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• W2130935296 abstract "Profilin and β/γ-actin from calf thymus were covalently linked using the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in combination withN-hydroxysuccinimide, yielding a single product with an apparent molecular mass of 60 kDa. Sequence analysis and x-ray crystallographic investigations showed that the cross-linked residues were glutamic acid 82 of profilin and lysine 113 of actin. The cross-linked complex was shown to bind with high affinity to deoxyribonuclease I and poly(l-proline). It also bound and exchanged ATP with kinetics close to that of unmodified profilin-actin and inhibited the intrinsic ATPase activity of actin. This inhibition occurred even in conditions where actin normally forms filaments. By these criteria the cross-linked profilin-actin complex retains the characteristics of unmodified profilin-actin. However, the cross-linked complex did not form filaments nor copolymerized with unmodified actin, but did interfere with elongation of actin filaments in a concentration-dependent manner. These results support a polymerization mechanism where the profilin-actin heterodimer binds to the (+)-end of actin filaments, followed by dissociation of profilin, and ATP hydrolysis and Pi release from the actin subunit as it assumes its stable conformation in the helical filament. Profilin and β/γ-actin from calf thymus were covalently linked using the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in combination withN-hydroxysuccinimide, yielding a single product with an apparent molecular mass of 60 kDa. Sequence analysis and x-ray crystallographic investigations showed that the cross-linked residues were glutamic acid 82 of profilin and lysine 113 of actin. The cross-linked complex was shown to bind with high affinity to deoxyribonuclease I and poly(l-proline). It also bound and exchanged ATP with kinetics close to that of unmodified profilin-actin and inhibited the intrinsic ATPase activity of actin. This inhibition occurred even in conditions where actin normally forms filaments. By these criteria the cross-linked profilin-actin complex retains the characteristics of unmodified profilin-actin. However, the cross-linked complex did not form filaments nor copolymerized with unmodified actin, but did interfere with elongation of actin filaments in a concentration-dependent manner. These results support a polymerization mechanism where the profilin-actin heterodimer binds to the (+)-end of actin filaments, followed by dissociation of profilin, and ATP hydrolysis and Pi release from the actin subunit as it assumes its stable conformation in the helical filament. covalently cross-linked profilin-β/γ-actin complex 1,N6-ethenoadenosine 5′-triphosphate 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide N-hydroxysuccinimide crystallography NMR software profilin-actin dithiothreitol high performance liquid chromatography poly(l-proline) Profilin, originally isolated as a 1:1 complex with β-actin (1Carlsson L. Nyström L.E. Lindberg U. Kannan K.K. Cid-Dresdner H. Lövgren S. J. Mol. Biol. 1976; 105: 353-366Crossref PubMed Scopus (110) Google Scholar,2Carlsson L. Nyström L.E. Sundkvist I. Markey F. Lindberg U. J. Mol. Biol. 1977; 115: 465-483Crossref PubMed Scopus (525) Google Scholar), is an essential actin-binding protein involved in the control of actin filament formation in vivo (see Refs. 3Sohn R.H. Goldschmidt-Clermont P.J. Bioessays. 1994; 16: 465-472Crossref PubMed Scopus (161) Google Scholar and 4Schlüter K. Jockusch B.M. Rothkegel M. Biochim. Biophys. Acta. 1997; 1359: 97-109Crossref PubMed Scopus (167) Google Scholar, and references therein). The profilin-actin complex is unable to nucleate filament formation in vitro, but is suggested to interact with the (+)-end (barbed end) of preexisting filaments (5Tilney L.G. Bonder E.M. Coluccio L.M. Mooseker M.S. J. Cell Biol. 1983; 97: 112-124Crossref PubMed Scopus (139) Google Scholar, 6Pollard T.D. Cooper J.A. Biochemistry. 1984; 23: 6631-6641Crossref PubMed Scopus (233) Google Scholar, 7Pring M. Weber A. Bubb M.R. Biochemistry. 1992; 31: 1827-1836Crossref PubMed Scopus (106) Google Scholar, 8Pantaloni D. Carlier M.F. Cell. 1993; 75: 1007-1014Abstract Full Text PDF PubMed Scopus (456) Google Scholar, 9Korenbaum E. Nordberg P. Björkegren-Sjögren C. Schutt C.E. Lindberg U. Karlsson R. Biochemistry. 1998; 37: 9274-9283Crossref PubMed Scopus (63) Google Scholar, 10Kang F. Purich D.L. Southwick F.S. J. Biol. Chem. 1999; 274: 36963-36972Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), resulting in the dissociation of the profilin-actin complex and incorporation of actin monomers into filaments. The (−)-end (pointed end) of actin filaments, does not bind profilin-actin (5Tilney L.G. Bonder E.M. Coluccio L.M. Mooseker M.S. J. Cell Biol. 1983; 97: 112-124Crossref PubMed Scopus (139) Google Scholar, 6Pollard T.D. Cooper J.A. Biochemistry. 1984; 23: 6631-6641Crossref PubMed Scopus (233) Google Scholar, 11Markey F. Larsson H. Weber K. Lindberg U. Biochim. Biophys. Acta. 1982; 704: 43-51Crossref PubMed Scopus (22) Google Scholar). This behavior of the complex is explained by the orientation of the actin protomers in the actin filament giving it polarity (12Huxley H.E. J. Mol. Biol. 1963; 7: 281-308Crossref PubMed Scopus (1016) Google Scholar), by the location of the profilin binding site on actin (13Schutt C.E. Myslik J.C. Rozycki M.D. Goonesekere N.C. Lindberg U. Nature. 1993; 365: 810-816Crossref PubMed Scopus (594) Google Scholar), and the strength of the profilin-actin interaction (6Pollard T.D. Cooper J.A. Biochemistry. 1984; 23: 6631-6641Crossref PubMed Scopus (233) Google Scholar, 9Korenbaum E. Nordberg P. Björkegren-Sjögren C. Schutt C.E. Lindberg U. Karlsson R. Biochemistry. 1998; 37: 9274-9283Crossref PubMed Scopus (63) Google Scholar). Profilin binds to the (+)-end of the actin monomer leaving the (−)-end free to interact with the (+)-end of actin nuclei or filaments. Consequently, in the presence of (+)-end capping agents like members of the gelsolin family, profilin efficiently sequesters actin monomers and causes depolymerization of actin filaments.Profilin greatly lowers the affinity for both ATP and divalent cation on actin, thereby increasing their exchange rates (14Mockrin S.C. Korn E.D. Biochemistry. 1980; 19: 5359-5362Crossref PubMed Scopus (195) Google Scholar, 15Goldschmidt-Clermont P.J. Machesky L.M. Doberstein S.K. Pollard T.D. J. Cell Biol. 1991; 113: 1081-1089Crossref PubMed Scopus (181) Google Scholar, 16Selden L.A. Kinosian H.J. Estes J.E. Gershman L.C. Biochemistry. 1999; 38: 2769-2778Crossref PubMed Scopus (66) Google Scholar). It has been suggested that this effect of profilin on actin might be importantin vivo during conditions of rapid filament turnover, when the exchange of ADP for ATP otherwise might be rate-limiting (16Selden L.A. Kinosian H.J. Estes J.E. Gershman L.C. Biochemistry. 1999; 38: 2769-2778Crossref PubMed Scopus (66) Google Scholar).To learn more about the nature of the profilin-actin complex and its significance in the actin polymerization process, a covalently cross-linked profilin-β/γ-actin complex (PxA)1 was produced. The value of PxA as a tool for in vivo studies of the profilin-actin complex was illustrated in a recent report describing the effects on the organization of the microfilament system of cultured cells by microinjected PxA (17Hajkova L. Nyman T. Lindberg U. Karlsson R. Exp. Cell Res. 2000; 256: 112-121Crossref PubMed Scopus (26) Google Scholar).The present study describes the preparation of PxA, the evaluation of its structural characteristics, and its use in studies of actin filament formation from profilin-actin in vitro. The PxA complex retained the capacities of wild type profilin-actin (PA) to bind DNase I and poly(l-proline) (PLP), and to bind and exchange nucleotide with kinetics close to that of PA. This indicates that the surface structure of PA was conserved all through the cross-linking reaction. The PxA complex did not hydrolyze ATP even under actin-polymerizing conditions (1 mmMgCl2, 100 mm KCl), and it could neither polymerize nor participate in filament formation from unmodified actin. It did, however, interfere with the formation of actin filaments, indicating that it retained the capacity to interact with the (+)-end of growing filaments. These results are discussed in comparison with a differently cross-linked profilin-actin complex (18Gutsche-Perelroizen I. Lepault J. Ott A. Carlier M.F. J. Biol. Chem. 1999; 274: 6234-6243Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Crystallographic analysis showed PxA to be closely similar to unmodified profilin-actin.DISCUSSIONAs shown here, the structure and biochemical characteristics of PxA are closely similar to those of unmodified PA with the exception that it does not polymerize.Intrinsic ATPase ActivityActin binds ATP tightly in complex with a divalent cation (38Valentin-Ranc C. Carlier M.F. J. Biol. Chem. 1989; 264: 20871-20880Abstract Full Text PDF PubMed Google Scholar, 39Kabsch W. Mannherz H.G. Suck D. Pai E.F. Holmes K.C. Nature. 1990; 347: 37-44Crossref PubMed Scopus (1522) Google Scholar). Under nonpolymerizing conditions, the ATP is slowly hydrolyzed, an activity that appears to be intrinsic to the actin monomer and not dependent on dimer formation (40Brenner S.L. Korn E.D. J. Biol. Chem. 1980; 255: 841-844Abstract Full Text PDF PubMed Google Scholar, 41Schüler H. The Molecular Dynamics of Actin.Ph.D. thesis. Stockholm University, Stockholm2000Google Scholar). Replacing Ca2+ by Mg2+ at the high affinity divalent cation binding site lowers the rate of nucleotide exchange and enhances the intrinsic ATPase activity (25Kinosian H.J. Selden L.A. Estes J.E. Gershman L.C. J. Biol. Chem. 1993; 268: 8683-8691Abstract Full Text PDF PubMed Google Scholar, 42Kinosian H.J. Selden L.A. Gershman L.C. Estes J.E. Biochemistry. 2000; 39: 13176-13188Crossref PubMed Scopus (29) Google Scholar, 43Chen X. Peng J. Pedram M. Swenson C.A. Rubenstein P.A. J. Biol. Chem. 1995; 270: 11415-11423Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). This may be related to the observation that Mg2+ induces a structural change, probably the closing of the interdomain cleft, that protects the region around Lys-68 in the interdomain cleft from proteolytic attack (44Strzelecka-Golaszewska H. Moraczewska J. Khaitlina S.Y. Mossakowska M. Eur. J. Biochem. 1993; 211: 731-742Crossref PubMed Scopus (126) Google Scholar).The binding of profilin to actin counteracts this Mg2+effect in that it greatly lowers the affinity for the nucleotide on actin, increasing its rate of exchange (14Mockrin S.C. Korn E.D. Biochemistry. 1980; 19: 5359-5362Crossref PubMed Scopus (195) Google Scholar, 15Goldschmidt-Clermont P.J. Machesky L.M. Doberstein S.K. Pollard T.D. J. Cell Biol. 1991; 113: 1081-1089Crossref PubMed Scopus (181) Google Scholar, 16Selden L.A. Kinosian H.J. Estes J.E. Gershman L.C. Biochemistry. 1999; 38: 2769-2778Crossref PubMed Scopus (66) Google Scholar). The explanation for this is found in the flexibility of actin in the interdomain region that allows opening and closing of the nucleotide-binding cleft (13Schutt C.E. Myslik J.C. Rozycki M.D. Goonesekere N.C. Lindberg U. Nature. 1993; 365: 810-816Crossref PubMed Scopus (594) Google Scholar,28Chik J.K. Lindberg U. Schutt C.E. J. Mol. Biol. 1996; 263: 607-623Crossref PubMed Scopus (184) Google Scholar). Shear motions involving the interdomain Gln-137–Ser-145 helix connecting subdomains 1 and 3 bring about a 2.8° rotation of subdomain 1 that results in an outward shift of the Asn-12–Cys-17 loop, exposing the ATP phosphate tail to solution (28Chik J.K. Lindberg U. Schutt C.E. J. Mol. Biol. 1996; 263: 607-623Crossref PubMed Scopus (184) Google Scholar, 45Page R. Lindberg U. Schutt C.E. J. Mol. Biol. 1998; 280: 463-474Crossref PubMed Scopus (83) Google Scholar). Importantly, the tight-to-open state transition disrupts divalent cation coordination with amino acid residues in the cleft: Asp-11 and Asp-154 in subdomains 1 and 3, respectively; and Gln-137 in the shearing helix. The profilin binding site on actin spans these subdomains on the (+)-end of the monomer, on the opposite side of the interdomain helix relative to the nucleotide binding cleft. This explains how the binding of ATP to actin depends on the divalent cation (25Kinosian H.J. Selden L.A. Estes J.E. Gershman L.C. J. Biol. Chem. 1993; 268: 8683-8691Abstract Full Text PDF PubMed Google Scholar, 46West J.J. Nagy B. Gergely J. J. Biol. Chem. 1967; 242: 1140-1145Abstract Full Text PDF PubMed Google Scholar), and how profilin might enhance nucleotide dissociation by disrupting cation coordination (42Kinosian H.J. Selden L.A. Gershman L.C. Estes J.E. Biochemistry. 2000; 39: 13176-13188Crossref PubMed Scopus (29) Google Scholar). The findings that profilin inhibits ATP hydrolysis on the actin monomer, under nonpolymerizing conditions (37Tobacman L.S. Korn E.D. J. Biol. Chem. 1982; 257: 4166-4170Abstract Full Text PDF PubMed Google Scholar) as well as under polymerizing conditions as shown here, supports the view that profilin-actin is in an open state conformation under physiological salt concentrations, even in the presence of Mg2+.Filament Formation from PA and the Structure of F-actinEarlier studies suggested the possibility that PA might interact directly with the (+)-end during filament formation (5Tilney L.G. Bonder E.M. Coluccio L.M. Mooseker M.S. J. Cell Biol. 1983; 97: 112-124Crossref PubMed Scopus (139) Google Scholar, 6Pollard T.D. Cooper J.A. Biochemistry. 1984; 23: 6631-6641Crossref PubMed Scopus (233) Google Scholar, 7Pring M. Weber A. Bubb M.R. Biochemistry. 1992; 31: 1827-1836Crossref PubMed Scopus (106) Google Scholar, 8Pantaloni D. Carlier M.F. Cell. 1993; 75: 1007-1014Abstract Full Text PDF PubMed Scopus (456) Google Scholar,47Kaiser D.A. Sato M. Ebert R.F. Pollard T.D. J. Cell Biol. 1986; 102: 221-226Crossref PubMed Scopus (49) Google Scholar). This view was supported by determination of free unpolymerized actin under steady state polymerizing conditions in the presence of mutant profilins with varying affinity for actin (9Korenbaum E. Nordberg P. Björkegren-Sjögren C. Schutt C.E. Lindberg U. Karlsson R. Biochemistry. 1998; 37: 9274-9283Crossref PubMed Scopus (63) Google Scholar). The present investigation demonstrates that PxA interferes with both nucleation and elongation phases of the polymerization reaction of actin in the presence of non-cross-linked material. This indicates that the PA complex can bind to the (+)-end of actin nuclei and filaments, and, unless profilin dissociates so as to allow the final annealing of its ferried actin monomer, the incorporation is aborted and the complex dissociates from the growing filament.Because profilin inhibits ATP hydrolysis on actin, profilin release during polymer formation from PA must occur prior to ATP hydrolysis. This implies that the initial binding of native PA to the (+)-end of actin filaments induces a conformational change in actin that promotes the dissociation of profilin. In this model, the ensuing ATP hydrolysis and Pi release are coupled to further structural changes during which the actin subunit adopts its final F-actin conformation.Recently, a covalently cross-linked complex comprising profilin and rabbit α-actin, denoted PAcov, was described (18Gutsche-Perelroizen I. Lepault J. Ott A. Carlier M.F. J. Biol. Chem. 1999; 274: 6234-6243Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). This complex was obtained by activating actin with the EDC/NHS reagent prior to the addition of profilin. Based on an earlier report on the covalent coupling of Acanthamoeba profilin and actin, shown to involve Lys-115 of profilin and Glu-364 of actin (30Vandekerckhove J.S. Kaiser D.A. Pollard T.D. J. Cell Biol. 1989; 109: 619-626Crossref PubMed Scopus (86) Google Scholar), the cross-link of PAcov was assumed to engage Lys-125 of profilin (corresponding to K115 in the amoeba profilin) and Glu-364 of actin. The alternative protocol used here linked Glu-82 of profilin to Lys-113 of actin. The two complexes, PAcov and PxA, behave differently in that PAcov can form filaments by itself, or together with uncomplexed actin, whereas PxA cannot. It was argued (18Gutsche-Perelroizen I. Lepault J. Ott A. Carlier M.F. J. Biol. Chem. 1999; 274: 6234-6243Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) that the formation of helical filaments from PAcov is not predicted by the Holmes/Lorenz F-actin model (48Holmes K.C. Popp D. Gebhard W. Kabsch W. Nature. 1990; 347: 44-49Crossref PubMed Scopus (1311) Google Scholar, 49Lorenz M. Popp D. Holmes K.C. J. Mol. Biol. 1993; 234: 826-836Crossref PubMed Scopus (445) Google Scholar) because PAcov would not only interfere with the formation of the long-pitch helix, but should cap actin filaments at their (+)-end. Alternatively, if the actin ribbon structure found in the profilin-actin crystals is an assembly intermediate in the formation of F-actin (13Schutt C.E. Myslik J.C. Rozycki M.D. Goonesekere N.C. Lindberg U. Nature. 1993; 365: 810-816Crossref PubMed Scopus (594) Google Scholar), converting to an F-actin helix upon release of profilin (50Cedergren-Zeppezauer E.S. Goonesekere N.C. Rozycki M.D. Myslik J.C. Dauter Z. Lindberg U. Schutt C.E. J. Mol. Biol. 1994; 240: 459-475Crossref PubMed Scopus (67) Google Scholar), then PAcov should form nonhelical profilin-actin ribbons, according to these authors (18Gutsche-Perelroizen I. Lepault J. Ott A. Carlier M.F. J. Biol. Chem. 1999; 274: 6234-6243Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Instead, it was found that PAcov formed filaments with the same helical periodicity as native F-actin.However, it should be noted that actin amino acid residue, Glu-364, involved in the PAcov cross-link is located in a turn near the actin C terminus at the outer “edge” of subdomain 1, in a region known to be flexible and particularly sensitive to polymerization conditions (Fig. 8). During incorporation of PAcov, at the (+)-end of an actin filament, it is conceivable that the tethered profilin detaches from its interfacial contact with actin and swings out to the side of the filament. This movement exposes the (+)-end of the filament for interaction with an incoming PAcov heterodimer. Thus, the Holmes/Lorenz model of F-actin and also profilin-actin ribbon-based models are compatible with the observation that PAcov can form normally appearing F-actin filaments.The difference between PAcov and PxA in the ability to form filaments most likely arises from differences in the freedom of profilin to shift in position relative to actin during the interaction with actin nuclei or polymers. As discussed above, in the case of PAcov, such shifts might enable normal polymerization reactions, involving the release of profilin from (+)-end assembly intermediates. In the case of PxA, the cross-linked residue, Lys-113, protrudes from the center of subdomain 1, a region not expected to be unusually flexible. Thus, for PxA, in contrast to PAcov,the position of the cross-link apparently prevents the dislocation of profilin and subsequent incorporation of the actin subunit into the filament, aborting the assembly at the intermediate stage.The observation that PAcov forms helical filaments with the same periodicity as native F-actin is most interesting, because it would seem to provide an opportunity to determine the orientation of the actin monomer in F-actin. This is important, because the monomer orientation in the Holmes/Lorenz model of F-actin differs from that in the actin ribbon found in the profilin-actin crystals. Reconstructions of PAcov filaments from electron micrographs may reveal the location of Glu-364 of actin relative to the filament axis accurately enough to discriminate between the two cases. Profilin, originally isolated as a 1:1 complex with β-actin (1Carlsson L. Nyström L.E. Lindberg U. Kannan K.K. Cid-Dresdner H. Lövgren S. J. Mol. Biol. 1976; 105: 353-366Crossref PubMed Scopus (110) Google Scholar,2Carlsson L. Nyström L.E. Sundkvist I. Markey F. Lindberg U. J. Mol. Biol. 1977; 115: 465-483Crossref PubMed Scopus (525) Google Scholar), is an essential actin-binding protein involved in the control of actin filament formation in vivo (see Refs. 3Sohn R.H. Goldschmidt-Clermont P.J. Bioessays. 1994; 16: 465-472Crossref PubMed Scopus (161) Google Scholar and 4Schlüter K. Jockusch B.M. Rothkegel M. Biochim. Biophys. Acta. 1997; 1359: 97-109Crossref PubMed Scopus (167) Google Scholar, and references therein). The profilin-actin complex is unable to nucleate filament formation in vitro, but is suggested to interact with the (+)-end (barbed end) of preexisting filaments (5Tilney L.G. Bonder E.M. Coluccio L.M. Mooseker M.S. J. Cell Biol. 1983; 97: 112-124Crossref PubMed Scopus (139) Google Scholar, 6Pollard T.D. Cooper J.A. Biochemistry. 1984; 23: 6631-6641Crossref PubMed Scopus (233) Google Scholar, 7Pring M. Weber A. Bubb M.R. Biochemistry. 1992; 31: 1827-1836Crossref PubMed Scopus (106) Google Scholar, 8Pantaloni D. Carlier M.F. Cell. 1993; 75: 1007-1014Abstract Full Text PDF PubMed Scopus (456) Google Scholar, 9Korenbaum E. Nordberg P. Björkegren-Sjögren C. Schutt C.E. Lindberg U. Karlsson R. Biochemistry. 1998; 37: 9274-9283Crossref PubMed Scopus (63) Google Scholar, 10Kang F. Purich D.L. Southwick F.S. J. Biol. Chem. 1999; 274: 36963-36972Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), resulting in the dissociation of the profilin-actin complex and incorporation of actin monomers into filaments. The (−)-end (pointed end) of actin filaments, does not bind profilin-actin (5Tilney L.G. Bonder E.M. Coluccio L.M. Mooseker M.S. J. Cell Biol. 1983; 97: 112-124Crossref PubMed Scopus (139) Google Scholar, 6Pollard T.D. Cooper J.A. Biochemistry. 1984; 23: 6631-6641Crossref PubMed Scopus (233) Google Scholar, 11Markey F. Larsson H. Weber K. Lindberg U. Biochim. Biophys. Acta. 1982; 704: 43-51Crossref PubMed Scopus (22) Google Scholar). This behavior of the complex is explained by the orientation of the actin protomers in the actin filament giving it polarity (12Huxley H.E. J. Mol. Biol. 1963; 7: 281-308Crossref PubMed Scopus (1016) Google Scholar), by the location of the profilin binding site on actin (13Schutt C.E. Myslik J.C. Rozycki M.D. Goonesekere N.C. Lindberg U. Nature. 1993; 365: 810-816Crossref PubMed Scopus (594) Google Scholar), and the strength of the profilin-actin interaction (6Pollard T.D. Cooper J.A. Biochemistry. 1984; 23: 6631-6641Crossref PubMed Scopus (233) Google Scholar, 9Korenbaum E. Nordberg P. Björkegren-Sjögren C. Schutt C.E. Lindberg U. Karlsson R. Biochemistry. 1998; 37: 9274-9283Crossref PubMed Scopus (63) Google Scholar). Profilin binds to the (+)-end of the actin monomer leaving the (−)-end free to interact with the (+)-end of actin nuclei or filaments. Consequently, in the presence of (+)-end capping agents like members of the gelsolin family, profilin efficiently sequesters actin monomers and causes depolymerization of actin filaments. Profilin greatly lowers the affinity for both ATP and divalent cation on actin, thereby increasing their exchange rates (14Mockrin S.C. Korn E.D. Biochemistry. 1980; 19: 5359-5362Crossref PubMed Scopus (195) Google Scholar, 15Goldschmidt-Clermont P.J. Machesky L.M. Doberstein S.K. Pollard T.D. J. Cell Biol. 1991; 113: 1081-1089Crossref PubMed Scopus (181) Google Scholar, 16Selden L.A. Kinosian H.J. Estes J.E. Gershman L.C. Biochemistry. 1999; 38: 2769-2778Crossref PubMed Scopus (66) Google Scholar). It has been suggested that this effect of profilin on actin might be importantin vivo during conditions of rapid filament turnover, when the exchange of ADP for ATP otherwise might be rate-limiting (16Selden L.A. Kinosian H.J. Estes J.E. Gershman L.C. Biochemistry. 1999; 38: 2769-2778Crossref PubMed Scopus (66) Google Scholar). To learn more about the nature of the profilin-actin complex and its significance in the actin polymerization process, a covalently cross-linked profilin-β/γ-actin complex (PxA)1 was produced. The value of PxA as a tool for in vivo studies of the profilin-actin complex was illustrated in a recent report describing the effects on the organization of the microfilament system of cultured cells by microinjected PxA (17Hajkova L. Nyman T. Lindberg U. Karlsson R. Exp. Cell Res. 2000; 256: 112-121Crossref PubMed Scopus (26) Google Scholar). The present study describes the preparation of PxA, the evaluation of its structural characteristics, and its use in studies of actin filament formation from profilin-actin in vitro. The PxA complex retained the capacities of wild type profilin-actin (PA) to bind DNase I and poly(l-proline) (PLP), and to bind and exchange nucleotide with kinetics close to that of PA. This indicates that the surface structure of PA was conserved all through the cross-linking reaction. The PxA complex did not hydrolyze ATP even under actin-polymerizing conditions (1 mmMgCl2, 100 mm KCl), and it could neither polymerize nor participate in filament formation from unmodified actin. It did, however, interfere with the formation of actin filaments, indicating that it retained the capacity to interact with the (+)-end of growing filaments. These results are discussed in comparison with a differently cross-linked profilin-actin complex (18Gutsche-Perelroizen I. Lepault J. Ott A. Carlier M.F. J. Biol. Chem. 1999; 274: 6234-6243Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Crystallographic analysis showed PxA to be closely similar to unmodified profilin-actin. DISCUSSIONAs shown here, the structure and biochemical characteristics of PxA are closely similar to those of unmodified PA with the exception that it does not polymerize.Intrinsic ATPase ActivityActin binds ATP tightly in complex with a divalent cation (38Valentin-Ranc C. Carlier M.F. J. Biol. Chem. 1989; 264: 20871-20880Abstract Full Text PDF PubMed Google Scholar, 39Kabsch W. Mannherz H.G. Suck D. Pai E.F. Holmes K.C. Nature. 1990; 347: 37-44Crossref PubMed Scopus (1522) Google Scholar). Under nonpolymerizing conditions, the ATP is slowly hydrolyzed, an activity that appears to be intrinsic to the actin monomer and not dependent on dimer formation (40Brenner S.L. Korn E.D. J. Biol. Chem. 1980; 255: 841-844Abstract Full Text PDF PubMed Google Scholar, 41Schüler H. The Molecular Dynamics of Actin.Ph.D. thesis. Stockholm University, Stockholm2000Google Scholar). Replacing Ca2+ by Mg2+ at the high affinity divalent cation binding site lowers the rate of nucleotide exchange and enhances the intrinsic ATPase activity (25Kinosian H.J. Selden L.A. Estes J.E. Gershman L.C. J. Biol. Chem. 1993; 268: 8683-8691Abstract Full Text PDF PubMed Google Scholar, 42Kinosian H.J. Selden L.A. Gershman L.C. Estes J.E. Biochemistry. 2000; 39: 13176-13188Crossref PubMed Scopus (29) Google Scholar, 43Chen X. Peng J. Pedram M. Swenson C.A. Rubenstein P.A. J. Biol. Chem. 1995; 270: 11415-11423Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). This may be related to the observation that Mg2+ induces a structural change, probably the closing of the interdomain cleft, that protects the region around Lys-68 in the interdomain cleft from proteolytic attack (44Strzelecka-Golaszewska H. Moraczewska J. Khaitlina S.Y. Mossakowska M. Eur. J. Biochem. 1993; 211: 731-742Crossref PubMed Scopus (126) Google Scholar).The binding of profilin to actin counteracts this Mg2+effect in that it greatly lowers the affinity for the nucleotide on actin, increasing its rate of exchange (14Mockrin S.C. Korn E.D. Biochemistry. 1980; 19: 5359-5362Crossref PubMed Scopus (195) Google Scholar, 15Goldschmidt-Clermont P.J. Machesky L.M. Doberstein S.K. Pollard T.D. J. Cell Biol. 1991; 113: 1081-1089Crossref PubMed Scopus (181) Google Scholar, 16Selden L.A. Kinosian H.J. Estes J.E. Gershman L.C. Biochemistry. 1999; 38: 2769-2778Crossref PubMed Scopus (66) Google Scholar). The explanation for this is found in the flexibility of actin in the interdomain region that allows opening and closing of the nucleotide-binding cleft (13Schutt C.E. Myslik J.C. Rozycki M.D. Goonesekere N.C. Lindberg U. Nature. 1993; 365: 810-816Crossref PubMed Scopus (594) Google Scholar,28Chik J.K. Lindberg U. Schutt C.E. J. Mol. Biol. 1996; 263: 607-623Crossref PubMed Scopus (184) Google Scholar). Shear motions involving the interdomain Gln-137–Ser-145 helix connecting subdomains 1 and 3 bring about a 2.8° rotation of subdomain 1 that results in an outward shift of the Asn-12–Cys-17 loop, exposing the ATP phosphate tail to solution (28Chik J.K. Lindberg U. Schutt C.E. J. Mol. Biol. 1996; 263: 607-623Crossref PubMed Scopus (184) Google Scholar, 45Page R. Lindberg U. Schutt C.E. J. Mol. Biol. 1998; 280: 463-474Crossref PubMed Scopus (83) Google Scholar). Importantly, the tight-to-open state transition disrupts divalent cation coordination with amino acid residues in the cleft: Asp-11 and Asp-154 in subdomains 1 and 3, respectively; and Gln-137 in the shearing helix. The profilin binding site on actin spans these subdomains on the (+)-end of the monomer, on the opposite side of the interdomain helix relative to the nucleotide binding cleft. This explains how the binding of ATP to actin depends on the divalent cation (25Kinosian H.J. Selden L.A. Estes J.E. Gershman L.C. J. Biol. Chem. 1993; 268: 8683-8691Abstract Full Text PDF PubMed Google Scholar, 46West J.J. Nagy B. Gergely J. J. Biol. Chem. 1967; 242: 1140-1145Abstract Full Text PDF PubMed Google Scholar), and how profilin might enhance nucleotide dissociation by disrupting cation coordination (42Kinosian H.J. Selden L.A. Gershman L.C. Estes J.E. Biochemistry. 2000; 39: 13176-13188Crossref PubMed Scopus (29) Google Scholar). The findings" @default.
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• W2130935296 title "A Cross-linked Profilin-Actin Heterodimer Interferes with Elongation at the Fast-growing End of F-actin" @default.
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