doi:10.1016/j.jmb.2004.01.038 J. Mol. Biol. (2004) 337, 521–534 Solution Structure of the KIX Domain of CBP Bound to the Transactivation Domain of c-Myb Tsaffrir Zor, Roberto N. De Guzman, H. Jane Dyson and Peter E. Wright* Department of Molecular The hematopoietic transcription factor c-Myb activates transcription of Biology and the Skaggs target genes through direct interactions with the KIX domain of the Institute for Chemical Biology co-activator CBP. The solution structure of the KIX domain in complex The Scripps Research Institute with the activation domain of c-Myb reveals a helical structure very simi- 10550 N. Torrey Pines Road, La lar to that adopted by KIX in complex with the phosphorylated kinase Jolla, CA 92037, USA inducible domain (pKID) of CREB. While pKID contains two helices, aA and aB, which interact with KIX, the structure of bound c-Myb reveals a single bent amphipathic helix that binds in the same hydrophobic groove as the aB helix of pKID. The affinity of c-Myb for KIX is lower than that of pKID, and relies more heavily on optimal interactions of the single helix of c-Myb with residues in the hydrophobic groove. In particular, a deep hydrophobic pocket in KIX accounts for more than half the inter- actions with c-Myb observed by NMR. A bend in the a-helix of c-Myb enables a critical leucine side-chain to penetrate into this pocket more deeply than the equivalent leucine residue of pKID. The components that mediate the higher affinity of pKID for KIX, i.e. the phosphate group and the aA helix, are absent from c-Myb. Results from isothermal titration calorimetry, together with the structural data, point to a key difference between the two complexes in optimal pH for binding, as a result of differential pH-dependent interactions with histidine residues of KIX. These results explain the structural and thermodynamic basis for the observed constitutive versus inducible activation properties of c-Myb and CREB. q 2004 Elsevier Ltd. All rights reserved. Keywords: CREB-binding protein; transcriptional activation; constitutive *Corresponding author activation; LXXLL motif Introduction general RNA polymerase II complex that binds the promoter.1 The key role of CBP and its homolog Transcriptional activation in eukaryotes involves p300 in transcription regulation is underscored by interactions between DNA-bound activators, the findings that mutations in the CBP gene have co-activators and components of the basal been described in various types of cancer and in transcription complex. The general co-activator the Rubinstein–Taybi syndrome, a haplo-insuffi- CREB-binding protein (CBP) functions as a bridge ciency disorder characterized by skeletal abnorm- between a number of transcription factors that alities, growth retardation and high incidence of bind specific enhancer DNA elements and the tumors.2 The first interaction to be described was between CBP and the kinase-inducible activation domain Present address: T. Zor, Genomics Institute of the (KID) of the cAMP-regulated transcription factor Novartis Foundation, La Jolla, CA 92121, USA. CREB. Phosphorylation of KID at Ser133 was Abbreviations used: CBP, CREB-binding protein; KID, shown to be essential for binding the KIX domain kinase-inducible activation domain; pKID, of CBP and for subsequent transcriptional phosphorylated KID; HSQC, heteronuclear single 3,4 quantum coherence; ITC, isothermal titration activation. The NMR structure of the phosphoryl- calorimetry; LPE, linked protonation effect. ated KID (pKID)–KIX complex showed that KIX E-mail address of the corresponding author: forms a helical bundle structure that is bound by [email protected] the two mutually perpendicular helices of pKID.5 0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. 522 Solution Structure of KIX:c-Myb Complex The phosphoserine residue is located at the was attributed mainly to favorable intermolecular N terminus of the aB helix of the KIX-bound interactions involving the phosphate moiety. Bio- pKID. This helix makes multiple hydrophobic physical and biochemical results indicated that interactions with the shallow hydrophobic groove unphosphorylated KID binds specifically to KIX formed by the a1 and a3 helices of KIX. NMR with a sevenfold lower affinity than that of chemical-shift mapping6 as well as mutagenesis c-Myb:KIX.6 This difference in binding affinity studies7 showed that the same hydrophobic groove enables c-Myb to be a constitutive transcription is the docking site for c-Myb, a constitutive tran- factor, while CREB has a very low basal transcrip- scriptional activator regulating cell growth and tional activity and has to be phosphorylated on differentiation of hematopoietic cells,8 whose aber- the KID domain in order to bind the KIX domain rant amplification or truncation has been observed of CBP with significant affinity.3,4 However, the in several types of leukemia.9 structural basis for the participation of KIX in both Although pKID and c-Myb share a common phosphorylation-dependent and phosphorylation- binding site on KIX, there is no obvious sequence independent interactions is unclear. Specifically, similarity in their binding regions. In the unbound the overall similarity in the binding mode of KIX state, the activation domains of both c-Myb and to c-Myb and to KID in the basal unphosphoryl- KID (either phosphorylated or unphosphorylated) ated state appears to be contradictory to the are only partly structured, and binding to KIX is different affinities and transcriptional outcome. coupled with folding to form an amphipathic To evaluate the structural basis for the different helix that binds the hydrophobic groove of KIX.6,10 binding affinities of c-Myb and CREB for KIX that The affinity of KIX for the phosphorylated KID is result in different regulation of transcription, we 20-fold higher than for c-Myb.6 This difference have determined the solution structure of the KIX Figure 1. Three-dimensional structure of the c-Myb:KIX complex. A, Stereoview of the backbone trace of a best-fit superposition of the family of 20 NMR structures. The backbone of KIX is shown in blue and that of c-Myb in red. Only the ordered parts of KIX (residues 589–665) and c-Myb (residues 291–310) are shown. B, Ribbon diagram of the lowest-energy structure, colored as in A. Solution Structure of KIX:c-Myb Complex 523 domain of CBP in complex with the activation Table 1. NMR structure statistics domain of c-Myb. The structure reveals optimal KIX: interactions of the single helix of c-Myb with NMR constraints KIX c-Myb c-Myb residues in the hydrophobic groove of KIX. In par- ticular, Leu302 of c-Myb is inserted deeply into a Distance constraints 1289 125 100 hydrophobic pocket in KIX, accounting for more Intra-residue 622 0 Sequential 252 75 than half of the interactions between the two Medium-range 226 50 proteins. A bend in the a-helix of c-Myb enables Long-range 189 0 Leu302 of c-Myb to penetrate into this pocket Ambiguous constraints 52 0 more deeply than the equivalent leucine of pKID. Torsion angle constraints f 58, 16 c The differences in the sequence and helical struc- 57 16 ture of the two transcription factors result in Structure statistics (20 structures) modulation of complementarity and enable the Violations statistics /structure KIX domain to distinguish between the con- NOE violations . 0.1 A˚ 7 ^ 2 stitutive c-Myb that activates transcription and the Maximum NOE violation (A˚ ) 0.30 ^ unphosphorylated form of CREB that should not Torsion angle constraint 0.3 0.4 violations . 08 (deg.) activate transcription. Maximum torsion angle 2.2 violation (deg.) Energies Results Mean constraint violation 9.7 ^ 1.7 (kcal mol21) ^ Structure determination Mean AMBER 21700 21 (kcal mol21) Multi-dimensional NMR experiments were used Mean deviations from ideal to assign the chemical shifts for the KIX domain of covalent geometry Bond lengths (A˚ ) 0.0058 ^ 0.0001 CBP (residues 586–672) complexed with a 25 Bond angles (deg.) 1.92 ^ 0.02 residue peptide derived from the c-Myb activation 1 15 domain (residues 291–315). H– N correlated PROCHECK statistics heteronuclear single quantum coherence (HSQC) Residues in most favored 90.3 spectra of both 15N-labeled KIX bound to unlabeled regions (%) c-Myb and 15N-labeled c-Myb bound to unlabeled Residues in allowed 9.1 regions (%) KIX showed that the domains studied adopt a Residues in generously 0.5 6 unique folded conformation. In addition, the mini- allowed regions (%) mal region of c-Myb retains binding affinity for Residues in disallowed 0.1 KIX similar to that of larger peptides.6 Solution regions (%) structures were calculated using torsion angle and inter-proton distance restraints. The 20 lowest- RMSD deviations from KIX c-Myb KIX: average structure c-Myb energy structures (Figure 1A) form a tight family Backbone atoms 0.60 0.63 0.61 with low RMSD values, good backbone confor- (N,Ca,C0)a (A˚ ) mations and no significant constraint violation All heavy atomsa (A˚ ) 1.10 1.01 1.08 (Table 1). a-Helices, backbone 0.40 0.63 0.46 atoms (N,Ca,C0)b (A˚ ) a-Helices, all heavy 1.01 1.01 1.01 b ˚ Structure of the KIX domain in the KIX:c-Myb atoms (A) complex a In ordered regions: KIX residues 589–665, c-Myb residues 293–309. b KIX residues 597–611, 623–640, 646–662, c-Myb residues The KIX domain of CBP is composed of three 293–309. mutually interacting helices, a1 (residues 597–611), a2 (residues 623–640) and a3 (residues 646–664), and two short 310 helices, G1 (residues 591–594) and G2 (617–621) (Figure 1B). The C ter- Structure of c-Myb minus of helix a3 of KIX is stabilized and extended in the c-Myb complex relative to the pKID complex,11 probably because a slightly longer KIX In the unbound state, residues 295–309 of c-Myb construct (87 residues, versus 81 residues) was populate a partially helical conformation, esti- used.