin II) located at the distal end of the upper 50-kDa subdomain (see Fig. 1) (3Rayment I. Rypniewski W.R. Schmidt B.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, 4Fisher A.J. Smith C.A. Thoden J.B. Smith R. Sutoh K. Holden H.M. Rayment I. Biochemistry. 1995; 34: 8960-8972Crossref PubMed Scopus (634) Google Scholar). Involvement of this β-turn-β structure in the actin-myosin interaction has been implied by two findings. First, mutation of Arg403 (equivalent to Arg397 inDictyostelium myosin II) to glutamine in human cardiac myosin is the cause of familial hypertrophic cardiomyopathy (5Geisterfer L.A. Kass S. Tanigawa G. Vosberg H.P. McKenna W. Seidman C.E. Seidman J.G. Cell. 1990; 62: 999-1006Abstract Full Text PDF PubMed Scopus (1030) Google Scholar) (thus, this β-turn-β structure is designated as the myopathy loop). In fact, R403Q mutation in cardiac myosin (6Sweeney H.L. Straceski A.J. Leinwand L.A. Tikunov B.A. Faust L. J. Biol. Chem. 1994; 269: 1603-1605Abstract Full Text PDF PubMed Google Scholar) and R397Q mutation inDictyostelium myosin II (7Fujita H. Sugiura S. Momomura S. Omata M. Sugi H. Sutoh K. J. Clin. Invest. 1997; 99: 1010-1015Crossref PubMed Scopus (61) Google Scholar) generated mutants with lower affinity to actin, suggesting the involvement of this conserved arginine residue in the actin-myosin interaction. Second, a threonine or serine residue of myosin I, whose location corresponds to a residue at the tip of the myopathy loop (Asp403 inDictyostelium myosin II), is phosphorylated by a myosin heavy-chain kinase (8Lee S.F. Cote G.P. J. Biol. Chem. 1995; 270: 11776-11782Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). This phosphorylation is required for the full activation of the actin-activated ATPase activity of myosin I. In the case of myosin II, however, the residue is glutamate or aspartate as inDictyostelium myosin II (TEDS rule (9Bement W.M. Mooseker M.S. Cell Motil. Cytoskeleton. 1995; 31: 87-92Crossref PubMed Scopus (147) Google Scholar), T or S in myosin I, and E or D in myosin II). In addition to this TEDS residue, the myopathy loop contains a cluster of hydrophobic residues (Ile398, Leu399, Leu404, and Val405) whose bulky side chains are fully exposed to the solvent (see Fig. 1), as if ready to interact with hydrophobic residues on actin. The second putative actin-binding site is located at the tip of the α-helix-loop-α-helix structure at the distal end of the lower 50-kDa subdomain, and it contains conserved hydrophobic residues (Fig. 1). Some of the hydrophobic side chains are fully exposed to the solvent and are likely to be in contact with hydrophobic residues located between subdomain 1 and subdomain 3 of actin (2Schroder R.R. Manstein D.J. Jahn W. Holden H. Rayment I. Holmes K.C. Spudich J.A. Nature. 1993; 364: 171-174Crossref PubMed Scopus (266) Google Scholar). These two hydrophobic actin-binding sites may be cooperatively involved in the rigor binding of actin and myosin in the absence of ATP as well as in their rigor-like binding in the presence of ADP, i.e. in the strong binding during the ATP hydrolysis cycle. The third putative site is the 50kDa-20kDa junction, a flexible loop easily recognized in almost all members of the myosin family (10Mooseker M.S. Cheney R.E. Annu. Rev. Cell Dev. Biol. 1995; 11: 633-675Crossref PubMed Scopus (386) Google Scholar). This loop, rich in basic residues and glycine residues, seems to be in contact with acidic residues of actin to form ionic bonds (1Milligan R.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 21-26Crossref PubMed Scopus (156) Google Scholar, 2Schroder R.R. Manstein D.J. Jahn W. Holden H. Rayment I. Holmes K.C. Spudich J.A. Nature. 1993; 364: 171-174Crossref PubMed Scopus (266) Google Scholar, 11Sutoh K. Ando M. Sutoh K. Toyoshima Y.Y. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7711-7714Crossref PubMed Scopus (109) Google Scholar,12Johara M. Toyoshima Y.Y. Ishijima A. Kojima H. Yanagida T. Sutoh K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2127-2131Crossref PubMed Scopus (70) Google Scholar). These ionic bonds are a dominant factor in maintaining the actin-myosin association in the weak-binding state during ATP hydrolysis. Dictyostelium discoideum cells have only one copy of the heavy-chain gene of myosin II (13Warrick H.M. De L.A. Leinwand L.A. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9433-9437Crossref PubMed Scopus (113) Google Scholar). Disruption of the gene generatesDictyostelium myosin-null cells with specific phenotypic defects (14De L.A. Spudich J.A. Science. 1987; 236: 1086-1091Crossref PubMed Scopus (752) Google Scholar, 15Manstein D.J. Titus M.A. De L.A. Spudich J.A. EMBO J. 1989; 8: 923-932Crossref PubMed Scopus (229) Google Scholar). The introduction of a mutant heavy-chain gene into these myosin-null cells generates transformants expressing the mutant myosin in place of the wild type. Using this Dictyosteliumexpression system developed by Spudich and co-workers (16O'Halloran T.J. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8110-8114Crossref PubMed Scopus (18) Google Scholar, 17Kubalek E.W. Uyeda T.Q. Spudich J.A. Mol. Biol. Cell. 1992; 3: 1455-1462Crossref PubMed Scopus (34) Google Scholar, 18Egelhoff T.T. Lee R.J. Spudich J.A. Cell. 1993; 75: 363-371Abstract Full Text PDF PubMed Scopus (241) Google Scholar, 19Uyeda T.Q. Spudich J.A. Science. 1993; 262: 1867-1870Crossref PubMed Scopus (103) Google Scholar, 20Ruppel K.M. Uyeda T.Q. Spudich J.A. J. Biol. Chem. 1994; 269: 18773-18780Abstract Full Text PDF PubMed Google Scholar, 21Uyeda T.Q. Ruppel K.M. Spudich J.A. Nature. 1994; 368: 567-569Crossref PubMed Scopus (188) Google Scholar), we investigated how the myopathy loop of Dictyostelium myosin II is involved in the actin-myosin association and the motor functions of myosin. DISCUSSIONIt has been proposed that the α-loop-α structure at the distal end of the lower 50-kDa subdomain interacts with the hydrophobic pocket formed by subdomain 1 and subdomain 3 of actin (2Schroder R.R. Manstein D.J. Jahn W. Holden H. Rayment I. Holmes K.C. Spudich J.A. Nature. 1993; 364: 171-174Crossref PubMed Scopus (266) Google Scholar). It has also been proposed that the β-turn-β structure at the distal end of the upper 50-kDa subdomain, the myopathy loop, is in contact with the upper surface of the ATP binding cleft of actin (2Schroder R.R. Manstein D.J. Jahn W. Holden H. Rayment I. Holmes K.C. Spudich J.A. Nature. 1993; 364: 171-174Crossref PubMed Scopus (266) Google Scholar). Thus, in the rigor binding, two distal ends of the upper and lower 50-kDa domains are likely to associate with actin through hydrophobic interactions and hold subdomain 1 of actin like two fingers holding a ball. Since deletion of one of the hydrophobic sites, i.e. the hydrophobic residues in the myopathy loop, abolished the rigor binding as shown here, cooperation between these two sites seems to be essential for the tight, hydrophobic association of actin and myosin. Consistent with this notion is the fact that replacement of one of the conserved hydrophobic residues in the α-loop-α structure (Phe535 in Dictyostelium myosin II) with alanine dramatically reduced the affinity of actin and myosin. 2N. Sasaki and K. Sutoh, unpublished result. It must be mentioned here that deletion of the loop abolished not only the rigor binding but also the rigor-like binding of actin and S1 in the presence of ADP, whereas replacement of Arg402 or Asp403 with alanine resulted in no or only slight destabilization of the complex under these conditions. The results show that the hydrophobic residues in this loop contribute to the hydrophobic interface to actin in the absence of nucleotide as well as in the presence of ADP,i.e. in the strong-binding state during ATPase cycle.Deletion of the loop did not completely abolish the motor functions of myosin. The basal MgATPase activity of Δ myosin was activated to some extent upon the addition of actin. Moreover, the mutant could support the slow sliding of actin filaments and exert a low level of force on them. The continuous, one-directional sliding of actin filaments driven by Δ myosin was disrupted upon increasing the concentration of KCl in the assay solvent from 25 to 75 mm. These results suggest that the hydrophobic actin-myosin binding in the strong-binding state is crucial for effective energy transduction for sliding and force generation but not essential for these motor functions. As far as weak ionic interactions between Δ myosin and actin being maintained in a low ionic strength solvent, Δ myosin could support the sliding of actin filaments and exert force on them, although at low levels. In view of the fact that the actin-Δ myosin interaction was highly dependent on the ionic strength of the solvent, this weak ionic association of Δ myosin and actin must have been disrupted in the living cells, consistent with the observation that Δ myosin was not functional in Dictyostelium myosin-null cells even though it retained a low level of in vitro motor functions.Removal of a negative charge from the TEDS residue by D403A mutation, which might mimic the dephosphorylation reaction, generated less severe defects than Δ mutation. Consistent with the result, the mutant myosin could completely complement the defects of myosin-null cells. Thus, the negative charge of the TEDS residue is not essential forin vivo and in vitro motor functions, although it is important for the full activation of ATPase activity of myosin and for efficient sliding of actin filaments. Removal of the positive charge in Arg402 rarely affected motor functions. It seems that the loss of the hydrophobic side chains was the main cause of defects observed for Δ myosin and Δ S1. Loss of the negative charge at Asp403 only partially contributed to the defects.In crystal structures of the motor domain of Dictyosteliummyosin II (4Fisher A.J. Smith C.A. Thoden J.B. Smith R. Sutoh K. Holden H.M. Rayment I. Biochemistry. 1995; 34: 8960-8972Crossref PubMed Scopus (634) Google Scholar, 31Smith C.A. Rayment I. Biochemistry. 1996; 35: 5404-5417Crossref PubMed Scopus (512) Google Scholar), the side chains of the hydrophobic residues in the myopathy loop are fully exposed to the solvent (Fig. 1). Chicken S1 has a similar cluster of exposed hydrophobic side chains at the corresponding location (3Rayment I. Rypniewski W.R. Schmidt B.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). Furthermore, almost all classes of myosins have a similar cluster of hydrophobic residues at a similar location along their sequences, although those amino acid residues are not conserved (10Mooseker M.S. Cheney R.E. Annu. Rev. Cell Dev. Biol. 1995; 11: 633-675Crossref PubMed Scopus (386) Google Scholar). This cluster of hydrophobic residues must be crucial for effective actin-activation of ATPase activity, sliding of actin filaments, and force generation. It has been proposed that the α-loop-α structure at the distal end of the lower 50-kDa subdomain interacts with the hydrophobic pocket formed by subdomain 1 and subdomain 3 of actin (2Schroder R.R. Manstein D.J. Jahn W. Holden H. Rayment I. Holmes K.C. Spudich J.A. Nature. 1993; 364: 171-174Crossref PubMed Scopus (266) Google Scholar). It has also been proposed that the β-turn-β structure at the distal end of the upper 50-kDa subdomain, the myopathy loop, is in contact with the upper surface of the ATP binding cleft of actin (2Schroder R.R. Manstein D.J. Jahn W. Holden H. Rayment I. Holmes K.C. Spudich J.A. Nature. 1993; 364: 171-174Crossref PubMed Scopus (266) Google Scholar). Thus, in the rigor binding, two distal ends of the upper and lower 50-kDa domains are likely to associate with actin through hydrophobic interactions and hold subdomain 1 of actin like two fingers holding a ball. Since deletion of one of the hydrophobic sites, i.e. the hydrophobic residues in the myopathy loop, abolished the rigor binding as shown here, cooperation between these two sites seems to be essential for the tight, hydrophobic association of actin and myosin. Consistent with this notion is the fact that replacement of one of the conserved hydrophobic residues in the α-loop-α structure (Phe535 in Dictyostelium myosin II) with alanine dramatically reduced the affinity of actin and myosin. 2N. Sasaki and K. Sutoh, unpublished result. It must be mentioned here that deletion of the loop abolished not only the rigor binding but also the rigor-like binding of actin and S1 in the presence of ADP, whereas replacement of Arg402 or Asp403 with alanine resulted in no or only slight destabilization of the complex under these conditions. The results show that the hydrophobic residues in this loop contribute to the hydrophobic interface to actin in the absence of nucleotide as well as in the presence of ADP,i.e. in the strong-binding state during ATPase cycle. Deletion of the loop did not completely abolish the motor functions of myosin. The basal MgATPase activity of Δ myosin was activated to some extent upon the addition of actin. Moreover, the mutant could support the slow sliding of actin filaments and exert a low level of force on them. The continuous, one-directional sliding of actin filaments driven by Δ myosin was disrupted upon increasing the concentration of KCl in the assay solvent from 25 to 75 mm. These results suggest that the hydrophobic actin-myosin binding in the strong-binding state is crucial for effective energy transduction for sliding and force generation but not essential for these motor functions. As far as weak ionic interactions between Δ myosin and actin being maintained in a low ionic strength solvent, Δ myosin could support the sliding of actin filaments and exert force on them, although at low levels. In view of the fact that the actin-Δ myosin interaction was highly dependent on the ionic strength of the solvent, this weak ionic association of Δ myosin and actin must have been disrupted in the living cells, consistent with the observation that Δ myosin was not functional in Dictyostelium myosin-null cells even though it retained a low level of in vitro motor functions. Removal of a negative charge from the TEDS residue by D403A mutation, which might mimic the dephosphorylation reaction, generated less severe defects than Δ mutation. Consistent with the result, the mutant myosin could completely complement the defects of myosin-null cells. Thus, the negative charge of the TEDS residue is not essential forin vivo and in vitro motor functions, although it is important for the full activation of ATPase activity of myosin and for efficient sliding of actin filaments. Removal of the positive charge in Arg402 rarely affected motor functions. It seems that the loss of the hydrophobic side chains was the main cause of defects observed for Δ myosin and Δ S1. Loss of the negative charge at Asp403 only partially contributed to the defects. In crystal structures of the motor domain of Dictyosteliummyosin II (4Fisher A.J. Smith C.A. Thoden J.B. Smith R. Sutoh K. Holden H.M. Rayment I. Biochemistry. 1995; 34: 8960-8972Crossref PubMed Scopus (634) Google Scholar, 31Smith C.A. Rayment I. Biochemistry. 1996; 35: 5404-5417Crossref PubMed Scopus (512) Google Scholar), the side chains of the hydrophobic residues in the myopathy loop are fully exposed to the solvent (Fig. 1). Chicken S1 has a similar cluster of exposed hydrophobic side chains at the corresponding location (3Rayment I. Rypniewski W.R. Schmidt B.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). Furthermore, almost all classes of myosins have a similar cluster of hydrophobic residues at a similar location along their sequences, although those amino acid residues are not conserved (10Mooseker M.S. Cheney R.E. Annu. Rev. Cell Dev. Biol. 1995; 11: 633-675Crossref PubMed Scopus (386) Google Scholar). This cluster of hydrophobic residues must be crucial for effective actin-activation of ATPase activity, sliding of actin filaments, and force generation. We thank Reiko Ohkura for her excellent technical assistance. We also thank Dr. Kazuhiro Oiwa (Kansai Advanced Research Center, Communication Research Laboratory, Japan) for providing us with Cy3-ATP." @default.
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• W2052806957 title "Deletion of the Myopathy Loop of Dictyostelium Myosin II and Its Impact on Motor Functions" @default.
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