Interaction of the TAZ1 Domain of the CREB-Binding Protein with the Activation Domain of CITED2
2004; Elsevier BV; Volume: 279; Issue: 4 Linguagem: Inglês
10.1074/jbc.m310348200
ISSN1083-351X
AutoresRoberto N. De Guzman, Maria A. Martinez‐Yamout, H. Jane Dyson, Peter E. Wright,
Tópico(s)Hippo pathway signaling and YAP/TAZ
ResumoThe TAZ1 domain of the homologous transcriptional coactivators CREB-binding protein (CBP) and p300 forms a complex with CITED2 (CBP/p300-interacting transactivator with ED-rich tail), inhibiting the activity of the hypoxia inducible factor (HIF-1α) and thereby attenuating the cellular response to low tissue oxygen concentration. We report the NMR structure of the CBP TAZ1 domain bound to the activation domain of CIT-ED2. The structure of TAZ1, consisting of four α-helices (α1-α4) stabilized by three zinc atoms, is very similar in the CITED2 and HIF-1α complexes. The activation domain of CITED2 is unstructured when free and folds upon binding, forming a helix (termed αA) and an extended structure that wraps around TAZ1. The CITED2 αA helix packs in the TAZ1 α1/α4 interface, a site that forms weak interactions with the poorly defined aminoterminal α-helix of HIF-1α. CITED2 and HIF-1α both contain a four residue motif, LP(E/Q)L, which binds in the TAZ1 α1/α2/α3 junction in each complex. The carboxyl-terminal region of CITED2 forms an extended structure with hydrophobic contacts in the TAZ1 α1/α3 interface in the site occupied by the HIF-1α αB helix. CITED2 does not bind at all to the TAZ1 site occupied by the HIF-1α carboxyl-terminal helix. The HIF-1α and CITED2 domains utilize partly overlapping surfaces of TAZ1 to achieve high affinity binding and to compete effectively with each other for interaction with CBP/p300; CITED2 and HIF-1α use these binding sites differently to maintain similar binding affinities in order to displace each other in a feedback loop during the hypoxic response. The TAZ1 domain of the homologous transcriptional coactivators CREB-binding protein (CBP) and p300 forms a complex with CITED2 (CBP/p300-interacting transactivator with ED-rich tail), inhibiting the activity of the hypoxia inducible factor (HIF-1α) and thereby attenuating the cellular response to low tissue oxygen concentration. We report the NMR structure of the CBP TAZ1 domain bound to the activation domain of CIT-ED2. The structure of TAZ1, consisting of four α-helices (α1-α4) stabilized by three zinc atoms, is very similar in the CITED2 and HIF-1α complexes. The activation domain of CITED2 is unstructured when free and folds upon binding, forming a helix (termed αA) and an extended structure that wraps around TAZ1. The CITED2 αA helix packs in the TAZ1 α1/α4 interface, a site that forms weak interactions with the poorly defined aminoterminal α-helix of HIF-1α. CITED2 and HIF-1α both contain a four residue motif, LP(E/Q)L, which binds in the TAZ1 α1/α2/α3 junction in each complex. The carboxyl-terminal region of CITED2 forms an extended structure with hydrophobic contacts in the TAZ1 α1/α3 interface in the site occupied by the HIF-1α αB helix. CITED2 does not bind at all to the TAZ1 site occupied by the HIF-1α carboxyl-terminal helix. The HIF-1α and CITED2 domains utilize partly overlapping surfaces of TAZ1 to achieve high affinity binding and to compete effectively with each other for interaction with CBP/p300; CITED2 and HIF-1α use these binding sites differently to maintain similar binding affinities in order to displace each other in a feedback loop during the hypoxic response. The cellular response to hypoxia in mammals involves a complex interplay of a number of transcriptional molecules. The primary response to hypoxia involves the heterodimeric transcription factor called hypoxia-inducible factor 1 (HIF-1), 1The abbreviations used are: HIF-1, hypoxia-inducible factor 1; CREB, cAMP-response element-binding protein; CBP, CREB-binding protein; CITED2, CBP/p300-interacting transactivator with ED-rich tail; CAD, carboxyl-terminal activation domain; TAZ1, transcription adaptor zinc-binding domain of CBP; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; HSQC, heteronuclear single quantum coherence; ITC, isothermal titration calorimetry. a major factor in the pathology of cancer, heart disease, and stroke (1.Semenza G.L. Genes Dev. 2000; 14: 1983-1991PubMed Google Scholar, 2.Semenza G.L. Trends Mol. Med. 2001; 7: 345-350Abstract Full Text Full Text PDF PubMed Scopus (800) Google Scholar, 3.Pugh C.W. Ratcliffe P.J. Nat. Med. 2003; 9: 677-684Crossref PubMed Scopus (1998) Google Scholar). Under conditions of oxygen deprivation, the HIF-1α subunit recruits the general transcriptional coactivators CBP or p300 to direct the expression of genes necessary for survival (4.Arany Z. Huang L.E. Eckner R. Bhattacharya S. Jiang C. Goldberg M.A. Bunn H.F. Livingston D.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12969-12973Crossref PubMed Scopus (636) Google Scholar). Deletion of HIF-1α in mice results in neural and cardiovascular developmental arrest and embryonic death (5.Iyer N.V. Kotch L.E. Agani F. Leung S.W. Laughner E. Wenger R.H. Gassmann M. Gearhart J.D. Lawler A.M. Yu A.Y. Semenza G.L. Genes Dev. 1998; 12: 149-162Crossref PubMed Scopus (2050) Google Scholar). Consistent with the central role of the HIF-1 system in the maintenance of cellular oxygen homeostasis, a variety of mechanisms, including oxidative degradation and transcriptional inactivation, closely regulate the activity of HIF-1α. Under normoxic conditions, low HIF-1α protein levels are maintained by a family of specific prolyl hydroxylases (PHD1-3) that target HIF-1α for ubiquitination and proteasome-mediated degradation. These hydroxylases modify two prolines within conserved LXXLAP motifs in HIF-1α to enhance interaction with the von Hippel-Lindau E3 ubiquitin ligase complex (6.Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4451) Google Scholar, 7.Ivan M. Kondo K. Yang H.F. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin Jr, W.G. Science. 2001; 292: 464-468Crossref PubMed Scopus (3886) Google Scholar, 8.Yu F. White S.B. Zhao Q. Lee F.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9630-9635Crossref PubMed Scopus (647) Google Scholar, 9.Bruick R.K. McKnight S.L. Science. 2001; 294: 1337-1340Crossref PubMed Scopus (2114) Google Scholar). The transcriptional activity of HIF-1α is directly dependent on the interaction of its carboxyl-terminal activation domain (CAD) with the TAZ1 domain, also referred to as cysteine/histidine-rich domain 1 (C/H1), of CBP/p300 (4.Arany Z. Huang L.E. Eckner R. Bhattacharya S. Jiang C. Goldberg M.A. Bunn H.F. Livingston D.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12969-12973Crossref PubMed Scopus (636) Google Scholar, 10.Kallio P.J. Okamoto K. O'Brien S. Carrero P. Makino Y. Tanaka H. Poellinger L. EMBO J. 1998; 17: 6573-6586Crossref PubMed Google Scholar). FIH-1 (factor inhibiting HIF-1) (11.Mahon P.C. Hirota K. Semenza G.L. Genes Dev. 2001; 15: 2675-2686Crossref PubMed Scopus (1127) Google Scholar), a second class of oxygen dependent HIF-1α specific regulator, hydroxylates an asparagine residue within the HIF-1α CAD to attenuate the interaction with the coactivator CBP/p300 (12.Lando D. Peet D.J. Whelan D.A. Gorman J.J. Whitelaw M.L. Science. 2002; 295: 858-861Crossref PubMed Scopus (1277) Google Scholar). Recent NMR structures from our laboratory and others highlight the exquisite specificity involved in the formation of the HIF-1α CAD·TAZ1 complex and the nature of the molecular interactions that define the hypoxic switch (13.Dames S.A. Martinez-Yamout M. De Guzman R.N. Dyson H.J. Wright P.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5271-5276Crossref PubMed Scopus (340) Google Scholar, 14.Freedman S.J. Sun Z.Y. Poy F. Kung A.L. Livingston D.M. Wagner G. Eck M.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5367-5372Crossref PubMed Scopus (363) Google Scholar). In the complex, the CBP TAZ1 domain is composed of four α-helices stabilized by three zinc atoms in a fashion similar to that of the isolated CBP TAZ2 domain (15.De Guzman R.N. Liu H.Y. Martinez-Yamout M. Dyson H.J. Wright P.E. J. Mol. Biol. 2000; 303: 243-253Crossref PubMed Scopus (99) Google Scholar). Upon binding to CBP, the HIF-1α CAD undergoes a folding transition to form three helices, namely a poorly defined aminoterminal helix (αA) that interacts weakly with TAZ1 and two tight-binding helices, αB and αC, all of which wrap around the TAZ1 domain in highly intimate fashion. The TAZ1 domain thus acts as a scaffold, providing extensive hydrophobic surface grooves to accommodate the transcription factor activation domain. Asn-803, the target of FIH-1, is deeply buried in a hydrophobic pocket in the CAD·TAZ1 complex and participates in an extended network of hydrogen bonds that stabilize the complex. In hypoxia, a third HIF-1α regulatory mechanism is proposed to be mediated by the protein CITED2 (CBP/p300 Interacting Transactivator with glutamate (E) and aspartate (D) rich tail, also termed MRG-1 and p35srj) (16.Bhattacharya S. Michels C.L. Leung M.K. Arany Z.P. Kung A.L. Livingston D.M. Genes Dev. 1999; 13: 64-75Crossref PubMed Scopus (327) Google Scholar). CITED2 is a ubiquitously expressed nuclear protein that competes with HIF-1α for binding to the CBP/p300 TAZ1 domain. In mice, deletion of CITED2 results in embryonic lethality due to severe cardiac and neural tube defects (17.Yin Z. Haynie J. Yang X. Han B. Kiatchoosakun S. Restivo J. Yuan S. Prabhakar N.R. Herrup K. Conlon R.A. Hoit B.D. Watanabe M. Yang Y.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10488-10493Crossref PubMed Scopus (168) Google Scholar). CITED2 knockouts also exhibit increased expression of vascular endothelial growth factor (VEGF) and other hypoxia-responsive gene products. The severe phenotype of CITED2-/- mice is consistent with the additional function of CITED2 as coactivator for AP-2 transcription factors, which are necessary for neural development (18.Bamforth S.D. Braganca J. Eloranta J.J. Murdoch J.N. Marques F.I. Kranc K.R. Farza H. Henderson D.J. Hurst H.C. Bhattacharya S. Nat. Genet. 2001; 29: 469-474Crossref PubMed Scopus (264) Google Scholar, 19.Braganca J. Eloranta J.J. Bamforth S.D. Ibbitt J.C. Hurst H.C. Bhattacharya S. J. Biol. Chem. 2003; 278: 16021Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Expression of CITED2 is directly induced by HIF-1, implicating CITED2 as a negative feedback regulator of HIF-1α and a fundamental component of the cellular mechanism for attenuation of the hypoxic response. Previous domain mapping and mutagenesis experiments have defined the region of interaction with CBP/p300 TAZ1 to be the carboxyl-terminal activation domain of CITED2 (16.Bhattacharya S. Michels C.L. Leung M.K. Arany Z.P. Kung A.L. Livingston D.M. Genes Dev. 1999; 13: 64-75Crossref PubMed Scopus (327) Google Scholar). As is typical of transcription factor activation domains, the CIT-ED2 CAD contains a relatively large number of residues with acidic and bulky hydrophobic side chains but has no obvious sequence homology with the HIF-1α CAD (Fig. 1). To determine the structural basis of HIF-1α inhibition by CITED2, we have determined the solution structure of the CBP TAZ1 domain bound to the activation domain of CITED2. The structure of TAZ1·CITED2 reveals the commonalities, as well as major differences, between the binding of CITED2 and HIF-1α to TAZ1. As this work was nearing completion, the solution structure of a homologous TAZ1 domain from the p300 coactivator, also bound to human CITED2, was reported (20.Freedman S.J. Sun Z.Y. Kung A.L. France D.S. Wagner G. Eck M.J. Nat. Struct. Biol. 2003; 10: 504-512Crossref PubMed Scopus (167) Google Scholar). Aside from the structural differences imposed by the sequence differences between the TAZ1 domains of CBP and p300, there are significant differences in the lengths of the domain constructs used in the two studies. The structures of the two complexes, TAZ1·HIF-1α and TAZ1·CITED2, together with NMR relaxation data that probe the backbone flexibility, suggest a mechanism of how CITED2 and HIF-1α displace one another from TAZ1. Protein Expression and Purification—A coexpression plasmid (21.Demarest S.J. Martinez-Yamout M. Chung J. Chen H. Xu W. Dyson H.J. Evans R.M. Wright P.E. Nature. 2002; 415: 549-553Crossref PubMed Scopus (354) Google Scholar) containing mouse CBP TAZ1 (residues 340-439) and human CITED2 (204-269, 220-269) was transformed into Escherichia coli BL21(DE3) (dnaY) and grown in 6-liter minimal medium containing 0.1 mm ZnCl2 and, for isotopic labeling, 15NH4Cl (1 g/liter) and/or 13C-glucose (2 g/liter). Cells were grown at 37 °C, induced with 1 mm isopropyl-β-d-thiogalactopyranoside at an OD600 of ∼0.8, and left at 15 °C for over-night expression. Cells were harvested and resuspended in 150 ml of buffer (10 mm Tris, pH 8, 50 μm ZnSO4, 20 mm dithiothreitol, and 6 m urea) and sonicated, and the cell lysate (160 ml) was diluted to 4 m urea and loaded unto a 10-ml SP-Sepharose attached to a 10 ml Q-Sepharose columns (Amersham Biosciences). TAZ1 was eluted from the SP-Sepharose column with a linear gradient of buffer (10 mm Tris-HCl, pH 8, 50 μm ZnSO4, and 1 m NaCl, 4 m urea) and further purified through a C4 and then through a C18 reverse phase high performance liquid chromatography (HPLC) column. CITED2 was eluted from the Q-Sepharose column and further purified through C4 and a C18 reverse phase HPLC columns in a similar manner. TAZ1 and HIF-1α CAD were prepared as described previously (13.Dames S.A. Martinez-Yamout M. De Guzman R.N. Dyson H.J. Wright P.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5271-5276Crossref PubMed Scopus (340) Google Scholar). Final NMR buffer was 10 mm Tris-d11, 10% 2H2O, and 2 mm NaN3, pH 6.8. NMR Sample Preparation—The lyophilized CITED2 was dissolved in 10 mm deuterated Tris-d11, adjusted to pH 7, and dialyzed in the same NMR buffer as TAZ1. To prepare the complex, CITED2 and TAZ1 were combined in 20-30 μl increments to give a final volume of 600 μl and a final molar ratio of 1:1.3 of labeled to unlabeled partner. Upon mixing TAZ1 and CITED2, precipitates formed in the NMR tube. The effective complex concentration was then estimated by comparing the NMR signal to a known sample of free protein. NMR samples contained ∼400-500 μm complex. NMR samples in 100%2H2O were prepared by lyophilizing the dialyzed TAZ1 and CITED2 and re-dissolving the proteins in 2H2O before mixing to form the complexes. Circular Dichroism Spectroscopy—CD spectra were collected at 25 °C using an Aviv model 202 CD spectrometer and a 0.2 cm cell. Samples contained 7 μm protein in 3 mm Tris-HCl buffer (pH 7.5). Isothermal Titration Calorimetry—ITC data were collected at 25 °C by using a Microcal MCS Titration calorimeter. Purified protein samples were dialyzed into ITC buffer (20 mm Tris, pH 6.8, containing 50 mm NaCl and 2 mm dithiothreitol) and centrifuged to remove aggregates. Protein concentrations were measured by a standard bicinchoninic acid protein assay. Titration data were fitted to a one-site model by using the Microcal data analysis software ORIGIN 2.3 (Microcal Software, Northampton, MA) and were not adjusted for buffer effects. ITC data were acquired at least three times using different batches of samples on different days to obtain an error estimate for the Kd values reported in Table I.Table IDissociation constants for the binding of TAZ1 with different HIF-1α and CITED2 constructs obtained by isothermal titration calorimetryTAZ1 binding partnerKdnmCITED2 (residues 220–269)13 ± 10HIF-1α (residues 776–826)10 ± 5HIF-1α (residues 790–826)47 ± 11 Open table in a new tab NMR Spectroscopy—NMR spectra were acquired at 25 °C on Bruker DRX600, DRX800, and AVANCE 900 MHz spectrometers, processed using NMRPipe (22.Delaglio F. Grzesiek S. Vuister G.W. Guang Z. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11561) Google Scholar), and analyzed using NMRView (23.Johnson B.A. Blevins R.A. J. Biomol. NMR. 1994; 4: 604-613Crossref Scopus (2678) Google Scholar). Resonance assignments for TAZ1 and CITED2 were obtained from heteronuclear (1H, 15N, 13C) and a multidimensional NMR experiment used previously in the TAZ1·HIF-1α complex (13.Dames S.A. Martinez-Yamout M. De Guzman R.N. Dyson H.J. Wright P.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5271-5276Crossref PubMed Scopus (340) Google Scholar). Distance restraints were obtained from three-dimensional 15N-edited NOESY-HSQC (τm, 120 ms) and three-dimensional 13C-edited NOESY-HSQC (τm, 120 ms) acquired for samples in 100%2H2O and 10%2H2O. Intermolecular distance restraints were obtained from 13C-edited/12C-filtered NOESY-HSQC (τm, 120 ms). The [1H]-15N heteronuclear steady-state NOE experiment for 15N-labeled CITED2 bound to unlabeled TAZ1 was acquired with water flipback for water suppression (24.Grzesiek S. Bax A. J. Am. Chem. Soc. 1993; 115: 12593-12594Crossref Scopus (1014) Google Scholar). Structure Calculations and Analysis—Interproton upper bound distance restraints were derived from cross peak volumes in the NOESY experiments and were assigned upper bounds of 2.7, 3.5, 4.5, and 5.5 Å and lower bounds of 1.8 Å. Torsion angle restraints for φ, Ψ, and χ1 were obtained from the chemical shift index and intraresidue NOE connectivities (25.Bax A. Vuister G.W. Grzesiek S. Delaglio F. Wang A.C. Tschudin R. Zhu G. Methods Enzymol. 1994; 239: 79-105Crossref PubMed Scopus (381) Google Scholar, 26.Wishart D.S. Nip A.M. Biochem. Cell Biol. 1998; 76: 153-163Crossref PubMed Scopus (177) Google Scholar, 27.Wüthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986Crossref Google Scholar). Initial structural models were calculated using 883 intraresidue and 126 intermolecular distance restraints obtained by manual assignment and 195 torsion angle restraints. Additional distance restraints were generated with the program SANE (28.Duggan B.M. Legge G.B. Dyson H.J. Wright P.E. J. Biomol. NMR. 2001; 19: 321-329Crossref PubMed Scopus (113) Google Scholar) and added during the structure calculation. The total numbers of distance and torsion angle restraints used are detailed in Table I. An initial set of 400 simulated annealing structures was generated using DYANA (29.Güntert P. Mumenthaler C. Wüthrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2555) Google Scholar), followed by variable target function minimization. The 100 DYANA structures with the lowest target function were further refined by restrained molecular dynamics in AMBER7 using the ff99 force field (30.Case, D. A., Pearlman, D. A., Caldwell, J. W., Cheatham, T. E., III, Wang, J., Ross, W. S., Simmerling, C. L., Darden, T. A., Merz, K. M., Stanton, R. V., Cheng, A. L., Vincent, J. J., Crowley, M., Tsui, V., Gohlke, H., Radmer, R. J., Duan, Y., Pitera, J., Massova, I., Seibel, G. L., Singh, U. C., Weiner, P. K., and Kollman, P. A. (2002) AMBER 7, University of California, San FranciscoGoogle Scholar). When the distance and dihedral angle violations dropped below 100 kcal/mol, refinement was performed using the generalized Born (GB) potential in AMBER7 to account for the effects of solvent (31.Tsui V. Case D.A. J. Am. Chem. Soc. 2000; 122: 2489-2498Crossref Scopus (395) Google Scholar). Twenty structures with the lowest combined distance and angle violations were sorted by AMBER energy and selected for analysis. Backbone analysis was performed using PROCHECK (32.Laskowski R.A. Rullmann J.A.C. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4427) Google Scholar), and graphics images were prepared using MOLMOL (33.Koradi R. Billeter M. Wüthrich K. J. Mol. Graphics. 1996; 14: 51-55Crossref PubMed Scopus (6489) Google Scholar). The free CITED2 activation domain, residues 220-269, is unstructured in solution as indicated by the absence of secondary structural elements in the circular dichroism spectra (Fig. 2A) and the narrow range of amide proton chemical shifts in the two-dimensional 1H-15N HSQC spectrum of 15N-labeled CITED2 (Fig. 2B). The CD spectrum of the complex (Fig. 2A) shows the formation of additional structure compared with the individual proteins, as indicated by a comparison of the spectrum of the complex with the sum of the spectra of the two components. Upon binding to TAZ1, there is increased dispersion of the CITED2 amide proton chemical shifts, indicating structure formation (Fig. 2B). A longer CITED2 construct, residues 204-269, binds to TAZ1 in a similar manner, as manifested by changes in the backbone amide peaks of 15N-labeled TAZ1 (data not shown), but has a tendency to precipitate upon complex formation. The shorter CITED2 construct (residues 220-269) showed high affinity to TAZ1 (Kd = 13 nm, Table I) with less tendency to precipitate upon binding to TAZ1 and was therefore used for structure determination. Binding is in slow exchange on the chemical shift time scale, as evidenced by the appearance of new CITED2 peaks in the bound form and the disappearance of free CITED2 peaks when 15N-labeled CIT-ED2 is added to unlabeled TAZ1. The changes in chemical shifts for the CITED2 protein upon binding indicate the formation of a helical structure in the N terminus, with an extended structure in the center of the sequence. This is in contrast to the observation for HIF-1α of the formation of helical structure in several locations throughout the sequence. Measurement of the heteronuclear [1H]-15N NOE for CITED2 in the complex (Fig. 3) shows that the carboxyl-terminal 10 residues are relatively mobile. As observed for the binding of HIF-1α to TAZ1 (13.Dames S.A. Martinez-Yamout M. De Guzman R.N. Dyson H.J. Wright P.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5271-5276Crossref PubMed Scopus (340) Google Scholar), a number of significant shifts in HSQC cross-peak positions were observed when unlabeled CITED2 was added to 15N-labeled TAZ1. Chemical shift mapping (Fig. 4) indicated that different regions of TAZ1 are perturbed by HIF-1α and CITED2, suggesting that the complexed structures of the HIF-1α and CITED2 are likely to be different.Fig. 4Chemical shift mapping of 15N-labeled TAZ1 titrated with unlabeled HIF-1α (top) and CITED2 (bottom). Chemical shift changes were calculated as ΔδHN,N = ¼√((δHN/2)2 + (δN/5)2). The four α-helices of TAZ1 are indicated above the graph, and the TAZ1 residues that interact with the CITED2 αA are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Table I shows the binding constants of TAZ1 with CITED2 and two HIF-1α constructs (residues 776-826 and 790-826) obtained by ITC. The two HIF-1α constructs were used for binding studies to correlate the measured affinities with structural results. The longer HIF-1α (residues 776-826) was used in the CBP complex (20.Freedman S.J. Sun Z.Y. Kung A.L. France D.S. Wagner G. Eck M.J. Nat. Struct. Biol. 2003; 10: 504-512Crossref PubMed Scopus (167) Google Scholar), and the shorter HIF-1α (residues 790-826) was used in the p300 complex (13.Dames S.A. Martinez-Yamout M. De Guzman R.N. Dyson H.J. Wright P.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5271-5276Crossref PubMed Scopus (340) Google Scholar). The structure of the CBP TAZ1·HIF-1α (residues 776-826) showed intermolecular interactions involving the amino-terminal extension of HIF-1α, which likely contribute to the tighter binding affinity of the longer HIF-1α (residues 776-826) compared with that of the shorter HIF-1α (residues 790-826). ITC results indicate that CITED2 binds to TAZ1 with comparable affinity to the longer HIF-1α construct, but it binds approximately four times more tightly than the shorter HIF-1α construct. Three-dimensional structures for the TAZ1·CITED2 complex were calculated using the program DYANA (29.Güntert P. Mumenthaler C. Wüthrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2555) Google Scholar) and refined by molecular dynamics and simulated annealing with the program AMBER (30.Case, D. A., Pearlman, D. A., Caldwell, J. W., Cheatham, T. E., III, Wang, J., Ross, W. S., Simmerling, C. L., Darden, T. A., Merz, K. M., Stanton, R. V., Cheng, A. L., Vincent, J. J., Crowley, M., Tsui, V., Gohlke, H., Radmer, R. J., Duan, Y., Pitera, J., Massova, I., Seibel, G. L., Singh, U. C., Weiner, P. K., and Kollman, P. A. (2002) AMBER 7, University of California, San FranciscoGoogle Scholar), using intramolecular and intermolecular NOEs and dihedral angle constraints as listed in Table II. A superposition of the 20 lowest energy structures of the TAZ1·CITED2 complex is shown in Fig. 5A, and a single member of the ensemble is shown in Fig. 5B. As was observed in the HIF-1α complex and in free TAZ1, 2R. DeGuzman, J. Wojciak, M. Martinez-Yamout, H. J. Dyson, and P. E. Wright, unpublished data. TAZ1 adopts a compact fold containing predominantly helical secondary structure stabilized by three zinc atoms. Upon complex formation, the activation domain of CITED2 wraps around TAZ1, and its folded structure is maintained by numerous intermolecular hydrophobic contacts. The structure of CITED2 is defined primarily by its association with TAZ1, as indicated by the much higher number of intermolecular NOEs (299; Table II) compared with only 13 intramolecular long-range NOEs (i - j > 4) for CIT-ED2 in the complex.Table IIExperimental restraints and structural statisticsNMR restraintsTAZ1CITED2Total distance restraints1052648Intraresidue457343Sequential250151Medium range (2 ≤ i – j ≥ 4)210141Long range (i – j > 4)13513Intermolecular distance restraints299Total dihedral angle restraints8787φ7540ψ5821χ15026Ensemble statistics (20 structures)Violation analysisMaximum distance violation (Å)0.20Maximum dihedral angle violation5.8EnergiesMean restraint violation energy (kcal mol–1)17mean AMBER energy (kcal mol–1)–6402Root mean square deviation from the average structureTAZ1-(345–435), CITED2-(222–257)Backbone atoms (N,Cα,C′) (Å)0.70 ± 0.11All heavy atoms (Å)1.16 ± 0.26Ordered residues onlyBackbone atoms (N,Cα,C′) (Å)0.49 ± 0.11All heavy atoms (Å)0.85 ± 0.12Deviation from idealized geometryBond lengths (Å)0.011 ± 0.01Bond angles (Å)2.4 ± 0.1Ramachandran plotMost favorable regions %81.6Additionally allowed regions %17.5Generously allowed regions %0.9Disallowed regions %0 Open table in a new tab The structure of the TAZ1 domain in the complex with CIT-ED2 is very similar to that in the HIF-1α complex (13.Dames S.A. Martinez-Yamout M. De Guzman R.N. Dyson H.J. Wright P.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5271-5276Crossref PubMed Scopus (340) Google Scholar, 14.Freedman S.J. Sun Z.Y. Poy F. Kung A.L. Livingston D.M. Wagner G. Eck M.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5367-5372Crossref PubMed Scopus (363) Google Scholar), consisting of four helices, namely α1 (Pro-347 to Ala-372), α2 (His-383 to His-396), α3 (Ala-406 to Cys-421), and α4 (Cys-429 to Asn-438), and three HCCC-type zinc-binding motifs, i.e. Zn1 (His-362, Cys-366, Cys-379, and Cys-384), Zn2 (His-393, Cys-397, Cys-403, and Cys-408), and Zn3 (His-417, Cys-421, Cys-426, and Cys-429). Each of the zinc-binding sites contains one histidine and three cysteine residues distributed across the ends of two helices and joined by a flexible loop. The two helices that form each zinc-binding site are packed at an angle against each other. The four helices are then packed against each other via hydrophobic interactions to enclose a hydrophobic core in the center of the molecule. Intermolecular interactions at the end of TAZ1 α4 and CITED2 (between residues Ala-435 and Lys-438 of TAZ1 and Val-227 of CITED2) stabilize the end of α4, making it significantly longer than the α4 helix in the TAZ1·HIF-1α (5 residues) (13.Dames S.A. Martinez-Yamout M. De Guzman R.N. Dyson H.J. Wright P.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5271-5276Crossref PubMed Scopus (340) Google Scholar) and p300 TAZ1·CITED2 complexes (5 residues) (20.Freedman S.J. Sun Z.Y. Kung A.L. France D.S. Wagner G. Eck M.J. Nat. Struct. Biol. 2003; 10: 504-512Crossref PubMed Scopus (167) Google Scholar). In the present complex, α4 forms a regular α-helix from Cys-429 to Ala-435 (7 residues) that becomes the 310-helix from Ala-435 to Lys-438. The structured portion of CITED2 in the complex begins at Phe-222, forming an extended structure with hydrophobic contacts with TAZ1 α1 and α4 (Fig. 6A). The aromatic ring of Phe-222 binds in a hydrophobic pocket formed by the TAZ1 α1 residues Leu-361, His-364, and Lys-365, whereas Ile-223 fits in the hydrophobic interface formed by α1 (Leu-361 and Lys-365) and α4 (Leu-432). The polar side chains of the highly conserved Asp-224 and Glu-226 residues are directed away from the interface and are close to the Lys-365, Lys-437, and Arg-438 of TAZ1. A well defined helix, αA, from Glu-225 to Met-235, fits in a groove formed by the TAZ1 α1/α4 interface and is stabilized by an extensive network of intermolecular hydrophobic contacts (Fig. 6A). Highly conserved nonpolar residues on αA are in direct hydrophobic contact with TAZ1 α1 and α4. Val-227 interacts with α4 residues Ala-435, Ser-436, and Lys-438, whereas Leu-228 is in a hydrophobic pocket formed by α1 residues Val-358, Leu-361, His-362, and Lys-365 and α4 residue Leu-432. The hydrophobic side chain of Met-229 fits in a cleft formed by the aromatic ring of His-362 and the methyl groups of Leu-381. The invariant Leu-231 interacts with α1 (Val-358) and α4 (Pro-431, Leu-432, and Ala-435) residues. At the end of αA, Val-232 interacts mainly with Val-358 and Leu-381, Ile-233 interacts with Leu-381, and Met-235 interacts with α1 residues Lys-351, Gln-354, and Val-358. The polar residues Glu-225, Glu-226, Ser-230, and Glu-234 are all located on the solvent-exposed face of αA The axis of CITED2 helix
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