The Globular Tail Domain of Myosin Va Functions as an Inhibitor of the Myosin Va Motor

Abstract

The actin-activated ATPase activity of full-length mammalian myosin Va is well regulated by Ca2+, whereas that of truncated myosin Va without the C-terminal globular tail domain (GTD) is not. Here, we have found that exogenous GTD is capable of inhibiting the actin-activated ATPase activity of GTD-deleted myosin Va. A series of truncated constructs of myosin Va further showed that the entire length of the first coiled-coil (coil-1) of the tail domain is critical for GTD-dependent regulation of myosin Va and that deletion of 58 residues from the C-terminal end of coil-1 markedly hampered regulation. Negative staining electron microscopy revealed that GTD-deleted myosin Va formed a “Y”-shaped structure, which was converted to a triangular shape, similar to the structure of full-length myosin Va in the inhibited state, by addition of exogenous GTD. In contrast, the triangular shape was not observed when the C-terminal 58 residues of coil-1 were deleted, even in the presence of exogenous GTD. Based on these results, we propose a model for the formation of the inhibited state of myosin Va. GTD binds to the C-terminal end of coil-1. The neck-tail junction of myosin Va is flexible, and the long neck enables the head domain to reach the GTD associated with the end of coil-1. Once the head interacts with the GTD, the triangular inhibited conformation is stabilized. Consistent with this model, we found that shortening of the neck of myosin Va by two IQ motifs abolished the regulation by GTD, whereas regulation was partially restored by shortening of coil-1 by an amount comparable to that of the two IQ motifs. The actin-activated ATPase activity of full-length mammalian myosin Va is well regulated by Ca2+, whereas that of truncated myosin Va without the C-terminal globular tail domain (GTD) is not. Here, we have found that exogenous GTD is capable of inhibiting the actin-activated ATPase activity of GTD-deleted myosin Va. A series of truncated constructs of myosin Va further showed that the entire length of the first coiled-coil (coil-1) of the tail domain is critical for GTD-dependent regulation of myosin Va and that deletion of 58 residues from the C-terminal end of coil-1 markedly hampered regulation. Negative staining electron microscopy revealed that GTD-deleted myosin Va formed a “Y”-shaped structure, which was converted to a triangular shape, similar to the structure of full-length myosin Va in the inhibited state, by addition of exogenous GTD. In contrast, the triangular shape was not observed when the C-terminal 58 residues of coil-1 were deleted, even in the presence of exogenous GTD. Based on these results, we propose a model for the formation of the inhibited state of myosin Va. GTD binds to the C-terminal end of coil-1. The neck-tail junction of myosin Va is flexible, and the long neck enables the head domain to reach the GTD associated with the end of coil-1. Once the head interacts with the GTD, the triangular inhibited conformation is stabilized. Consistent with this model, we found that shortening of the neck of myosin Va by two IQ motifs abolished the regulation by GTD, whereas regulation was partially restored by shortening of coil-1 by an amount comparable to that of the two IQ motifs. Class V myosin is considered one of the oldest classes of myosin, being distributed from low eukaryotes, such as yeast, to vertebrate cells (1Berg J.S. Powell B.C. Cheney R.E. Mol. Biol. Cell. 2001; 12: 780-794Crossref PubMed Scopus (616) Google Scholar, 2Richards T.A. Cavalier-Smith T. Nature. 2005; 436: 1113-1118Crossref PubMed Scopus (316) Google Scholar). In mammalian cells, there are three distinct subclasses of myosin V, named myosin Va, Vb, and Vc (3Cheney R.E. O'Shea M.K. Heuser J.E. Coelho M.V. Wolenski J.S. Espreafico E.M. Forscher P. Larson R.E. Mooseker M.S. Cell. 1993; 75: 13-23Abstract Full Text PDF PubMed Scopus (379) Google Scholar, 4Rodriguez O.C. Cheney R.E. J. Cell Sci. 2002; 115: 991-1004Crossref PubMed Google Scholar, 5Zhao L.P. Koslovsky J.S. Reinhard J. Bahler M. Witt A.E. Provance Jr., D.W. Mercer J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10826-10831Crossref PubMed Scopus (76) Google Scholar). Myosin Va is one of the best characterized motor proteins of the myosin superfamily. Myosin Va is a processive motor, which undergoes multiple catalytic cycles coupled to multiple mechanical advances for each diffusional encounter with its actin track (6Mehta A.D. Rock R.S. Rief M. Spudich J.A. Mooseker M.S. Cheney R.E. Nature. 1999; 400: 590-593Crossref PubMed Scopus (673) Google Scholar, 7Yildiz A. Forkey J.N. McKinney S.A. Ha T. Goldman Y.E. Selvin P.R. Science. 2003; 300: 2061-2065Crossref PubMed Scopus (1545) Google Scholar, 8Forkey J.N. Quinlan M.E. Shaw M.A. Corrie J.E. Goldman Y.E. Nature. 2003; 422: 399-404Crossref PubMed Scopus (380) Google Scholar, 9Burgess S. Walker M. Wang F. Sellers J.R. White H.D. Knight P.J. Trinick J. J. Cell Biol. 2002; 159: 983-991Crossref PubMed Scopus (103) Google Scholar, 10Walker M.L. Burgess S.A. Sellers J.R. Wang F. Hammer 3rd, J.A. Trinick J. Knight P.J. Nature. 2000; 405: 804-807Crossref PubMed Scopus (282) Google Scholar). This property allows single or a few myosin Va molecules to support movements of an organelle along actin filaments with a large step approximating the 36-nm pseudo repeat of the track. Myosin Va consists of two identical heavy chains that dimerize through the formation of a coiled-coil structure to form a homodimer. At the N terminus is the motor domain containing the ATP and actin binding sites. The motor domain is followed by a neck that consists of six IQ motifs with the consensus sequence IQXXXRGXXXR, which act as the binding sites for calmodulins or myosin light chains. The next ∼500 amino acids are predicted to form a series of coiled-coils separated by several flexible regions. The last ∼400 amino acids form a globular tail domain (3Cheney R.E. O'Shea M.K. Heuser J.E. Coelho M.V. Wolenski J.S. Espreafico E.M. Forscher P. Larson R.E. Mooseker M.S. Cell. 1993; 75: 13-23Abstract Full Text PDF PubMed Scopus (379) Google Scholar). This C-terminal globular tail domain (GTD), 2The abbreviations used are: GTD, C-terminal globular tail domain of myosin Va; DTT, dithiothreitol; McM5, melanocyte-type myosin Va; PEP, phosphoenol pyruvate; TCEP, Tris [2-carboxyethyl]phosphine; Ni-NTA, nickelnitrilotriacetic acid; aa, amino acids; MOPS, 4-morpholinepropanesulfonic acid.2The abbreviations used are: GTD, C-terminal globular tail domain of myosin Va; DTT, dithiothreitol; McM5, melanocyte-type myosin Va; PEP, phosphoenol pyruvate; TCEP, Tris [2-carboxyethyl]phosphine; Ni-NTA, nickelnitrilotriacetic acid; aa, amino acids; MOPS, 4-morpholinepropanesulfonic acid. in conjunction with a portion of the coiled-coil region, mediates myosin Va binding to specific membrane-bound organelles such as melanosomes (11Wu X. Bowers B. Wei Q. Kocher B. Hammer 3rd., J.A. J. Cell Sci. 1997; 110: 847-859Crossref PubMed Google Scholar). One of the most important questions is how the motor function of myosin Va is regulated. If myosin Va were constantly active, then the high concentrations of myosin Va found in brain (3Cheney R.E. O'Shea M.K. Heuser J.E. Coelho M.V. Wolenski J.S. Espreafico E.M. Forscher P. Larson R.E. Mooseker M.S. Cell. 1993; 75: 13-23Abstract Full Text PDF PubMed Scopus (379) Google Scholar) would consume substantial amounts of ATP. Therefore, it is desirable that the mechanoenzymatic activities of myosin Va is regulated in vivo. However, the molecular mechanism of myosin Va regulation is not well understood. The ATPase activities of tissue-isolated myosin Va (3Cheney R.E. O'Shea M.K. Heuser J.E. Coelho M.V. Wolenski J.S. Espreafico E.M. Forscher P. Larson R.E. Mooseker M.S. Cell. 1993; 75: 13-23Abstract Full Text PDF PubMed Scopus (379) Google Scholar, 12Wang F. Thirumurugan K. Stafford W.F. Hammer 3rd, J.A. Knight P.J. Sellers J.R. J. Biol. Chem. 2004; 279: 2333-2336Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) and baculovirus-expressed full-length myosin Va (13Li X.D. Mabuchi K. Ikebe R. Ikebe M. Biochem. Biophys. Res. Commun. 2004; 315: PubMed Scopus Google Scholar, J. Cell Biol. 2004; PubMed Scopus Google Scholar) are well the actin-activated ATPase activity of myosin Va being by concentrations of showed that the myosin Va undergoes a from to F. Thirumurugan K. Stafford W.F. Hammer 3rd, J.A. Knight P.J. Sellers J.R. J. Biol. Chem. 2004; 279: 2333-2336Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, X.D. Mabuchi K. Ikebe R. Ikebe M. Biochem. Biophys. Res. Commun. 2004; 315: PubMed Scopus Google Scholar, J. Cell Biol. 2004; PubMed Scopus Google Scholar). microscopy revealed that low myosin Va an conformation in high whereas it a shape in the presence of in which the tail domain is the head F. Thirumurugan K. Stafford W.F. Hammer 3rd, J.A. Knight P.J. Sellers J.R. J. Biol. Chem. 2004; 279: 2333-2336Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). The is with of the actin-activated ATPase activity of myosin Va (13Li X.D. Mabuchi K. Ikebe R. Ikebe M. Biochem. Biophys. Res. Commun. 2004; 315: PubMed Scopus Google Scholar). the truncated myosin Va without the globular tail domain is not regulated by K. J. Ikebe R. Ikebe M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus (76) Google Scholar, F. Bowers B. Hammer 3rd, J.A. Sellers J.R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) and not a large full-length myosin Va (13Li X.D. Mabuchi K. Ikebe R. Ikebe M. Biochem. Biophys. Res. Commun. 2004; 315: PubMed Scopus Google Scholar, J. Cell Biol. 2004; PubMed Scopus Google Scholar). that the tail domain a in the regulation of myosin Va, by inhibiting the ATPase of the head F. Thirumurugan K. Stafford W.F. Hammer 3rd, J.A. Knight P.J. Sellers J.R. J. Biol. Chem. 2004; 279: 2333-2336Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, X.D. Mabuchi K. Ikebe R. Ikebe M. Biochem. Biophys. Res. Commun. 2004; 315: PubMed Scopus Google Scholar, J. Cell Biol. 2004; PubMed Scopus Google Scholar). In this model, myosin Va in the inhibited state is in a conformation such that the tail domain interacts with and the myosin Va motor high Ca2+, the the head and tail its Consistent with this model, we found that the actin-activated ATPase activity of melanocyte-type myosin Va is by the binding of X.D. Ikebe R. Ikebe M. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google an organelle for myosin Va in M. K. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, K. Wang F. Sellers J.R. Hammer 3rd., J.A. Cell Biol. 2002; PubMed Scopus (380) Google Scholar, K. S. M. K. T. T. 2002; PubMed Scopus Google Scholar, M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). myosin Va binds to through its tail we that this with the the tail and head of myosin Va, the of the tail domain X.D. Ikebe R. Ikebe M. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). In this we further the molecular mechanism of the regulation of the motor activity of myosin Va. that the C-terminal globular tail domain of myosin Va binds to the head inhibiting motor that the of the neck domain and the first long coiled-coil domain the binding the head and the tail and are critical for the regulation of myosin Va motor and were from was from were by was from to Spudich and J.A. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of Mol. Cell. Biol. PubMed Scopus Google Scholar) was and to a M. M. A. Biochem. PubMed Scopus Google Scholar). and were from phosphoenol and were from and were from an the C-terminal amino residues of myosin Va, was by of of J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Myosin Va melanocyte-type myosin Va was as (13Li X.D. Mabuchi K. Ikebe R. Ikebe M. Biochem. Biophys. Res. Commun. 2004; 315: PubMed Scopus Google Scholar, X.D. Ikebe R. Ikebe M. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). the an containing a sequence of and was to myosin Va were by a sites of was by the last residues of with the residues of by GTD-deleted myosin Va with and were by as X.D. Ikebe R. Ikebe M. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). The the GTD was by and to containing was to GTD. were as (13Li X.D. Mabuchi K. Ikebe R. Ikebe M. Biochem. Biophys. Res. Commun. 2004; 315: PubMed Scopus Google Scholar). and of Myosin Va was in cells and by and GTD, of cells was with GTD, and in of for and with in and the were with in of and for the was with of in a on a in a for The was then on a and with of A and The to the was with in A. showed that the from the two with molecular of and The molecular of GTD is that the and in and GTD, showed that were by and the two the from was to of the from was a with The were and to The containing high concentrations of GTD were The of GTD was by the to of GTD in one The of the is not there are residues in GTD, we that GTD have by through However, the of the in not even when it was with high concentrations of DTT, or TCEP, not In the GTD of of that it an of GTD. the of the were with the GTD. myosin Va and the truncated were in cells by with myosin Va and myosin Va was by as (13Li X.D. Mabuchi K. Ikebe R. Ikebe M. Biochem. Biophys. Res. Commun. 2004; 315: PubMed Scopus Google that the myosin Va were DTT, and on The myosin Va in an of were in and The of myosin Va was by staining of myosin heavy as a as (13Li X.D. Mabuchi K. Ikebe R. Ikebe M. Biochem. Biophys. Res. Commun. 2004; 315: PubMed Scopus Google Scholar). the of the the molecular were and electron microscopy of myosin Va was as (13Li X.D. Mabuchi K. Ikebe R. Ikebe M. Biochem. Biophys. Res. Commun. 2004; 315: PubMed Scopus Google Scholar). myosin Va to in were a for proteins were and then the was by to K. J. Biol. PubMed Scopus Google Scholar) full-length or truncated myosin Va was to with of the were to or and with S.A. Walker M.L. Thirumurugan K. Trinick J. Knight P.J. J. Biol. 2004; PubMed Scopus Google Scholar). The was used for the by truncated myosin Va with GTD for were in a electron with an by and were on an a of ATPase activity of myosin Va was an ATP as (13Li X.D. Mabuchi K. Ikebe R. Ikebe M. Biochem. Biophys. Res. Commun. 2004; 315: PubMed Scopus Google Scholar). was and for ATP to the and were as (13Li X.D. Mabuchi K. Ikebe R. Ikebe M. Biochem. Biophys. Res. Commun. 2004; 315: PubMed Scopus Google Scholar). GTD the ATPase of GTD-deleted Myosin Va in the the actin-activated ATPase activity of truncated myosin Va without the C-terminal GTD is not regulated by Ca2+, whereas that of full-length myosin Va is K. J. Ikebe R. Ikebe M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus (76) Google Scholar, F. Bowers B. Hammer 3rd, J.A. Sellers J.R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). is abolished by the GTD of J. Cell Biol. 2004; PubMed Scopus Google Scholar). that the GTD is to the actin-activated ATPase activity of myosin Va. exogenous GTD the function of the myosin Va motor we the actin-activated ATPase activity of in the presence of exogenous GTD. The of ATPase activity was and the and GTD was The actin-activated ATPase activity of in the presence of GTD was similar to that of full-length myosin Va which was similar (13Li X.D. Mabuchi K. Ikebe R. Ikebe M. Biochem. Biophys. Res. Commun. 2004; 315: PubMed Scopus Google that was to an inhibited state in the presence of exogenous GTD. to full-length myosin Va, the activity of was not inhibited by GTD high The of GTD on was to in with an of from to the ATPase activity of in the of GTD whereas that of in the presence of GTD was with an in the of GTD on with an in the is GTD that GTD-dependent of the ATPase activity of on The for of the ATPase of Myosin Va motor domain and neck of myosin Va are to the GTD by a series of coiled-coil by a of flexible The and length of predicted coiled-coils of myosin Va. McM5, coiled-coils separated by flexible are predicted by B. T. M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). which portion of the tail domain is for regulation by GTD, we a series of C-terminal of myosin Va and the of GTD on the actin-activated ATPase activity of the in the ATPase activity of was inhibited by GTD with an of which is similar to that of The of the actin-activated ATPase activity of by GTD was that of contrast, the actin-activated ATPase activities of and were not inhibited by GTD. that the first long coiled-coil (coil-1) is critical for whereas the tail to is not for by GTD. in three The first is that the specific sequence of the C-terminal portion of coil-1 is for regulation by GTD. the structure is critical for regulation and deletion of the C-terminal portion of coil-1 the the length of coil-1 is critical for of myosin Va. the regulation of truncated myosin Va is to the state, we the molecules by electron found that the of the molecules of and were whereas of and most of were that myosin Va is not regulated. the and are GTD markedly inhibited the actin-activated ATPase activity of inhibited that of that the structure is not for the regulation by electron microscopy of truncated myosin Va. truncated myosin Va constructs were to by and The in A the globular domain the end of the tail of The length of the of is and that of the tail of is further the of the structure, we an myosin Va by the C-terminal amino residues of with amino residues of a coiled-coil in the of molecules are However, the actin-activated ATPase activity of is not inhibited by GTD, being similar to that of that the structure is not for the regulation of myosin Va by GTD. the of the on the regulation of myosin Va, we three constructs with in this of the of GTD on the ATPase whereas deletion of or the of GTD The support the that the C-terminal portion of coil-1 is important for of ATPase In the of GTD, Myosin Va a to Myosin was that full-length myosin Va a triangular shape low and low F. Thirumurugan K. Stafford W.F. Hammer 3rd, J.A. Knight P.J. Sellers J.R. J. Biol. Chem. 2004; 279: 2333-2336Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). This that GTD the motor inhibiting its motor exogenous GTD inhibited the ATPase activity of we exogenous GTD and formed this triangular in a or in the of GTD formed a triangular shape in its presence similar to that of full-length myosin Va, to formed a triangular shape in the presence of GTD a or in its In contrast, we not a triangular shape for or even in the presence of GTD the not results, in conjunction with the that the ATPase activities of and are inhibited by GTD, whereas of and are that the triangular shape of myosin Va an inhibited state and that GTD interacts with the head to the motor function of myosin Va. The of the and the for the of Myosin the that the triangular form the state, we that the length of the neck domain and the first long as well as the of the tail and the head are critical for by the tail binding to the this we a of constructs with of the neck or coil-1 and regulation by GTD. in a myosin Va with its neck by the and IQ motifs was not inhibited by GTD. This that the and IQ are for or that the neck is important for regulation. these we and by and or and of myosin Va, to the actin-activated ATPase activity of was inhibited by GTD not that the deletion of two IQ motifs of neck the the motor domain and GTD. The deletion of two IQ motifs the neck by amino residues for each in the One is that the shortening of the neck it for GTD, which is from the of the two to with the motor If deletion of an length of the of coil-1 the the motor domain and GTD. in full-length myosin Va a triangular shape with an of the two Therefore, shortening of the coiled-coil by would the triangular shape the motor domain and GTD. The length of is to amino residues in an the of the coiled-coil is amino deletion of or residues would a Therefore, we and of the coil-1 region, and found that deletion of partially restored the regulation of by GTD whereas deletion of not not In contrast, the deletion of of markedly the regulation of by GTD support the that the length of neck and coil-1 of myosin Va are critical for the motor domain and GTD, and the regulation of myosin Va. The of the of Myosin the structure of truncated constructs of myosin Va by electron One of the most is that the tail length is that predicted a series of coiled-coil in the The portion of the tail of myosin Va are predicted to coiled-coils separated by flexible by B. T. M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The length of coil-1 of is predicted to and of the coiled-coil to are and If these coiled-coil are to form a long the length the of the to GTD would the observed length of the of myosin Va and R.E. O'Shea M.K. Heuser J.E. Coelho M.V. Wolenski J.S. Espreafico E.M. Forscher P. Larson R.E. Mooseker M.S. Cell. 1993; 75: 13-23Abstract Full Text PDF PubMed Scopus (379) Google Scholar). found a globular structure the end of which is not found for the tail of or The length of the of is to that of tail that the not as a the globular This is with the of myosin Va by electron Cheney (3Cheney R.E. O'Shea M.K. Heuser J.E. Coelho M.V. Wolenski J.S. Espreafico E.M. Forscher P. Larson R.E. Mooseker M.S. Cell. 1993; 75: 13-23Abstract Full Text PDF PubMed Scopus (379) Google Scholar) observed a the globular tail domain in electron of brain myosin Va, and that this structure to residues of of brain myosin Va to of (3Cheney R.E. O'Shea M.K. Heuser J.E. Coelho M.V. Wolenski J.S. Espreafico E.M. Forscher P. Larson R.E. Mooseker M.S. Cell. 1993; 75: 13-23Abstract Full Text PDF PubMed Scopus (379) Google Scholar). observed that the length of tail of myosin Va is of myosin Va observed by electron microscopy showed the length from the junction to the end of GTD to from to a of (3Cheney R.E. O'Shea M.K. Heuser J.E. Coelho M.V. Wolenski J.S. Espreafico E.M. Forscher P. Larson R.E. Mooseker M.S. Cell. 1993; 75: 13-23Abstract Full Text PDF PubMed Scopus (379) Google Scholar). of myosin Va that the the junction and GTD is in low in high J. Cell Biol. 2004; PubMed Scopus Google Scholar). that the globular domain formed by to The of the of Myosin the we found that exogenous GTD the actin-activated ATPase activity of myosin Va The that GTD as an domain of myosin Va and is to the of full-length myosin Va motor activity F. Thirumurugan K. Stafford W.F. Hammer 3rd, J.A. Knight P.J. Sellers J.R. J. Biol. Chem. 2004; 279: 2333-2336Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, X.D. Mabuchi K. Ikebe R. Ikebe M. Biochem. Biophys. Res. Commun. 2004; 315: PubMed Scopus Google Scholar, J. Cell Biol. 2004; PubMed Scopus Google Scholar). The GTD-deleted constructs such as and formed a or structure in the of GTD, these molecules a triangular shape in the presence of GTD that is similar to the conformation of full-length myosin Va in the inhibited The formation of the triangular conformation was with the GTD-dependent of the actin-activated ATPase activity of the truncated myosin Va. support the that the triangular conformation the inhibited form of myosin Va F. Thirumurugan K. Stafford W.F. Hammer 3rd, J.A. Knight P.J. Sellers J.R. J. Biol. Chem. 2004; 279: 2333-2336Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). This is further by the that the triangular conformation in the presence of exogenous GTD, and this is by the of by GTD. The that GTD binds to the C-terminal of coil-1. The of coil-1 and the head domain of myosin Va are A triangular conformation GTD binds to the terminus of coil-1 and the head If the length of the neck and coil-1 would critical for the inhibited state of myosin Va. This is by the that myosin Va constructs with a neck or are not inhibited by GTD. If GTD binds to the terminus of the head domain not reach the GTD when the neck length is myosin Va with a coil-1 as is not inhibited by GTD. This the neck length is to reach the GTD the C-terminal end of the the neck domain and the coil-1 are and the of the length of the two is critical for the the motor domain and the GTD the motor this the of by GTD was partially restored by amino residues of coil-1 in The of regulation of the GTD and the motor domain in addition to the length of the coil-1 and the head Based the of the we propose a model for the formation of the inhibited state of myosin Va low in as GTD binds to the C-terminal end of coil-1. The neck-tail junction of myosin Va is flexible, and the long neck enables the head domain to reach the GTD associated the end of coil-1. Once the with the GTD, the conformation is stabilized. The binding of the head domain to GTD its ATP or with inhibiting the actin-activated ATPase activity of the motor domain are two to the inhibited binding to the tail domain of myosin Va in the inhibited state the GTD and the head of myosin Va, the the This is by the that the actin-activated ATPase activity of myosin Va was by its X.D. Ikebe R. Ikebe M. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). a of molecules to myosin Va, it is that these molecules the inhibited a for the of such that concentrations of the actin-activated ATPase activity and an conformation of myosin Va F. Thirumurugan K. Stafford W.F. Hammer 3rd, J.A. Knight P.J. Sellers J.R. J. Biol. Chem. 2004; 279: 2333-2336Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, X.D. Mabuchi K. Ikebe R. Ikebe M. Biochem. Biophys. Res. Commun. 2004; 315: PubMed Scopus Google Scholar, J. Cell Biol. 2004; PubMed Scopus Google Scholar). The by is through to the IQ motifs. is that the neck region, the to the IQ is in the binding of GTD to the the conformation of this its with GTD. the triangular conformation of myosin Va the calmodulins to the IQ the of the each which is by high is that high the of from IQ the structure of neck and the triangular are to this the of three of myosin Va, the motor domain A. J. 2004; PubMed Scopus Google Scholar, J. A. Nature. 2003; PubMed Scopus Google the neck M. Stafford W.F. R. J. 2003; PubMed Scopus Google Scholar, M. R. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google and GTD S. J. PubMed Scopus Google have high The of these the triangular conformation of myosin Va on the inhibited state of myosin Va. for for the of the for in for the and Wang for