The Transactivation Domain within Cysteine/Histidine-rich Region 1 of CBP Comprises Two Novel Zinc-binding Modules

Abstract

cAMP-response element-binding protein-binding protein (CBP) is a transcriptional coactivator that interacts with a number of DNA-binding proteins and cofactor proteins involved in the regulation of transcription. Relatively little is known about the structure of CBP, but it has been noted that it contains three domains that are rich in cysteine and histidine (CH1, CH2, and CH3). The sequence of CH2 conforms to that of a leukemia-associated protein domain (PHD finger), and it has been postulated that this and both CH1 and CH3 may be zinc finger domains. This has not, however, been demonstrated experimentally. We have studied CH1 and show that it is composed of two novel zinc-binding modules, which we term “zinc bundles.” Each bundle contains the sequence Cys-X 4-Cys-X 8-His-X 3-Cys, and we show that a synthetic peptide comprising one zinc bundle from CH1 can fold in a zinc-dependent manner. CH3 also appears to contain two zinc bundles, one with the variant sequence Cys-X 2-Cys-X 9-His-X 3-Cys, and we demonstrate that this variant motif also undergoes Zn(II)-induced folding. CH1 acts as a transcriptional activation domain in cellular assays. We show that mutations in any of the four zinc-chelating residues in either zinc bundle of CH1 significantly impair this activity and that these mutations also interfere with certain protein-protein interactions mediated by CH1. Our results indicate that CBP is a genuine zinc-binding protein and introduce zinc bundles as novel protein interaction domains. cAMP-response element-binding protein-binding protein (CBP) is a transcriptional coactivator that interacts with a number of DNA-binding proteins and cofactor proteins involved in the regulation of transcription. Relatively little is known about the structure of CBP, but it has been noted that it contains three domains that are rich in cysteine and histidine (CH1, CH2, and CH3). The sequence of CH2 conforms to that of a leukemia-associated protein domain (PHD finger), and it has been postulated that this and both CH1 and CH3 may be zinc finger domains. This has not, however, been demonstrated experimentally. We have studied CH1 and show that it is composed of two novel zinc-binding modules, which we term “zinc bundles.” Each bundle contains the sequence Cys-X 4-Cys-X 8-His-X 3-Cys, and we show that a synthetic peptide comprising one zinc bundle from CH1 can fold in a zinc-dependent manner. CH3 also appears to contain two zinc bundles, one with the variant sequence Cys-X 2-Cys-X 9-His-X 3-Cys, and we demonstrate that this variant motif also undergoes Zn(II)-induced folding. CH1 acts as a transcriptional activation domain in cellular assays. We show that mutations in any of the four zinc-chelating residues in either zinc bundle of CH1 significantly impair this activity and that these mutations also interfere with certain protein-protein interactions mediated by CH1. Our results indicate that CBP is a genuine zinc-binding protein and introduce zinc bundles as novel protein interaction domains. cAMP-response element-binding protein-binding protein d 4-(trimethylsilyl)propionic acid 1,3-bis[tris(hydroxymethyl)methlamine]propane The regulation of transcription is coordinated primarily by sequence-specific DNA-binding proteins that recognize elements within the promoters and enhancers of their target genes. Although the mechanisms by which DNA-binding proteins activate or repress transcription are not completely understood, these proteins often work by recruiting coactivators such as cAMP-response element-binding protein-binding protein (CBP)1 and p300 (1.Chrivia J.C. Kwok R.P. Lamb N. Hagiwara M. Montminy M.R. Goodman R.H. Nature. 1993; 365: 855-859Crossref PubMed Scopus (1775) Google Scholar, 2.Eckner R. Ewen M.E. Newsome D. Gerdes M. DeCaprio J.A. Lawrence J.B. Livingston D.M. Genes Dev. 1994; 8: 869-884Crossref PubMed Scopus (932) Google Scholar, 3.Shikama N. Lyon J. La Thangue N.B. Trends Cell Biol. 1997; 7: 230-236Abstract Full Text PDF PubMed Scopus (436) Google Scholar). CBP was first isolated as a coactivator that is recruited by the phosphorylated form of cAMP-response element-binding protein (1.Chrivia J.C. Kwok R.P. Lamb N. Hagiwara M. Montminy M.R. Goodman R.H. Nature. 1993; 365: 855-859Crossref PubMed Scopus (1775) Google Scholar). It is now clear that CBP is capable of interacting with a wide array of other DNA-bound transcription factors and that it mediates or modulates the transcriptional activity of these proteins (4.Horvai A.E. Xu L. Korzus E. Brard G. Kalafus D. Mullen T.M. Rose D.W. Rosenfeld M.G. Glass C.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1074-1079Crossref PubMed Scopus (388) Google Scholar, 5.Xu L. Lavinsky R.M. Dasen J.S. Flynn S.E. McInerney E.M. Mullen T.M. Heinzel T. Szeto D. Korzus E. Kurokawa R. Aggarwal A.K. Rose D.W. Glass C.K. Rosenfeld M.G. Nature. 1997; 395: 301-306Crossref Scopus (251) Google Scholar, 6.Boyes J. Byfield P. Nakatani Y. Ogryzko V. Nature. 1998; 396: 594-598Crossref PubMed Scopus (636) Google Scholar, 7.Zhang W. Bieker J.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9855-9860Crossref PubMed Scopus (328) Google Scholar, 8.Gingras S. Simard J. Groner B. Pfitzner E. Nucleic Acids Res. 1999; 27: 2722-2729Crossref PubMed Scopus (110) Google Scholar, 9.Hung H.L. Lau J. Kim A.Y. Weiss M.J. Blobel G.A. Mol. Cell. Biol. 1999; 19: 3496-3505Crossref PubMed Scopus (221) Google Scholar). The molecular mode of action of CBP is complex. It has been shown that it possesses intrinsic histone acetylase activity and that it is able to recruit P/CAF, another acetyl transferase (10.Yang X.J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1325) Google Scholar, 11.Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1546) Google Scholar). These results suggest that CBP is involved in the remodeling of chromatin, since it has been shown that the degree of histone acetylation significantly alters chromatin structure (12.Wolffe A.P. Hayes J.J. Nucleic Acids Res. 1999; 27: 711-720Crossref PubMed Scopus (441) Google Scholar). Furthermore, CBP can interact directly with the basal transcriptional machinery (13.Nakajima T. Uchida C. Anderson S.F. Lee C.G. Hurwitz J. Parvin J.D. Montminy M. Cell. 1997; 90: 1107-1112Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar, 14.Cho H. Orphanides G. Sun X. Yang X.J. Ogryzko V. Lees E. Nakatani Y. Reinberg D. Mol. Cell. Biol. 1998; 18: 5355-5363Crossref PubMed Scopus (249) Google Scholar), underlining its central role as a transcriptional adaptor. The intracellular concentration of CBP is thought to be limiting, and it has consequently been proposed that CBP may be instrumental in coordinating different signaling pathways. Thus, for example, the inhibitory effects of nuclear hormone receptors on AP-1 activity have been attributed to the competition between hormone receptors and Jun/Fos for CBP (15.Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.C. Heyman R.A. Rose D.W. Glass C.K. Rosenfeld M.G. Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (1930) Google Scholar). CBP has consequently been termed a transcriptional cointegrator (15.Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.C. Heyman R.A. Rose D.W. Glass C.K. Rosenfeld M.G. Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (1930) Google Scholar). One of the most remarkable properties of CBP is its ability to physically associate with and modulate the activity of so many different and apparently unrelated DNA-bound transcription factors. A diagram of CBP and some of its partner proteins is shown in Fig. 1. A substantial number of these partner proteins associate with either CH1 or CH3 or both. Almost nothing is known about the molecular nature of these CH1/CH3-mediated interactions, although other interactions made by CBP, such as that made with cAMP-response element-binding protein (which does not involve the Cys/His-rich domains), have been studied in some detail (1.Chrivia J.C. Kwok R.P. Lamb N. Hagiwara M. Montminy M.R. Goodman R.H. Nature. 1993; 365: 855-859Crossref PubMed Scopus (1775) Google Scholar, 16.Kwok R.P. Lundblad J.R. Chrivia J.C. Richards J.P. Bachinger H.P. Brennan R.G. Roberts S.G. Green M.R. Goodman R.H. Nature. 1994; 370: 223-226Crossref PubMed Scopus (1288) Google Scholar, 17.Parker D. Ferreri K. Nakajima T. LaMorte V.J. Evans R. Koerber S.C. Hoeger C. Montminy M.R. Mol. Cell. Biol. 1996; 16: 694-703Crossref PubMed Scopus (349) Google Scholar). In this case the solution structure of the complex formed between the KIX domain of CBP and the cAMP-response element-binding protein KID domain has been determined (18.Radhakrishnan I. Perez-Alvarado G.C. Parker D. Dyson H.J. Montminy M.R. Wright P.E. Cell. 1997; 91: 741-752Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar). The fact that CH1 and CH3 contain a large number of residues that are capable of chelating zinc ions has led to the suggestion that these domains may constitute zinc fingers (1.Chrivia J.C. Kwok R.P. Lamb N. Hagiwara M. Montminy M.R. Goodman R.H. Nature. 1993; 365: 855-859Crossref PubMed Scopus (1775) Google Scholar, 2.Eckner R. Ewen M.E. Newsome D. Gerdes M. DeCaprio J.A. Lawrence J.B. Livingston D.M. Genes Dev. 1994; 8: 869-884Crossref PubMed Scopus (932) Google Scholar). Many classes of zinc finger domains have been described and ascribed roles in nucleic acid binding, protein recognition, or both (19.Schwabe J.W. Klug A. Nat. Struct. Biol. 1994; 1: 345-349Crossref PubMed Scopus (243) Google Scholar, 20.Mackay J.P. Crossley M. Trends Biochem. Sci. 1998; 23: 1-4Abstract Full Text PDF PubMed Scopus (390) Google Scholar). However, neither CH1 nor CH3 resemble any previously reported zinc-binding modules. In this study, we show that the CH1/CH3 regions of CBP proteins contain a highly conserved arrangement of cysteine and histidine residues. These regions can be broken down into discrete modules that each contain four putative metal-ligating residues and share the consensus sequence Cys-X 2–4-Cys-X 8–9-His-X 3-Cys. We have termed these modules “zinc bundles.” We show that chemically synthesized peptides corresponding to individual zinc bundles from both CH1 and CH3 are capable of forming stable secondary structure in the presence of Zn(II) and that this structure appears to contain a substantial degree of helical character. These results are consistent with 1H NMR studies of the first zinc bundle of CH1 (CH11); Hα secondary chemical shifts suggest that the domain is largely helical in nature. Finally, we demonstrate that the integrity of both zinc bundles in the CH1 domain of CBP is essential for its interaction with some partner proteins and for the transactivation activity of CH1 in cellular assays. Taken together, these results indicate that the zinc bundles of CBP constitute novel protein domains that mediate protein-protein interactions. Peptides were synthesized by Auspep Pty. Ltd. (Victoria, Australia). All other reagents were of analytical grade. Samples of CH11 and CH32 were prepared by dissolving each of the lyophilized peptides (as provided) in a solution containing 1 mm ZnSO4 and 0.5 mmtris-(carboxyethyl)phosphine, to a final concentration of 30–40 μm peptide. The pH was then adjusted slowly to either 5.0 (CH32) or 6.5 (CH11) using 0.01 mNaOH. Spectra were collected on a Jasco J-720 spectropolarimeter, using a 1-mm path length cell, and the temperature was controlled with a water-jacketed cell holder. Spectra were collected at 20 °C, over the wavelength range 184–260 nm, using a resolution of 0.5 nm and a bandwidth of 1 nm. Final spectra were the sum of five scans accumulated at a speed of 20 nm min−1 with a response time of 1 s. Spectra were also base line-corrected. For the thermal denaturation of CH11, the ellipticity at 195 nm was monitored over a temperature range of 5–95 °C, using a resolution of 0.2 °C, a bandwidth of 1 nm, and a temperature gradient of 1 °C min−1. Sedimentation equilibrium experiments were carried out at 20 °C using a Beckman Optima with and at of and CH11 was in a solution containing mm 0.5 mm 1 and mm so that the of CH11 was at nm. A of the was to the equilibrium experiments in to that the peptide was of this and were also were in three as scans were collected at and with that the of the was carried out using the J.J. J. Full Text PDF PubMed Scopus Google by out of the to a number of different The final were determined by a to a a The of was determined by of the from the The of CH11 was from the acid sequence using the T. J. of Scholar). The presence of a of Zn(II) was for using the for the of zinc The was from for each solution that the of was by that of using T. J. of Scholar). CH11 was in a solution containing μm 20 μm d 4-(trimethylsilyl)propionic acid and pH The pH was then slowly by of it NMR spectra were at °C on a with a and Spectra were to d at 1H NMR spectra and A. J. Scholar), nuclear A. K. Biochem. Res. 95: PubMed Scopus Google Scholar), and U. J. Scopus Google experiments were as described previously K. R. Crossley M. J.P. J. 1999; PubMed Scopus Google Scholar). were by in each and either or to was using either or as described previously R. J.A. K. in Scholar). and were in the of and are on All cell were carried out using J. T. A Scholar). were with of the hormone and 20 of residues and using the J. T. A Scholar). hormone were carried out using to the The are the of three experiments and have been to from with the In interactions between CH1 and either and were using the and of CH1 residues were in and of the in The the activation domain of the regions for or were with the and were on by at °C for were then containing mm and monitored for at °C for to We are in the molecular mechanisms which the CH1 and CH3 domains of CBP recognize such array of transcription factors a first these regions of CBP, we for domains in sequence a of CH1 from the CBP we were able to with CBP and p300 as as with proteins in and these domains are also rich in the zinc-chelating residues cysteine and but a of these residues is conserved in of the domains The conserved residues to the consensus Cys-X 4-Cys-X 8-His-X this sequence in the CH1 The CH3 domain also contains a Cys-X 4-Cys-X 8-His-X motif as as the sequence Cys-X 2-Cys-X 9-His-X These not to any zinc-binding domains of known that constitute a novel protein We termed these “zinc bundles.” In to the zinc bundles of CBP are protein peptides corresponding to one bundle from CH1 (CH11) and one from CH3 Cys-X 2-Cys-X 9-His-X were synthesized using peptide spectra of each of these peptides were a range of solution pH A and these spectra both show of largely at nm and at The of Zn(II) and in the pH in substantial in secondary structure for both as demonstrated by a of the to nm, the of a at nm, and the of a at nm A and that neither the in pH nor the of Zn(II) is to the of secondary structure not The of these spectra are consistent with the presence of a substantial of the of mm and in a of the secondary consistent with a The thermal of CH11 was also by the 195 with the temperature range 5–95 °C, was that of not These results suggest that the zinc bundle is a stable protein domain capable of of the of the that have been a number of that zinc-binding domains are capable of as protein a 20.Mackay J.P. Crossley M. Trends Biochem. Sci. 1998; 23: 1-4Abstract Full Text PDF PubMed Scopus (390) Google Scholar), we to the of Fig. from equilibrium experiments on with of these to a described by a and the from that three This the to the a solution molecular of with of and This is in with the molecular of CH11 that is some both the and the of the zinc on the of the peptide. These indicate that the CH11 domain does not in that CH11 does not in we NMR to its in A a NMR of The chemical and are consistent with the peptide in a and we were able to the K. NMR of and Nucleic Google to to in A of chemical Richards PubMed Scopus Google for the Hα of CH11 number is in B. The is a in which Hα that are from their by are a of Hα that are by are a of 1. Hα are a of of three with of are of the presence of of three or with of 1 are consistent with a of may be within these so as the of does not over any these we can that are two helical regions in CH11, residues and residues In it is that residues in a helical The of 1 between the helical regions most the CBP is a large protein that appears to activate transcription by of a number of In it contains a histone domain and it is thought that CBP may the effects of chromatin by (10.Yang X.J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1325) Google Scholar, 11.Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1546) Google Scholar). CBP also contains a number of domains that to to its ability to activate transcription. In CH1 has been shown to as a activation domain to a DNA-binding domain such as the DNA-binding We to the integrity of zinc bundles CH11 and was for the transcriptional activation of CH1. We first a CH11 and and in with the DNA-binding domain in a and then this for its ability to activate a in experiments in shown in and consistent with S. S. R.P. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, Chrivia J.C. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, E. Nakajima T. K. A. 1998; PubMed Scopus Google Scholar), the protein was a of transcription. We then prepared a of each containing a acid of a putative on CH11, we each of the four residues in the Cys-X 4-Cys-X 8-His-X motif to We also prepared proteins containing mutations in other residues within the domain and residues that have the to zinc but not within the zinc bundle consensus The results are shown in Fig. A. containing of putative residues in CH11 not activate transcription. mutations in histidine residues the bundle consensus (which are not to be as for the not significantly interfere with the activity of this was in a and of residues in the bundle consensus significantly the ability of the domain to activate mutations of other residues little These results are in that suggest that the zinc-binding residues are in and are consistent with the that each bundle zinc the motif Cys-X 4-Cys-X 8-His-X 3-Cys. these indicate that both bundles are for the transcriptional activity of CH1. Many zinc-binding modules either in or protein We carried out a CH1 and three previously reported partner R. S. C. Livingston D.M. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar), M.E. A.J. S. Y. T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar), and S. Livingston D.M. Genes Dev. 1999; PubMed Scopus Google Scholar). and are DNA-binding proteins involved in transcriptional activation J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, D. T. Cell. Full Text PDF PubMed Scopus Google Scholar). has been as a transcriptional coactivator but has also been shown to the activity of CBP in certain S. Livingston D.M. Genes Dev. 1999; PubMed Scopus Google Scholar, R.A. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). We of for to these proteins to the zinc bundle domains were for interaction with these The results are shown in In the case of and of any of the putative residues in CH11 or binding, mutations in residues not in the consensus This that the integrity of both bundles is for interaction with these In a different its ability to interact with of the with mutations that either or and These results indicate that the zinc bundles are directly involved in the interactions made by some but not proteins that to the as CH1. It is clear that the mechanisms by which CBP modulates are complex. In this study, we have the of individual domains within CBP, the regions CH1 and A sequence of proteins from a range of has a conserved motif that appears in CH1 and this motif conforms to the consensus Cys-X 4-Cys-X 8-His-X 3-Cys. CH3 contains one motif that this of cysteine and histidine residues and has to CH11, and a motif (CH32) that has the form Cys-X 2-Cys-X 9-His-X 3-Cys. The and of residues that are known to be capable of ions that these domains zinc-binding modules. A number of zinc-binding modules with different consensus and have previously been described (19.Schwabe J.W. Klug A. Nat. Struct. Biol. 1994; 1: 345-349Crossref PubMed Scopus (243) Google Scholar), but the domains within CBP CH1 and CH3 to be unrelated to previously zinc-binding domains. from both and 1H NMR however, that the secondary structure of peptides corresponding to CH11 and the variant motif CH32 is on the presence of chemical shifts for the Hα of CH11 as the chemical and suggest that the secondary structure of this domain is The fold two or three helical regions by and largely of a Zn(II) These in with the novel consensus to that these domains a protein which we have termed a zinc previously described zinc-binding modules, such as zinc are with nucleic acid binding, it is now clear that classes of zinc-binding domains are able to other These domains Cell. 1994; Full Text PDF PubMed Scopus Google Scholar), fingers A.J. Trends Biochem. Sci. 1996; Full Text PDF PubMed Scopus Google Scholar), and the fingers of proteins C. M. K. J. Crossley M. J. 1999; 18: PubMed Scopus Google Scholar), and results indicate that zinc bundles also mediate protein-protein interactions. CH1 and CH3 have been in interactions with a large array of different It has also been noted that CH1 can as a transactivation The transactivation ability of this domain is by mutations to the putative residues that the zinc bundle consensus The by which CH1 to transcription from promoters is not It is that CH1 acts in by recruiting transcriptional proteins as shown in Fig. or coactivators or with the basal transcriptional machinery at the It has been reported that one known partner protein of can protein and as a coactivator for the domain protein R.A. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). It has also been demonstrated that the as a coactivator for the of transcription factors T. H. T. Roberts Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). In the of these one that be the coactivator that mediates the transcriptional activity of CH1. Our however, suggest that a of this is not since the transcriptional activity of CH1 does not with its ability to It has been can to of CBP to and repress the response S. Livingston D.M. Genes Dev. 1999; PubMed Scopus Google Scholar), and it was shown that and to within CH1 and with each other for to It be however, that the for and are not appears to interact with the zinc bundles does not the bundle although it appears to recognize a that these domains and not A large number of different and apparently unrelated proteins CBP in the of CH1 or CH3 However, in most the of interaction have not been and it is not to the zinc bundles in CH1 or CH3 are directly involved in the partner that the of the zinc bundles have been it is that involve in some which zinc bundles are involved in each The of CBP partner proteins that interact with both CH1 and CH3 may since it is that in some these proteins recognize elements within Although the zinc bundles of CH1 and CH3 are not the individual bundles CH11 and in to the zinc bundle Cys-X 4-Cys-X 8-His-X 3-Cys. also contains the bundle consensus but little CH32 is the most in that it contains the variant zinc-binding motif Cys-X 2-Cys-X 9-His-X 3-Cys. It be to the of different proteins that recognize different bundles within Finally, it be that CBP contains such as CH2 that also contain a number of cysteine and histidine residues. The sequence of CH2 conforms to the but consensus of a leukemia-associated protein domain V. T. A. P. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google or finger R. Trends Biochem. Sci. Full Text PDF PubMed Scopus Google Scholar). These domains are often in but their and have to be It is that the CH2 domain of CBP is also a zinc-binding that is instrumental in protein-protein interactions. is and motif of and its properties also The of the of and the other zinc-binding modules within CBP and of their protein properties into these domains associate with such a array of partner We are to Blobel and to of for and of this