Mechanism of Transcription Factor Recruitment by Acidic Activators

Many transcriptional activators are intrinsically unstructured yet display unique, defined conformations when bound to target proteins. Target-induced folding provides a mechanism by which activators could form specific interactions with an array of structurally unrelated target proteins. Evidence for such a binding mechanism has been reported previously in the context of the interaction between the cancer-related c-Myc protein and the TATA-binding protein, which can be modeled as a two-step process in which a rapidly forming, low affinity complex slowly converts to a more stable form, consistent with a coupled binding and folding reaction. To test the generality of the target-induced folding model, we investigated the binding of two widely studied acidic activators, Gal4 and VP16, to a set of target proteins, including TATA-binding protein and the Swi1 and Snf5 subunits of the Swi/Snf chromatin remodeling complex. Using surface plasmon resonance, we show that these activator-target combinations also display bi-phasic kinetics suggesting two distinct steps. A fast initial binding phase that is inhibited by high ionic strength is followed by a slow phase that is favored by increased temperature. In all cases, overall affinity increases with temperature and, in most cases, with increased ionic strength. These results are consistent with a general mechanism for recruitment of transcriptional components to promoters by naturally occurring acidic activators, by which the initial contact is mediated predominantly through electrostatic interactions, whereas subsequent target-induced folding of the activator results in a stable complex. Many transcriptional activators are intrinsically unstructured yet display unique, defined conformations when bound to target proteins. Target-induced folding provides a mechanism by which activators could form specific interactions with an array of structurally unrelated target proteins. Evidence for such a binding mechanism has been reported previously in the context of the interaction between the cancer-related c-Myc protein and the TATA-binding protein, which can be modeled as a two-step process in which a rapidly forming, low affinity complex slowly converts to a more stable form, consistent with a coupled binding and folding reaction. To test the generality of the target-induced folding model, we investigated the binding of two widely studied acidic activators, Gal4 and VP16, to a set of target proteins, including TATA-binding protein and the Swi1 and Snf5 subunits of the Swi/Snf chromatin remodeling complex. Using surface plasmon resonance, we show that these activator-target combinations also display bi-phasic kinetics suggesting two distinct steps. A fast initial binding phase that is inhibited by high ionic strength is followed by a slow phase that is favored by increased temperature. In all cases, overall affinity increases with temperature and, in most cases, with increased ionic strength. These results are consistent with a general mechanism for recruitment of transcriptional components to promoters by naturally occurring acidic activators, by which the initial contact is mediated predominantly through electrostatic interactions, whereas subsequent target-induced folding of the activator results in a stable complex. Transcriptional activators function by binding specific DNA sequences in the promoter regions of target genes and subsequently recruiting components of the transcriptional machinery via their activation domains (ADs). 1The abbreviations used are: AD, activation domain; ABD, activator binding domain; TBP, TATA-box binding protein; GST, glutathione S-transferase; SPR, surface plasmon resonance; RU, resonance unit(s). Activation domains have to be able to interact with multiple structurally unrelated target proteins because eukaryotic genes require successive recruitment of different co-activator proteins (1Cosma M.P. Tanaka T. Nasmyth K. Cell. 1999; 97: 299-311Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar), and different target genes utilize different repertoires of co-activators, even if they are regulated by the same activator (2Gregory P.D. Schmid A. Zavari M. Munsterkotter M. Horz W. EMBO J. 1999; 18: 6407-6414Crossref PubMed Scopus (119) Google Scholar, 3Gregory P.D. Schmid A. Zavari M. Lui L. Berger S.L. Horz W. Mol. Cell. 1998; 1: 495-505Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The mechanism by which activator proteins make specific interactions with structurally distinct target proteins is, however, poorly understood. Many activation domains are known to be intrinsically unstructured but are found in a folded conformation when bound to target proteins (for review, see Ref. 4Dyson H.J. Wright P.E. Curr. Opin. Struct. Biol. 2002; 12: 54-60Crossref PubMed Scopus (1132) Google Scholar). Whereas the structures of the initial and final states of some systems have been characterized, the determinants of complementarity, as well as the mechanism of the coupled folding and binding event, remain unknown. Support for the physiological significance of this transition has been provided by studies showing the unstructured nature of activation domains, even in the context of intact activator proteins (5Grossmann J.G. Sharff A.J. O'Hare P. Luisi B. Biochemistry. 2001; 40: 6267-6274Crossref PubMed Scopus (32) Google Scholar, 6Ayed A. Mulder F.A.A. Yi G.S. Lu Y. Kay L.E. Arrowsmith C.H. Nat. Struct. Biol. 2001; 8: 756-760Crossref PubMed Scopus (236) Google Scholar). In addition, mutations, causing substitutions of hydrophobic residues that might be expected to affect folding of the glucocorticoid receptor tau1 activation domain have been shown to cause changes in the activity of the intact receptor protein (7Almlöf T. Gustafsson J Å. Wright A.P.H. Mol. Cell. Biol. 1997; 17: 934-945Crossref PubMed Scopus (63) Google Scholar). We have previously reported that the activation domain from the cancer-related c-Myc protein interacts with the TATA-binding protein (TBP) in two steps, such that folding of the c-Myc activation domain is coupled to its interaction with the surface of TBP (8Hermann S. Berndt K.D. Wright A.P. J. Biol. Chem. 2001; 276: 40127-40132Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Coupled binding and folding could explain how activators are able to interact specifically with a large set of structurally different targets in the transcriptional machinery and recruit them to promoters. The c-Myc activation domain is unusual because it is divided in two protein segments that are interspersed with sequences that destabilize c-Myc by targeting its proteasome-mediated degradation (9Flinn E.M. Busch C.M. Wright A.P. Mol. Cell. Biol. 1998; 18: 5961-5969Crossref PubMed Scopus (104) Google Scholar, 10Flinn E.M. Wallberg A.E. Hermann S. Grant P.A. Workman J.L. Wright A.P. J. Biol. Chem. 2002; 277: 23399-23406Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Unlike the c-Myc activation domain, many activation domains consist of short protein segments that are rich in acidic amino acids. Two examples of acidic transcriptional activators that display extensive structural flexibility in their activation domains are Gal4 and VP16 (5Grossmann J.G. Sharff A.J. O'Hare P. Luisi B. Biochemistry. 2001; 40: 6267-6274Crossref PubMed Scopus (32) Google Scholar, 11Leuther K.K. Salmeron J.M. Johnston S.A. Cell. 1993; 72: 575-585Abstract Full Text PDF PubMed Scopus (105) Google Scholar, 12Uesugi M. Nyanguile O. Lu H. Levine A.J. Verdine G.L. Science. 1997; 277: 1310-1313Crossref PubMed Scopus (273) Google Scholar). Gal4 regulates the genes required for galactose metabolism in yeast (13Johnston M. Microbiol. Rev. 1987; 51: 458-476Crossref PubMed Google Scholar). The C-terminal activation domain of Gal4 also contains the interaction site for Gal80, which inhibits Gal4 activity under non-inducing conditions (14Ma J. Ptashne M. Cell. 1987; 48: 847-853Abstract Full Text PDF PubMed Scopus (604) Google Scholar, 15Ma J. Ptashne M. Cell. 1987; 50: 137-142Abstract Full Text PDF PubMed Scopus (218) Google Scholar, 16Johnston M. Flick J.S. Pexton T. Mol. Cell. Biol. 1994; 14: 3834-3841Crossref PubMed Scopus (200) Google Scholar, 17Rohde J.R. Trinh J. Sadowski I. Mol. Cell. Biol. 2000; 20: 3880-3886Crossref PubMed Scopus (57) Google Scholar). VP16 is a co-activator involved in activation of immediate early viral genes in herpes simplex-infected cells, and the C-terminal domain has been identified as an activation domain (18Triezenberg S.J. Kingsbury R.C. Mcknight S.L. Genes Dev. 1988; 2: 718-729Crossref PubMed Scopus (596) Google Scholar, 19Preston C.M. Frame M.C. Campbell M.E.M. Cell. 1988; 52: 425-434Abstract Full Text PDF PubMed Scopus (218) Google Scholar). Acidic activators have been proposed to interact with target proteins by complementary electrostatic interactions mediated via their acidic residues, without adopting defined structures (20Sigler P.B. Nature. 1988; 333: 210-212Crossref PubMed Scopus (300) Google Scholar, 21Ansari A.Z. Reece R.J. Ptashne M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13543-13548Crossref PubMed Scopus (48) Google Scholar). However, it has also been demonstrated that critical hydrophobic amino acids are essential for both target factor binding and the activating potential of acidic activation domains, whereas mutating individual acidic residues has little or no effect (7Almlöf T. Gustafsson J Å. Wright A.P.H. Mol. Cell. Biol. 1997; 17: 934-945Crossref PubMed Scopus (63) Google Scholar, 11Leuther K.K. Salmeron J.M. Johnston S.A. Cell. 1993; 72: 575-585Abstract Full Text PDF PubMed Scopus (105) Google Scholar, 22Almlöf T. Wright A.P.H. Gustafsson J. Å. J. Biol. Chem. 1995; 270: 17535-17540Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 23Cress W.D. Triezenberg S.J. Science. 1991; 251: 87-90Crossref PubMed Scopus (324) Google Scholar, 24Lin J.Y. Chen J.D. Elenbaas B. Levine A.J. Genes Dev. 1994; 8: 1235-1246Crossref PubMed Scopus (581) Google Scholar, 25Regier J.L. Shen F. Triezenberg S.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 883-887Crossref PubMed Scopus (226) Google Scholar, 26Sullivan S.M. Horn P.J. Olson V.A. Koop A.H. Niu W. Ebright R.H. Triezenberg S.J. Nucleic Acids Res. 1998; 26: 4487-4496Crossref PubMed Scopus (57) Google Scholar). These results underscore the importance of critical hydrophobic residues in the target interaction that could be coupled to the conformational changes referred to earlier. Indeed, the Gal4 and Gcn4 acidic activation domains have been shown to adopt β-sheet conformation under acidic conditions in vitro, and mutational analysis indicates that this is a structural requirement for the activation function of Gal4 (11Leuther K.K. Salmeron J.M. Johnston S.A. Cell. 1993; 72: 575-585Abstract Full Text PDF PubMed Scopus (105) Google Scholar, 27Van Hoy M. Leuther K.K. Kodadek T. Johnston S.A. Cell. 1993; 72: 587-594Abstract Full Text PDF PubMed Scopus (121) Google Scholar). However, a relatively recent report indicates that whereas conformational changes may be important for interaction with Gal80, they may not be significant for either the activation activity of Gal4 or its interaction with TBP (21Ansari A.Z. Reece R.J. Ptashne M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13543-13548Crossref PubMed Scopus (48) Google Scholar). Both the Gal4 and VP16 activation domains interact with TBP with an affinity that correlates with their potency (26Sullivan S.M. Horn P.J. Olson V.A. Koop A.H. Niu W. Ebright R.H. Triezenberg S.J. Nucleic Acids Res. 1998; 26: 4487-4496Crossref PubMed Scopus (57) Google Scholar, 28Ingles C.J. Shales M. Cress W.D. Triezenberg S.J. Greenblatt J. Nature. 1991; 351: 588-590Crossref PubMed Scopus (227) Google Scholar, 29Stringer K.F. Ingles C.J. Greenblatt J. Nature. 1990; 345: 783-786Crossref PubMed Scopus (408) Google Scholar, 30Melcher K. Johnston S.A. Mol. Cell. Biol. 1995; 15: 2839-2848Crossref PubMed Scopus (146) Google Scholar, 31Nedialkov Y.A. Triezenberg S.J. Arch. Biochem. Biophys. 2004; 425: 77-86Crossref PubMed Scopus (15) Google Scholar). We have recently shown that they can also recruit the Swi/Snf chromatin remodeling complex to activated promoters via interactions with specific segments within the Swi1 and Snf5 subunits of the complex (32Neely K.E. Hassan A.H. Wallberg A.E. Steger D.J. Cairns B.R. Wright A.P.H. Workman J.L. Mol. Cell. 1999; 4: 649-655Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 33Neely K.E. Hassan A.H. Brown C.E. Howe L. Workman J.L. Mol. Cell. Biol. 2002; 22: 1615-1625Crossref PubMed Scopus (146) Google Scholar, 34Prochasson P. Neely K.E. Hassan A.H. Li B. Workman J.L. Mol. Cell. 2003; 12: 983-990Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). These activator-interacting segments are not required for the overall integrity of the Swi/Snf complex, but when both are deleted, interaction with activators is severely reduced. This reduction correlates with a similar reduction in the activation potential of Gal4 in yeast strains containing the defective Swi/Snf complex (34Prochasson P. Neely K.E. Hassan A.H. Li B. Workman J.L. Mol. Cell. 2003; 12: 983-990Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The mechanisms by which these segments of the Swi/Snf complex interact with activator proteins have not been studied. Here we investigate whether the target-induced folding model for the mechanism of activator-target protein interactions that we previously suggested in the context of the interaction between c-Myc and TBP could be a general mechanism for the interaction of acidic transcriptional activators with a broader group of target proteins. Using surface plasmon resonance (SPR), we have focused on binding of the two highly acidic natural activators Gal4 and VP16 to the yeast target proteins TBP, Swi1, and Snf5. The results demonstrate that the target-induced folding model is applicable to a broader range of activator-target protein interactions. Plasmids and Proteins—pRSET-Snf5, which encodes amino acids 1–334 of yeast Snf5 with an N-terminal His6 tag, has been described previously (34Prochasson P. Neely K.E. Hassan A.H. Li B. Workman J.L. Mol. Cell. 2003; 12: 983-990Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) (referred to hereafter as Snf5ABD). pRSET-Swi1 encodes amino acids 329–547 of yeast Swi1 with an N-terminal His6 tag and was constructed as described for pRSET-Snf5 (34Prochasson P. Neely K.E. Hassan A.H. Li B. Workman J.L. Mol. Cell. 2003; 12: 983-990Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) (Swi1ABD). pT7yD encodes full-length yeast TBP (35Hoopes B.C. Leblanc J.F. Hawley D.K. J. Biol. Chem. 1992; 267: 11539-11547Abstract Full Text PDF PubMed Google Scholar). pGST-VP16 encodes amino acids 413–490 of VP16 (VP16AD), deletion mutant pGST-VP16Δ456 additionally lacks residues 456–490 (VP16Δ456) (18Triezenberg S.J. Kingsbury R.C. Mcknight S.L. Genes Dev. 1988; 2: 718-729Crossref PubMed Scopus (596) Google Scholar), and pGST-Gal4 encodes amino acids 769–881 of Gal4 (Gal4AD) (32Neely K.E. Hassan A.H. Wallberg A.E. Steger D.J. Cairns B.R. Wright A.P.H. Workman J.L. Mol. Cell. 1999; 4: 649-655Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 36Carrozza M.J. John S. Sil A.K. Workman J.L. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). has an N-terminal proteins in this by in by with and from in and in and with and by and by and and the was by affinity to in was used for to with was and and was and and proteins of and in in and with subsequently as described that the was by affinity was used for to with glutathione in and proteins TBP was and as described previously (8Hermann S. Berndt K.D. Wright A.P. J. Biol. Chem. 2001; 276: 40127-40132Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). proteins and subsequently proteins as by on a under of the and from The is to all resonance of of to the binding of was by with as and the activators the which of target protein was a of by two of a of the and and by of binding to the affinity tag as well as for by of was and a two-step model as previously described (8Hermann S. Berndt K.D. Wright A.P. J. Biol. Chem. 2001; 276: 40127-40132Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). of and mutant was on of the interaction was in on cells, and of was to of target protein and used for of the and interactions was on for the with the of target and and and of to of of target proteins, which followed by a of was used for The was with and to to on the described containing was as by the on different and on different by for the fast for the slow and for overall on in results by for the fast for the slow and for overall Acidic in Two used to the interaction of target proteins with activation domains that on the surface of a Using this it is to the of target proteins in the with activation domains as they the because the of target protein can be for it is to the kinetics of that have been on the The are to analysis in to the and to a binding To whether acidic activators to target proteins by a mechanism similar to that reported for the we the kinetics of binding to TBP as well as protein segments the of Swi1 and Snf5. the bi-phasic of with all target proteins. The mutant which has transcriptional activity (18Triezenberg S.J. Kingsbury R.C. Mcknight S.L. Genes Dev. 1988; 2: 718-729Crossref PubMed Scopus (596) Google Scholar, S.L. Cress W.D. Cress A. Triezenberg S.J. L. Cell. 1990; Full Text PDF PubMed Scopus Google Scholar) and severely affinity for Swi1 and Snf5 K.E. Hassan A.H. Brown C.E. Howe L. Workman J.L. Mol. Cell. Biol. 2002; 22: 1615-1625Crossref PubMed Scopus (146) Google Scholar, 34Prochasson P. Neely K.E. Hassan A.H. Li B. Workman J.L. Mol. Cell. 2003; 12: 983-990Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), is as a this mutant also a A two-step model the well and that activator-target complex is consistent with an initial fast followed by a subsequent the but the kinetics of activator binding to a of target proteins was as a function of temperature. provides an of the binding by showing for the of the target proteins with activator the of temperature activator-target combinations interact with bi-phasic kinetics under all conditions temperature increases the of the slow phase but not affect the or the fast in an increased of complex To investigate the nature of the interaction affinity for initial and subsequent as a function of temperature and to a temperature a in the slow phase for all activator-target and the of the indicates that the slow phase is The fast phase was not by in the of binding to in which the fast phase a that is to that of the slow phase from shown in in a the but the kinetics of activator binding to a of target proteins was as a function of of the binding is shown by the for the of the target proteins with activator the of shown for the effect of temperature the interaction kinetics of of the activator-target combinations bi-phasic under all the conditions studied. ionic strength the of the overall without the of the fast and slow as be expected if the initial was electrostatic in as the natural of the affinity for binding phase as a function of the demonstrate that ionic strength results in a reduction of the of the initial fast phase for all activator-target combinations to this effect on the initial fast ionic strength favored the subsequent slow of and on the of Acidic for different nature of electrostatic and hydrophobic interactions it to the effect of increased ionic strength and temperature on the overall affinity of activator-target interactions. that the overall for all the interactions with increased temperature. The of these a for The overall is also The effect of ionic strength on overall affinity is more an increased overall affinity for both TBP and with ionic strength. when binding and The interaction is similar to the with both in the of binding to the in overall affinity with increased ionic strength is However, the interaction of with TBP is to ionic strength and a to that for the interactions. Here we have the generality of the target-induced folding model, the activation domains of two naturally occurring acidic activators, Gal4 and VP16, which are used as in studies of activator In to the binding TBP, two recently defined domains within the Swi1 and Snf5 subunits of the Swi/Snf chromatin remodeling complex also studied. The target-induced folding model that we proposed previously to for the interaction between c-Myc and TBP the interaction between activator and target in two steps. the initial is and on electrostatic interactions between the proteins. the interaction is and suggesting the of protein of these conditions are by all the interactions between the acidic activators and target proteins We that the target-induced protein folding model is applicable to a range of activator-target interactions. has been suggested that activation domains of acidic activators, including VP16 and function as that recruit target proteins to promoters via low electrostatic interactions (20Sigler P.B. Nature. 1988; 333: 210-212Crossref PubMed Scopus (300) Google Scholar, 21Ansari A.Z. Reece R.J. Ptashne M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13543-13548Crossref PubMed Scopus (48) Google Scholar). results a more context for the by electrostatic in interactions between acidic activation domains and the target they to model, electrostatic interactions the initial binding but they have a in the subsequent of the final complex on hydrophobic interactions. This is consistent with that increased which increases the of the of the final complex. model is consistent with studies showing both the importance of overall and the by specific individual hydrophobic but not individual acidic residues in activation domain function (7Almlöf T. Gustafsson J Å. Wright A.P.H. Mol. Cell. Biol. 1997; 17: 934-945Crossref PubMed Scopus (63) Google Scholar, 22Almlöf T. Wright A.P.H. Gustafsson J. Å. J. Biol. Chem. 1995; 270: 17535-17540Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 23Cress W.D. Triezenberg S.J. Science. 1991; 251: 87-90Crossref PubMed Scopus (324) Google Scholar, 30Melcher K. Johnston S.A. Mol. Cell. Biol. 1995; 15: 2839-2848Crossref PubMed Scopus (146) Google Scholar). We that whereas electrostatic interactions might the of complex the affinity of interactions between acidic activators and the they recruit on hydrophobic interactions that are to be with folding of the activation The target-induced folding model that target proteins a on the of the activator-target complex, and might to see binding with some target proteins. a was for TBP, for which all changes in the same on the binding of both and and This that the activators may have similar on TBP, a that is by studies K. Johnston S.A. Mol. Cell. Biol. 1995; 15: 2839-2848Crossref PubMed Scopus (146) Google Scholar, S. R.J. M. Nature. 1994; PubMed Scopus Google Scholar). In of these a of a relatively TBP a surface in activator all the interactions studied to the two-step target-induced folding interaction model, of electrostatic and hydrophobic interactions could be for some between the different activator-target interactions. is the different of the overall for the and in to ionic strength. This correlates with affinity for TBP, K.F. Ingles C.J. Greenblatt J. Nature. 1990; 345: 783-786Crossref PubMed Scopus (408) Google Scholar, 30Melcher K. Johnston S.A. Mol. Cell. Biol. 1995; 15: 2839-2848Crossref PubMed Scopus (146) Google Scholar). Unlike increased overall affinity for both target proteins in increased ionic which that may have some electrostatic interactions to complex with the that but not TBP, can an increased overall affinity with both of these activators high ionic strength to the individual of a target on activator binding some interactions. whereas the effect of ionic strength on overall affinity to on both activator and target protein, the of the effect of temperature to on the target protein A and temperature has a effect on the activator with Swi1 it with These binding of Swi1 could be as the surface more hydrophobic in activator electrostatic interactions with However, because are multiple that both electrostatic and hydrophobic interactions could the interaction the between activator-target it is to a the nature of the interaction In results that the acidic activator proteins Gal4 and VP16 a target protein binding mechanism that is consistent with folding of the We in the of the acidic residues of such activators is to a initial contact and that on hydrophobic interactions that the of the final complex and the with which it However, as shown the of electrostatic and hydrophobic interactions to complex for activator-target The model we provides a that the previously demonstrated importance of both acidic and hydrophobic amino residues for target recruitment and the activation potential of acidic activators (11Leuther K.K. Salmeron J.M. Johnston S.A. Cell. 1993; 72: 575-585Abstract Full Text PDF PubMed Scopus (105) Google Scholar, 23Cress W.D. Triezenberg S.J. Science. 1991; 251: 87-90Crossref PubMed Scopus (324) Google Scholar, 25Regier J.L. Shen F. Triezenberg S.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 883-887Crossref PubMed Scopus (226) Google Scholar, 26Sullivan S.M. Horn P.J. Olson V.A. Koop A.H. Niu W. Ebright R.H. Triezenberg S.J. Nucleic Acids Res. 1998; 26: 4487-4496Crossref PubMed Scopus (57) Google Scholar, 30Melcher K. Johnston S.A. Mol. Cell. Biol. 1995; 15: 2839-2848Crossref PubMed Scopus (146) Google Scholar). These results that target-induced folding may be a general mechanism by which transcriptional components are to promoters by naturally occurring We for and