Actin-Latrunculin A Structure and Function

Latrunculin A is used extensively as an agent to sequester monomeric actin in living cells. We hypothesize that additional activities of latrunculin A may be important for its biological activity. Our data are consistent with the formation of a 1:1 stoichiometric complex with an equilibrium dissociation constant of 0.2 to 0.4 μm and provide no evidence that the actin-latrunculin A complex participates in the elongation of actin filaments. Profilin and latrunculin A bind independently to actin, whereas binding of thymosin β4 to actin is inhibited by latrunculin A. Potential implications of this differential effect on actin-binding proteins are discussed. From a structural perspective, if latrunculin A binds to actin at a site that sterically influences binding by thymosin β4, then the observation that latrunculin A inhibits nucleotide exchange on actin implies an allosteric effect on the nucleotide binding cleft. Alternatively, if, as previously postulated, latrunculin A binds in the nucleotide cleft of actin, then its ability to inhibit binding by thymosin β4 is a surprising result that suggests that significant allosteric changes affect the thymosin β4 binding site. We show that latrunculin A and actin form a crystalline structure with orthorhombic space group P212121and diffraction to 3.10 Å. A high resolution structure with optimized crystallization conditions should provide insight regarding these remarkable allosteric properties. Latrunculin A is used extensively as an agent to sequester monomeric actin in living cells. We hypothesize that additional activities of latrunculin A may be important for its biological activity. Our data are consistent with the formation of a 1:1 stoichiometric complex with an equilibrium dissociation constant of 0.2 to 0.4 μm and provide no evidence that the actin-latrunculin A complex participates in the elongation of actin filaments. Profilin and latrunculin A bind independently to actin, whereas binding of thymosin β4 to actin is inhibited by latrunculin A. Potential implications of this differential effect on actin-binding proteins are discussed. From a structural perspective, if latrunculin A binds to actin at a site that sterically influences binding by thymosin β4, then the observation that latrunculin A inhibits nucleotide exchange on actin implies an allosteric effect on the nucleotide binding cleft. Alternatively, if, as previously postulated, latrunculin A binds in the nucleotide cleft of actin, then its ability to inhibit binding by thymosin β4 is a surprising result that suggests that significant allosteric changes affect the thymosin β4 binding site. We show that latrunculin A and actin form a crystalline structure with orthorhombic space group P212121and diffraction to 3.10 Å. A high resolution structure with optimized crystallization conditions should provide insight regarding these remarkable allosteric properties. Latrunculin A, isolated from the Red Sea sponge Negombata magnifica, was initially identified as an inhibitor of actin polymerization by its morphological effects and by the effects it had on actin filament distribution in cultured nonmuscle cells (1Spector I. Shochet N.R. Kashman Y. Groweiss A. Science. 1983; 214: 493-495Crossref Scopus (625) Google Scholar). Based on the effects of latrunculin A on the steady state level of F-actinin vitro, the effects of the drug were thought to be consistent with sequestration of monomeric actin in a 1:1 molar complex with equilibrium dissociation constant of 0.2 μm (2Coué M. Brenner S.L. Spector I. Korn E.D. FEBS Lett. 1987; 213: 316-318Crossref PubMed Scopus (661) Google Scholar). The binding site of latrunculin has not been conclusively identified, but based on the study of the effects of specific mutations of yeast actin on latrunculin A binding, it has been inferred that latrunculin A may bind to actin near or in its nucleotide binding cleft (3Ayscough K.R. Stryker J. Pokala N. Sanders M. Crews P. Drubin D.G. J. Cell Biol. 1997; 137: 399-416Crossref PubMed Scopus (644) Google Scholar, 4Belmont L.D. Patterson G.M.L. Drubin D.G. J. Cell Sci. 1999; 112: 1325-1336PubMed Google Scholar). The observation that latrunculin affects nucleotide exchange has been offered as support of this conclusion (3Ayscough K.R. Stryker J. Pokala N. Sanders M. Crews P. Drubin D.G. J. Cell Biol. 1997; 137: 399-416Crossref PubMed Scopus (644) Google Scholar). These data, however, are inconclusive in light of the fact that many actin-binding proteins with binding sites that are spatially distant from the nucleotide cleft are also able to affect nucleotide exchange (5Safer D. Sosnick T.R. Elzinga M. Biochemistry. 1997; 36: 5806-5816Crossref PubMed Scopus (97) Google Scholar) and that actin demonstrates several additional allosteric properties that serve as a precedent for the transmission of structural alterations to distant sites (6Kuznetsova I. Antropova O. Turoverov K. Khaitlina S. FEBS Lett. 1996; 383: 105-108Crossref PubMed Scopus (44) Google Scholar, 7Prochniewicz E. Thomas D.D. Biochemistry. 1997; 36: 12845-12853Crossref PubMed Scopus (44) Google Scholar, 8De La Cruz E.M. Ostap E.M. Brundage R.A. Reddy K.S. Sweeney H.L. Safer D. Biophys. J. 2000; 78: 2516-2527Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). When latrunculin A is employed in studies of cell biology, the observed effects are consistent with depolymerization of actin filaments consequent to sequestration of monomeric actin by latrunculin (9Spector I. Shocet N.R. Blasberger D. Kashman Y. Cell Motil. Cytoskeleton. 1989; 13: 127-144Crossref PubMed Scopus (487) Google Scholar). A previous preliminary report (2Coué M. Brenner S.L. Spector I. Korn E.D. FEBS Lett. 1987; 213: 316-318Crossref PubMed Scopus (661) Google Scholar) did not rule out the possibility that latrunculin A has effects related to the polymerization of actin in addition to monomer sequestration, and these possibilities are explored in our current studies. Other effects of latrunculin A on the cytoskeleton are possible, however, and evidence has been reported that latrunculin can affect the expression of actin and possibly of other actin-binding proteins by a feedback mechanism that may sense the cellular concentration of actin monomers, resulting in more complicated outcomes than that predicted by monomer sequestration alone (10Bershadsky A.D. Gluck U. Denisenko O.N. Sklyarova T.V. Spector I. Ben-Zéev A. J. Cell Sci. 1995; 108: 1183-1193Crossref PubMed Google Scholar). To characterize the surface interactions of latrunculin A and actin, we examined whether latrunculin A affected the interaction of actin with other actin-monomer-binding proteins. To our surprise, latrunculin A inhibited binding by thymosin β4 but not binding by profilin or DNase I. Because thymosin β4 has been postulated to perform functions related to wound healing (11Frohm M. Gunne H. Bergman A.C. Agerberth B. Bergman T. Boman A. Liden S. Jornvall H. Boman H.G. Eur. J. Biochem. 1996; 237: 86-89Crossref PubMed Scopus (195) Google Scholar), apoptosis (12Niu M. Nachmias V.T. Cell Adhes. Commun. 2000; 7: 311-320Crossref PubMed Scopus (36) Google Scholar), and the inflammatory response (13Young J.D. Lawrence A.J. MacLean A.G. Leung B.P. McInnes I.B. Canas B. Pappin D.J.C. Stevenson R.D. Nat. Med. 1999; 5: 1424-1427Crossref PubMed Scopus (171) Google Scholar), augmentation of the concentration of free thymosin β4 by latrunculin A could potentiate these responses. Our results imply that actin-binding marine natural products may have effects other than those predicted solely by their effects on actin polymerization and, by inference, that marine natural products may exist that affect actin-binding protein function without directly affecting actin polymerization. Finally, our results illustrate a novel mechanism by which pharmacological agents that bind actin could be used to modulate the function of actin-binding proteins. Rabbit skeletal muscle actin was prepared from frozen muscle (Pel-Freez, Rogers, AR) in Buffer G (5.0 mmTris, 0.2 mm ATP, 0.2 mm dithiothreitol, 0.1 mm CaCl2, and 0.01% sodium Biochemistry. 1997; 36: PubMed Scopus Google Scholar), and on was prepared with of of protein the of and T. K. Eur. J. Biochem. PubMed Scopus Google Scholar). profilin was as previously Biochemistry. 1997; 36: PubMed Scopus Google Scholar). DNase was from thymosin for an to that of thymosin in a was a from Nachmias of of were as to a to the and of the products were to the an expression the was with Latrunculin A was as a or mm in and was to μm in Buffer G for the in or thymosin β4 were at in and with Cell were in for in and for at The was to with and to the of to with the was to for on for for at and on a The thymosin β4 was with a of in mm The were with as as and mm mm 0.2 sodium was the protein thymosin β4 was in mm sodium and then was in to a molar of to thymosin β4 of of at the was on and The was by addition of dithiothreitol, and the was β4 was then with and the concentration of thymosin by protein concentration of was for of and of the with addition of an has previously been not to affect the actin binding properties of thymosin β4 D. I. J. J. I. H. Y. D. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). was to by the addition of μm and and it was by the addition of to a concentration of at mm and latrunculin steady state were prepared by of μm without a in and steady state were at as previously M. Spector I. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). dissociation were that the the of actin, as monomer or as a complex of latrunculin A and The that is to A polymerization was used to elongation of as previously M. Spector I. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). data that the of polymerization was to the concentration of and to the concentration of free free was exchange as previously Korn E.D. Biochemistry. PubMed Scopus Google Scholar). and profilin were in a with Buffer G without and of latrunculin A. A of and μm and was to the were in and the of changes was Biochemistry. 1997; 36: PubMed Scopus Google Scholar). were by the to a were then to the equilibrium dissociation for profilin to for latrunculin A to actin, for profilin to the complex of actin and latrunculin A, and also to the of dissociation from actin, actin-latrunculin A, and actin-latrunculin at a concentration of μm was in Buffer G with or without μm thymosin β4 in the and of μm latrunculin A. were for on were in 0.1 mm CaCl2, 0.01% sodium 0.2 mm ATP, 0.2 mm dithiothreitol, and mm with thymosin β4, the of the was with were on a thymosin β4 was with light at The and of the light were at of thymosin β4 in Buffer G were with in the or of a constant of latrunculin A with latrunculin A in the or of a constant of were as by La Cruz E.M. Biochemistry. PubMed Scopus Google Scholar), with the of a for the formation of a complex actin, and the equilibrium dissociation for thymosin β4 to actin for latrunculin A to actin and for thymosin β4 to the complex of actin and latrunculin A and the the of free thymosin β4 and the of the complex of thymosin β4 with actin or with actin-latrunculin A complex that the concentration of free is to for the observed can be as a function of the actin, and latrunculin A, as is free actin equilibrium were with data at for thymosin in a μm thymosin of in Buffer G equilibrium in at initially to at Buffer was by and specific were as previously reported for actin or from for thymosin β4 Biochemistry. PubMed Scopus Google Scholar). The was to a of as previously Korn E.D. J. Biol. Full Text PDF PubMed Google Scholar). at thymosin β4 has a The other are at this the at is directly to the of the concentration of thymosin a of these thymosin β4, thymosin β4 to actin, and thymosin A The are to be in at as by equilibrium dissociation is by the concentration of and the the dissociation and the concentration of at an Korn E.D. J. Biol. Full Text PDF PubMed Google Scholar). DNase was with actin with of latrunculin A for at The was in mm 0.1 mm mm 0.1 mm ATP, and mm were on and the were with were in mm with actin concentration at and a of 1:1 or of latrunculin A to at crystallization conditions in of with 0.4 0.4 mm were on at The data were from a mm The was with an and data were at a of mm with an of were and the of the and 1997; PubMed Scopus Google Scholar). or in crystallization prepared to E.M. PubMed Scopus Google Scholar, were in the with crystallization to of The of was from the of 0.4 of as with a was used to the for a concentration of The on the that but not the of the were on of a of in a and at for the of were the was for an additional to for changes in from to were in for at to were for at the were constant for several as a function of the specific of the was then as previously E.M. PubMed Scopus Google Scholar). The concentration of latrunculin complex was to the concentration of latrunculin A, consistent with monomer sequestration The equilibrium dissociation constant is to that previously reported 0.2 μm in 0.1 mm and (2Coué M. Brenner S.L. Spector I. Korn E.D. FEBS Lett. 1987; 213: 316-318Crossref PubMed Scopus (661) Google Scholar). Latrunculin A did not significant in the of the for actin concentration to consistent with the of Spector I. T. J. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), and binding to and actin Korn E.D. J. Biol. Full Text PDF PubMed Google Scholar). The of in at implies that interactions to binding the actin-monomer-binding elongation data for actin in the of latrunculin A can be by monomer sequestration alone of μm the data that monomer sequestration is the for these data, to that latrunculin complex not in as of a of more complicated are in which the complex and in a exchange on actin was used to the interaction of latrunculin A with actin in the or of profilin by nucleotide exchange on actin Korn E.D. Biochemistry. PubMed Scopus Google Latrunculin A alone inhibited nucleotide exchange on μm as previously reported (3Ayscough K.R. Stryker J. Pokala N. Sanders M. Crews P. Drubin D.G. J. Cell Biol. 1997; 137: 399-416Crossref PubMed Scopus (644) Google Scholar). Latrunculin A also inhibited nucleotide exchange in the of profilin that latrunculin A with profilin to actin or that nucleotide exchange on actin was inhibited in the complex of latrunculin A, and of the data the possibility that binding was with data not profilin binds to actin with equilibrium dissociation of 0.1 μm these and a of binding, the data could not be by of binding of latrunculin A to actin and exchange for the of and latrunculin the an for latrunculin with the other results reported a in which profilin and latrunculin A independently to actin a to the data and a for latrunculin in the in the nucleotide exchange to the other reported is to the of the to the data was latrunculin A and profilin were to with actin, that the of latrunculin A for actin was by a of profilin was The by this of of was not in to a more binding We that the data rule out binding, but the of or no can the dissociation by of from not state polymerization exchange polymerization dissociation are for binding latrunculin A to actin thymosin β4 to actin thymosin β4 to actin with latrunculin A and profilin to actin used for of constant are in the are based on a with from not in a dissociation are for binding latrunculin A to actin thymosin β4 to actin thymosin β4 to actin with latrunculin A and profilin to actin used for of constant are in the are based on a with the nucleotide exchange profilin concentration was constant and not the exchange with no latrunculin A is not was as the to the binding, the for the exchange was with A and for A are consistent with previous Biochemistry. 1999; PubMed Scopus Google Scholar). The are to changes and and these be from by the The addition of latrunculin A to of thymosin β4 and actin actin to to a to a high complex of thymosin β4 and actin, that the complex is by latrunculin A thymosin β4 to the to this complex in the of latrunculin A results have that the of binding in this may not the for thymosin β4 and actin D. Nachmias V.T. Biochemistry. Scholar), of but changes in the of protein are of the of formation of a thymosin The data also show that thymosin β4 as to actin as thymosin β4 and that binding to actin was inhibited by latrunculin A to the as thymosin β4 of thymosin β4 from free to with The was in the of latrunculin A than in its at actin that latrunculin A inhibits binding of thymosin β4 to actin A, latrunculin A at actin concentration dissociation of thymosin β4 from actin A, if binding was if is to the equilibrium dissociation constant for binding of thymosin β4 to latrunculin then these be the to data was with and this implies by latrunculin A, with of thymosin β4 for actin latrunculin A is to of the elongation actin polymerization additional regarding the interaction of latrunculin A, thymosin β4, and actin The data for of latrunculin A and thymosin β4 were that the effects of thymosin β4 on filament those of latrunculin A, can be by a of monomer sequestration and that the binding are The observation that latrunculin A and thymosin β4 have an effect on actin polymerization is in consistent with binding by the on binding of the then the effect of a of be than the effect of alone at a concentration to the of the of the elongation data that a binding but not an binding the data for The to the data, binding with a concentration of these data not a more complicated that a to a the results are to and a more binding equilibrium also illustrate of the thymosin β4 by latrunculin A were with thymosin β4 with or without and μm latrunculin A or an of of thymosin for and changes in the of thymosin β4 binding to the The observation of a in a of thymosin β4 with actin than for thymosin β4 alone is to the formation of an β4 complex and binding of the thymosin β4 and latrunculin A on actin then the data with or without latrunculin A be binding of thymosin β4 to actin was inhibited by latrunculin A, with the for equilibrium dissociation and The data for thymosin β4 alone a of from the consistent with a of protein to an not thymosin β4 has been reported to be monomeric S. M. H.L. J. Biol. Full Text PDF PubMed Google Scholar), this of may have previously or be a result related to expression of protein or of thymosin β4 the observed in the for the data that actin, with a of thymosin β4 binding to actin at and high actin The results of for latrunculin interactions are in I. The of reported equilibrium dissociation in of the and related to binding to and and in but it is also that changes that The of DNase is inhibited by of the of DNase by of has previously been used to show that the actin-binding protein can actin from DNase K. Biochemistry. 1997; 36: PubMed Scopus Google Scholar). the we were to in the of DNase in the of to of latrunculin A not A DNase with or without latrunculin A out the possibility that latrunculin A was by an inhibitor of DNase I. The high interaction actin and DNase a of K. Biochemistry. 1997; 36: PubMed Scopus Google the of changes in DNase and A in with the conditions DNase and be predicted to the of free DNase from to that the conditions could a of this we that latrunculin A not inhibit DNase or if it the is were to latrunculin A and actin by and diffraction in a data that was for the with an of and for the resolution the was with an The to the orthorhombic space group with cell of a were consistent with the space group of for and for were for of the of the data are in The to 3.10 or at however, a data could be to examined however, in data consistent with orthorhombic space group and cell was consistent with cell of the for a with A of 1:1 in the of with if are to be in the J. Biol. PubMed Scopus Google Scholar). These are the observed for other proteins. We are the function to the observed and to for other for of latrunculin A and in cell of of in a Our current data show that in the of latrunculin A, profilin binds to actin or with latrunculin A had no effect on binding of thymosin β4 to actin is inhibited by latrunculin A. reported that latrunculin A to actin in the cleft and of actin, at a site to the nucleotide binding site (3Ayscough K.R. Stryker J. Pokala N. Sanders M. Crews P. Drubin D.G. J. Cell Biol. 1997; 137: 399-416Crossref PubMed Scopus (644) Google Scholar). We that latrunculin A inhibits nucleotide exchange on as DNase which the cleft and the of nucleotide exchange on actin B. E. H.G. Eur. J. Biochem. 1989; PubMed Scopus Google Scholar), latrunculin A may the of the cleft and resulting in of nucleotide of the binding of thymosin β4 to actin by latrunculin A not with the postulated of latrunculin A to the cleft of the data the data are consistent with which but that latrunculin A inhibits thymosin β4 of thymosin β4 binding to actin by latrunculin A has several structural Latrunculin A could bind near or at the binding site of thymosin β4, but not be to a effect that binding by thymosin Because evidence has been reported that thymosin β4 may bind to actin in an with interactions on actin at sites (5Safer D. Sosnick T.R. Elzinga M. Biochemistry. 1997; 36: 5806-5816Crossref PubMed Scopus (97) Google Scholar), a possibility is that latrunculin A sterically at site of and the of thymosin β4 in the of latrunculin A is to interactions actin and thymosin β4 at other The possibility is that latrunculin A inhibits binding by an allosteric with a binding site in the nucleotide binding cleft of actin, as previously The of thymosin β4 to actin show that thymosin β4 is more than from the nucleotide binding cleft (5Safer D. Sosnick T.R. Elzinga M. Biochemistry. 1997; 36: 5806-5816Crossref PubMed Scopus (97) Google Scholar, 8De La Cruz E.M. Ostap E.M. Brundage R.A. Reddy K.S. Sweeney H.L. Safer D. Biophys. J. 2000; 78: 2516-2527Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). these evidence that thymosin β4 could be to A. D. H. H. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) was to be an by than nucleotide to thymosin evidence the of an interaction of actin and thymosin β4 (5Safer D. Sosnick T.R. Elzinga M. Biochemistry. 1997; 36: 5806-5816Crossref PubMed Scopus (97) Google Scholar, A. D. H. H. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, E. E. T. H.G. Biochem. J. 1997; PubMed Scopus Google Scholar), regarding whether thymosin directly to of actin (5Safer D. Sosnick T.R. Elzinga M. Biochemistry. 1997; 36: 5806-5816Crossref PubMed Scopus (97) Google Scholar) or whether the effects on can be by an allosteric mechanism E. E. T. H.G. Biochem. J. 1997; PubMed Scopus Google Scholar). To if latrunculin A bind in the cleft of actin, then effects on thymosin β4 are the result of allosteric effects on latrunculin A may bind at or near of the thymosin β4 binding sites on actin and, thymosin β4, affect the of nucleotide that bind to actin may actin filament and may have functions of their these functions may be by their interaction with actin filaments or Profilin and thymosin to be for which of functions has been by the of complex functions that in be on actin binding activity. The of by profilin and the of thymosin β4 in wound and illustrate the of functions by proteins (12Niu M. Nachmias V.T. Cell Adhes. Commun. 2000; 7: 311-320Crossref PubMed Scopus (36) Google Scholar, J.D. Lawrence A.J. MacLean A.G. Leung B.P. McInnes I.B. Canas B. Pappin D.J.C. Stevenson R.D. Nat. Med. 1999; 5: 1424-1427Crossref PubMed Scopus (171) Google Scholar, M. Gunne H. Bergman A.C. Agerberth B. Bergman T. Boman A. Liden S. Jornvall H. Boman H.G. Eur. J. Biochem. 1996; 237: 86-89Crossref PubMed Scopus (195) Google Scholar, T. E. A. T. H.G. E. FEBS Lett. 1999; PubMed Scopus Google Scholar). latrunculin A, we have that a drug that binds to actin may have differential effects on actin-binding proteins. the functions of these actin-binding the effects of actin-binding may have these effects may be significant the are employed to cell biological the of addition of latrunculin A to cells in or in may be the result of other than monomer our preliminary with more than of latrunculin A suggests that may independently properties of monomer sequestration and of thymosin β4 Finally, it may be to these effects in a to the effects of thymosin β4 on thymosin β4 to be an for the effect of (13Young J.D. Lawrence A.J. MacLean A.G. Leung B.P. McInnes I.B. Canas B. Pappin D.J.C. Stevenson R.D. Nat. Med. 1999; 5: 1424-1427Crossref PubMed Scopus (171) Google Scholar). is not thymosin β4 or to the but it is that free thymosin participates in or of these latrunculin A may the function of thymosin β4 by free thymosin actin-binding may effects by a novel Because several cell as protein and their are spatially by to La Cruz E.M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), the and of these proteins by actin-binding suggests that this mechanism in other have implications with complicated Latrunculin A the of actin for thymosin β4 by of this is not a it is to be significant in living cells. a cell as a with μm thymosin β4 Safer D. Nachmias V.T. J. Cell Biol. PubMed Scopus Google Scholar), the addition of of latrunculin A result in a in the equilibrium dissociation constant for thymosin β4 from to μm that the concentration of actin in a cell is at μm by of this be to an in the of free thymosin β4 by conditions in the the of these interactions by a of which is a Biol. 2000; PubMed Scopus Google Scholar), then the concentration of free thymosin β4 latrunculin A. as actin and the complex of actin-latrunculin A to this complex a for monomer those that bind to the complex as as bind to free at steady free profilin and free thymosin be addition of latrunculin A, but of the differential effect of latrunculin A, the in profilin concentration be than that for thymosin The actin is it is the actin to to this resolution in the of other actin-binding proteins. is also in its and The of a marine natural inhibitor of actin polymerization to actin for of crystallization may have other in which of an actin in complex with other proteins or of has been to the addition of latrunculin A or marine natural may