Solution Structure of the KIX Domain of CBP Bound to the Transactivation Domain of c-Myb

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 Biology and the Skaggs Institute for Chemical Biology The Scripps Research Institute 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA

The hematopoietic transcription factor c-Myb activates transcription of target genes through direct interactions with the KIX domain of the co-activator CBP. The solution structure of the KIX domain in complex with the activation domain of c-Myb reveals a helical structure very similar to that adopted by KIX in complex with the phosphorylated kinase 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 interactions 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.

*Corresponding author

Keywords: CREB-binding protein; transcriptional activation; constitutive activation; LXXLL motif

Introduction Transcriptional activation in eukaryotes involves interactions between DNA-bound activators, co-activators and components of the basal transcription complex. The general co-activator CREB-binding protein (CBP) functions as a bridge between a number of transcription factors that bind specific enhancer DNA elements and the Present address: T. Zor, Genomics Institute of the Novartis Foundation, La Jolla, CA 92121, USA. Abbreviations used: CBP, CREB-binding protein; KID, kinase-inducible activation domain; pKID, phosphorylated KID; HSQC, heteronuclear single quantum coherence; ITC, isothermal titration calorimetry; LPE, linked protonation effect. E-mail address of the corresponding author: [email protected]

general RNA polymerase II complex that binds the promoter.1 The key role of CBP and its homolog p300 in transcription regulation is underscored by the findings that mutations in the CBP gene have been described in various types of cancer and in the Rubinstein – Taybi syndrome, a haplo-insufficiency disorder characterized by skeletal abnormalities, growth retardation and high incidence of tumors.2 The first interaction to be described was between CBP and the kinase-inducible activation domain (KID) of the cAMP-regulated transcription factor CREB. Phosphorylation of KID at Ser133 was shown to be essential for binding the KIX domain of CBP and for subsequent transcriptional activation.3,4 The NMR structure of the phosphorylated KID (pKID) – KIX complex showed that KIX forms a helical bundle structure that is bound by the two mutually perpendicular helices of pKID.5

0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.

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The phosphoserine residue is located at the N terminus of the aB helix of the KIX-bound pKID. This helix makes multiple hydrophobic interactions with the shallow hydrophobic groove formed by the a1 and a3 helices of KIX. NMR chemical-shift mapping6 as well as mutagenesis studies7 showed that the same hydrophobic groove is the docking site for c-Myb, a constitutive transcriptional activator regulating cell growth and differentiation of hematopoietic cells,8 whose aberrant amplification or truncation has been observed in several types of leukemia.9 Although pKID and c-Myb share a common binding site on KIX, there is no obvious sequence similarity in their binding regions. In the unbound state, the activation domains of both c-Myb and KID (either phosphorylated or unphosphorylated) are only partly structured, and binding to KIX is coupled with folding to form an amphipathic helix that binds the hydrophobic groove of KIX.6,10 The affinity of KIX for the phosphorylated KID is 20-fold higher than for c-Myb.6 This difference

Solution Structure of KIX:c-Myb Complex

was attributed mainly to favorable intermolecular interactions involving the phosphate moiety. Biophysical and biochemical results indicated that unphosphorylated KID binds specifically to KIX with a sevenfold lower affinity than that of c-Myb:KIX.6 This difference in binding affinity enables c-Myb to be a constitutive transcription factor, while CREB has a very low basal transcriptional activity and has to be phosphorylated on the KID domain in order to bind the KIX domain of CBP with significant affinity.3,4 However, the structural basis for the participation of KIX in both phosphorylation-dependent and phosphorylationindependent interactions is unclear. Specifically, the overall similarity in the binding mode of KIX to c-Myb and to KID in the basal unphosphorylated state appears to be contradictory to the different affinities and transcriptional outcome. To evaluate the structural basis for the different binding affinities of c-Myb and CREB for KIX that result in different regulation of transcription, we 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.

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Solution Structure of KIX:c-Myb Complex

domain of CBP in complex with the activation domain of c-Myb. The structure reveals optimal interactions of the single helix of c-Myb with residues in the hydrophobic groove of KIX. In particular, Leu302 of c-Myb is inserted deeply into a hydrophobic pocket in KIX, accounting for more than half of the interactions between the two proteins. A bend in the a-helix of c-Myb enables Leu302 of c-Myb to penetrate into this pocket more deeply than the equivalent leucine of pKID. The differences in the sequence and helical structure of the two transcription factors result in modulation of complementarity and enable the KIX domain to distinguish between the constitutive c-Myb that activates transcription and the unphosphorylated form of CREB that should not activate transcription.

Results Structure determination Multi-dimensional NMR experiments were used to assign the chemical shifts for the KIX domain of CBP (residues 586– 672) complexed with a 25 residue peptide derived from the c-Myb activation domain (residues 291– 315). 1H – 15N correlated heteronuclear single quantum coherence (HSQC) spectra of both 15N-labeled KIX bound to unlabeled c-Myb and 15N-labeled c-Myb bound to unlabeled KIX showed that the domains studied adopt a unique folded conformation.6 In addition, the minimal region of c-Myb retains binding affinity for KIX similar to that of larger peptides.6 Solution structures were calculated using torsion angle and inter-proton distance restraints. The 20 lowestenergy structures (Figure 1A) form a tight family with low RMSD values, good backbone conformations and no significant constraint violation (Table 1). Structure of the KIX domain in the KIX:c-Myb complex The KIX domain of CBP is composed of three 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 terminus 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 construct (87 residues, versus 81 residues) was used. A shallow hydrophobic groove is formed by helices a1 and a3, which pack at an angle of 17(^ 2)8, while helices a1 and a2 pack at an angle of 56(^ 2)8. The secondary structural elements define a compact structural domain with an extensive hydrophobic core. The core interactions reported for KIX in complex with pKID were all observed in the KIX:c-Myb complex.5

Table 1. NMR structure statistics NMR constraints

KIX

c-Myb

Distance constraints Intra-residue Sequential Medium-range Long-range Ambiguous constraints Torsion angle constraints

1289 622 252 226 189 52 f 58, c 57

125 0 75 50 0 0 16 16

Structure statistics (20 structures) Violations statistics /structure ˚ NOE violations . 0.1 A ˚) Maximum NOE violation (A Torsion angle constraint violations . 08 (deg.) Maximum torsion angle violation (deg.)

100

7^2 0.30 0.3 ^ 0.4 2.2

Energies Mean constraint violation (kcal mol21) Mean AMBER (kcal mol21)

9.7 ^ 1.7 21700 ^ 21

Mean deviations from ideal covalent geometry ˚) Bond lengths (A Bond angles (deg.)

0.0058 ^ 0.0001 1.92 ^ 0.02

PROCHECK statistics Residues in most favored regions (%) Residues in allowed regions (%) Residues in generously allowed regions (%) Residues in disallowed regions (%) RMSD deviations from average structure Backbone atoms ˚) (N,Ca,C0 )a (A ˚) All heavy atomsa (A a-Helices, backbone ˚) atoms (N,Ca,C0 )b (A a-Helices, all heavy ˚) atomsb(A

KIX: c-Myb

90.3 9.1 0.5 0.1

KIX

c-Myb

0.60

0.63

KIX: c-Myb 0.61

1.10 0.40

1.01 0.63

1.08 0.46

1.01

1.01

1.01

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 293–309.

Structure of c-Myb In the unbound state, residues 295– 309 of c-Myb populate a partially helical conformation, estimated to have 25 – 30% helical content.6 Binding of KIX to the 25-mer c-Myb peptide leads to a significant stabilization of the c-Myb helix. In the bound state, the helix extends from Lys293 to Leu309 or Lys310. Inter-molecular interactions with the hydrophobic groove of KIX serve to stabilize the helical conformation of c-Myb. Outside the helix boundaries, residues 291– 292 and 311 –315 are

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unstructured, as indicated by the lack of NOEs (Cand N termini) and sharp resonances (C terminus). While the backbone f and c angles indicate a continuous helix that spans 17 –18 residues, analysis of intra-helical distances and angles required for i,i þ 4 hydrogen bonds reveals a bend with an angle of 50(^ 9)8 that is centered at Ser304. Strong i,i þ 4 hydrogen bonds, characteristic of a helices, are observed in the segment 291 –304 as well as in the segment 304 – 311. Only a single i,i þ 3 hydrogen bond crosses the bend from the backbone carbonyl group of Leu302 to the backbone amide group of Thr305. The KIX:c-Myb interface The single a helix of c-Myb binds in the shallow hydrophobic groove formed by the a1 and a3 helices of KIX (Figure 2A). This groove is formed by the side-chains of Leu599, His602, Leu603, the aliphatic region of Lys606, Leu607 and Ala610, all located on the a1 helix, together with Tyr650, Leu653, Ala654, Ile657, Tyr658, Gln661 and the aliphatic portion of Lys662, on the a3 helix (Figure 2B). The borders of the hydrophobic groove are formed by the charged side-chains of Asp598 and Arg646 on one side and Lys662 and Glu665 on the other side. The groove serves as the docking surface for the non-polar face of the amphipathic helix of c-Myb, formed by the side-chains of Ile295, Leu298, Leu301, Leu302, Met303, Thr305 and Leu309. The charged side-chain of Arg294 is Glu298 are located in the N-terminal side of the binding region and those of Glu306 and Glu308 are in the C-terminal part. The orientation of c-Myb in the hydrophobic groove is determined by the electrostatic interactions of Arg294 with Glu665 on one side and Glu306 with Arg646 on the other side. In the center of the shallow hydrophobic groove, there is a deep hydrophobic pocket lined by the side-chains of Leu603, Lys606, Leu607, Tyr650, Leu653, Ala654 and Ile657 of KIX. The side-chain of Leu302 of c-Myb is inserted into the center of the pocket, where it is completely buried. pH-dependence of the interaction between KIX and c-Myb The NMR experiments for the structure determination were recorded at pH 5.5 in order to increase the quality of the spectra by lowering amide proton exchange with the solvent. The previous thermodynamic analysis was carried out at a more physiological pH 7.0.6 To correlate between the structural and thermodynamic data, we performed an isothermal titration calorimetry (ITC) study at the two pH values. The results are shown in Figure 3A, and the affinities and enthalpy changes are shown in Table 2. Both of the interactions show a significant pH-dependence. For c-Myb, the affinity for KIX is about sixfold higher at pH 5.5 compared to pH 7.0, while for pKID, the

Solution Structure of KIX:c-Myb Complex

affinity is approximately threefold lower at pH 5.5 compared to pH 7.0. These differences can be related directly to changes in the enthalpy (Table 2), which is more favorable for c-Myb at pH 5.5 and for pKID at pH 7. Rather than being related directly to differences in the mechanics of binding, the observed enthalpy differences may arise from a linked protonation effect (LPE), which may occur due to a change in the pKa of a protein side-chain upon complex formation.12 An experimental test for LPE is to measure the observed enthalpy change (DHobs) in the presence of buffers with very different enthalpies of ionization (DHion), and to calculate the change in the number of bound protons (DNHþ) upon complex formation, according to: DHobs ¼ DH0 þ DNHþ DHion

ð1Þ

ITC measurements were made for the c-Myb:KIX and pKID:KIX complex formation at pH 7.0 using Tris buffer (DHion ¼ 11:35 kcal mol21) or phosphate buffer (DHion ¼ 0:8 kcal mol21) (1 cal ¼ 4.184 J). The results are shown in Figure 3B. The calculated thermodynamic parameters of binding are shown in Table 2. As expected, the affinity was not dependent on buffer identity, but the enthalpy of binding was affected. A large LPE was observed for c-Myb: KIX interaction: binding was significantly more enthalpy-driven in the presence of the phosphate buffer (with its negligible ionization enthalpy) relative to Tris buffer (which has a strong unfavorable ionization enthalpy). In contrast, a small LPE in the opposite direction was observed for pKID: KIX complex formation. Calculation according to equation (1) yields DNHþ of þ 0.75 for KIX binding to c-Myb and 2 0.07 for KIX binding to pKID. The buffer-independent enthalpy ðDH0 Þ at pH 7.0, obtained from these values according to equation (1), is 2 12.2 kcal mol21 for KIX binding to c-Myb and 2 14.0 kcal mol21 for KIX binding to pKID.

Discussion Comparison of the c-Myb:KIX and pKID:KIX complexes Structures of the KIX (residues 586– 666) complex with the KID domain of CREB (residues 101 –160) phosphorylated at Ser133 have been previously determined in this laboratory.5 Our current data show that the structure of KIX is virtually identical in the c-Myb and pKID complexes. This is illustrated in Figure 4A, where the two structure families are overlaid. The backbone RMSD between the mean of the two sets of KIX structures ˚ (0.447 A ˚ if only the three helices are is 0.502 A superimposed). A representative pair of structures from the overlay is shown in Figure 4B. It is clear that the hydrophobic groove formed by the a1 and a3 helices of KIX can accommodate either the helix of the bound c-Myb or the aB helix of pKID, but the orientation of these helices in the groove is

Solution Structure of KIX:c-Myb Complex

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Figure 2. Complementarity between KIX and c-Myb. A, KIX is shown as a surface. The backbone of c-Myb is shown as a red ribbon and the side-chains of c-Myb that interact with KIX are shown in yellow. B, Stereoview of interacting side-chains along the hydrophobic groove: c-Myb (red) and KIX (blue). The side-chains at the ends of the hydrophobic groove are shown in black. The side-chain of A654 is shown but not labeled.

significantly different. The minor binding site on KIX for the aA helix of pKID is solvent-accessible in the c-Myb:KIX complex, since c-Myb does not have a second helix that can wrap around KIX. Upon complex formation, KIX and c-Myb bury ˚ 2 of solvent-accessible surface, which is pre1480 A

dominantly hydrophobic. The size of the surface is ˚ 2) but all comparable to that of KIX:pKID (1460 A of it resides in the hydrophobic groove, while only about 80% of the pKID:KIX interface is in the hydrophobic groove; the aA helix of pKID accounts for the remaining 20%.

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Solution Structure of KIX:c-Myb Complex

Figure 3. Sensitivity of KIX:c-Myb and KIX:pKID complex formation to pH and buffer variation. A, Sensitivity to pH. ITC measurements of KIX binding to pKID29 (circles) and to c-Myb (squares) at pH 7.0 (filled) and at pH 5.5 (open). Concentrations of KIX were 0.145 mM and 0.125 mM for the pKID29 titrations at pH 7.0 and at pH 5.5, respectively, and 0.225 mM and 0.170 mM for the corresponding c-Myb titrations. B, Sensitivity to buffer. ITC measurements at pH 7.0 of KIX binding to pKID34 (circles) and to c-Myb (squares) in Tris buffer (filled) and in phosphate buffer (open). Concentrations of KIX were 0.050 mM and 0.040 mM for the pKID34 titration in Tris buffer and in phosphate buffer, respectively. Concentrations of KIX were 0.225 mM and 0.215 mM for the c-Myb titration in Tris buffer and in phosphate buffer, respectively. Thermodynamic parameters of complex formation are in Table 2.

Table 2. Thermodynamic formation

parameters

of

complex

Kd a (mM)

DHb (kcal mol21)

Tris Tris

15.0 2.5

23.8 29.4

7.0 5.5

Tris Tris

3.2 8.3

214.3 212.0

c-Myb–KIX c-Myb–KIX

7.0 7.0

Tris Phosphate

15.0 12.5

23.8 211.6

pKID34–KIX pKID34–KIX

7.0 7.0

Tris Phosphate

1.3 1.0

214.7 214.0

Complex

pH

c-Myb–KIX c-Myb–KIX

7.0 5.5

pKID29c –KIX pKID29–KIX

Buffer

a The variation in Kd in duplicate measurements was typically 15% or better. b The reproducibility of DH for duplicate measurements was typically 0.2 kcal mol21 or better. c pKID29 ¼ residues 119–147; pKID34 ¼ residues 116–149.

The location of the bend in the structure of c-Myb bound to KIX is precisely at the position of Leu302, the side-chain of which is buried in a deep hydrophobic pocket on KIX. This is shown in close-up in Figure 4C. The equivalent Leu141 of pKID is inserted into the same pocket, but at a less optimal orientation. The bend of the c-Myb helix enables the side-chain of Leu302 to project ˚ deeper than the sideinto the pocket about 1 A chain of Leu141 of pKID. Therefore, Leu302 of c-Myb (but not Leu141 of pKID) is able to make hydrophobic contacts with the side-chain of Leu607 at the bottom of the deep pocket. This structural difference between the two complexes is consistent with differences in KIX chemical shifts upon complex formation in each case.6 Optimal packing of Leu302 in the deep pocket is helped by the interactions of the adjacent Leu301 and Met303 (Figure 2). Leu301 interacts with the hydrophobic portion of Lys606 and with Ala610, while the equivalent Asp140 of pKID can interact only with Lys606. Met303 makes extensive hydrophobic contacts with His651, Ala654 and Tyr650. The equivalent Ser142 of pKID is too far from His651 and Ala654, and its polar hydroxyl group is close to the hydrophobic aromatic ring of Tyr650, presumably a relatively unfavorable interaction. In the c-Myb:KIX complex, the side-chains of Leu298 and Glu299, located one helical turn from Leu302, pack against a shallower region of the hydrophobic groove, making extensive van der Waals contacts with Ile657 and Tyr658, as well as Ala654 (Glu299 only) or Ala610 (Leu298 only). In addition, Leu298 interacts with Gln661 and in half of the structures also with Lys662. Leu298 and Glu299 are replaced in the pKID:KIX complex by Ile137 and Leu138 of pKID, respectively. The interactions of these side-chains with KIX are in general similar, except for some differences that result from the bend in the c-Myb helix. The charged sidechain of Glu299 (c-Myb) is more accessible to solvent than the equivalent Leu138 (pKID). Thr305 is also located one helical turn from Leu302 and is completely buried in a secondary pocket of KIX, formed by the side-chains of Leu599, His602, Leu603 and Lys606. The hydroxyl group of Thr305 is able to hydrogen-bond to the side-chain of Lys606. In contrast, the equivalent Asp144 of pKID, although it interacts also with Lys606, remains largely solvent-exposed in the pKID:KIX complex. Glu306 of c-Myb makes an electrostatic interaction with Arg646 of KIX and van der Waals contacts with Leu599 and Tyr650, while the equivalent Ala145 of pKID is unable to complement the charge of Arg646. Electrostatic interactions between Glu308 of c-Myb, located on the second helical turn from Leu302, and His602 were observed in most structures and with Lys606 of KIX in about half of the structures. In contrast, the equivalent Gly147 of pKID is disordered, since its size and uncharged nature do not support these interactions. Finally, further hydrophobic contacts are made between

Solution Structure of KIX:c-Myb Complex

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Figure 4. Superposition of pKID:KIX and c-Myb:KIX complexes. A, Stereoview of a superposition of pKID:KIX5 and c-Myb:KIX families of structures. The backbone of KIX in complex with pKID (yellow) is colored dark blue, and the KIX backbone in complex with c-Myb (red) is colored light blue. B, Ribbon diagram of representative structures from A. C, Close-up view of hydrophobic pocket interactions. The c-Myb:KIX complex is superimposed on the pKID:KIX complex. The KIX backbone and side-chains are shown in dark blue for the pKID complex and in light blue for the c-Myb complex. The backbone of c-Myb is in red and that of pKID is in yellow. The side-chain of the penetrating leucine residue is shown in the corresponding color.

the side-chain of Leu309 from c-Myb and Leu599 and His602 from KIX. In the pKID:KIX complex, these two KIX residues interact with Pro146 of pKID. The different spacing of Leu309 (c-Myb) and Pro146 (pKID) from the deep pocket-inserted leucine residue (Leu302 and Leu141, respectively) is the result of the bend in the a helix of c-Myb. The interactions of the conserved leucine residue

and the C-terminal end of the bound helix of c-Myb in the KIX complex are optimized for binding, consistent with the constitutive binding of c-Myb. In contrast, the equivalent interactions in the pKID:KIX complex are not optimal, consistent with the inducible binding mode and the low affinity of unphosphorylated KID for KIX. Another major difference in the interactions of

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c-Myb and pKID with KIX occurs at the N-terminal end of the helix. Arg294 of c-Myb is located on the second helical turn from Leu302, and makes van der Waals contacts with Gln661 and Lys662, and an electrostatic interaction with Glu665 of KIX, while the adjacent Ile295 interacts with Tyr658 and Lys662. In the pKID:KIX complex, these two KIX residues interact with the pSer133 of pKID, and Tyr658 interacts additionally with Tyr134 and Leu128. These favorable interactions between KIX and the phosphorylated form of KID are absent for c-Myb, which lacks the highly charged phosphoserine residue. The interactions would, of course, be absent from unphosphorylated KID. These differences in specific interactions at the amino acid side-chain level account for the observed difference in transcriptional activation mode (constitutive for c-Myb and inducible for CREB), and can explain differences between CREB and c-Myb in affinity for KIX and tolerance of mutations in KIX.4 Thus, differences in the C-terminal half of the helix explain the difference in the basal levels of binding between c-Myb and KID, while differences in the N-terminal half explain the inducible nature of the pKID – KIX interaction. Structural basis for transcriptional activation mechanism The KIX domain of the co-activator CBP utilizes the hydrophobic groove to bind both the constitutive activator c-Myb and the kinase inducible domain (KID) of CREB. The minimal requirement for interaction with the hydrophobic groove of KIX is binding-coupled stabilization of an amphipathic helix, which we have shown to be sufficient for the generation of a low-affinity complex. The affinities of KIX for unphosphorylated KID, c-Myb and pKID are 108 mM, 15 mM and 0.7 mM, respectively.6 The results presented here explain the structural basis for these large differences in affinity. Induction of CREB by phosphorylation increases its affinity for KIX by two orders of magnitude, mainly via formation of intermolecular interactions between the phosphoserine residue of pKID and Tyr658 and Lys662 of KIX. The constitutive activator c-Myb does not have a residue that can substitute for the highly charged phosphoserine residue of pKID. Glu299 of c-Myb is relatively close to Tyr658 (Figure 2B), but its position does not allow formation of a hydrogen bond with Tyr658 or electrostatic interaction with Lys662. Substitution of the phosphate-acceptor Ser133 in CREB by the negatively charged residues aspartate or glutamate does not lead to constitutive CREB activity,13 further indicating that both the specific position and charge density of the phosphoserine residue are crucial for high-affinity complex formation. Consistent with this notion, mutation of either Tyr658 or Lys662 of KIX had a drastic effect on its binding to pKID but only a modest effect on c-Myb binding.4 The affinity of KIX for c-Myb is almost an order of magnitude higher than its affinity for unphosphorylated KID, although both specifically bind to

Solution Structure of KIX:c-Myb Complex

the hydrophobic groove of KIX.6 The c-Myb:KIX structure provides insight into the differences that are mandated in a constitutive transcriptional activator by the absence of the phosphate group. Analysis of the structure indicates that the interactions at the common binding surface are more extensive for c-Myb. In particular, insertion of Leu302 of c-Myb into the deep hydrophobic pocket on KIX provides a major driving-force for binding. Mutation of Leu302 of c-Myb abrogates binding, and mutations of hydrophobic pocket residues in KIX have a larger effect on binding of c-Myb than on binding of pKID.4 The bend in the helix of c-Myb enables Leu302 to penetrate into the pocket more deeply than the equivalent Leu141 of KID, thereby increasing the number and strength of interactions with KIX residues. Notably, Leu607, which defines the bottom of the hydrophobic pocket, interacts only with Leu302 of c-Myb and not with Leu141 of KID. Consistent with this finding, Shaywitz et al. showed that the L607F KIX mutant interacted more efficiently than wild-type KIX with unphosphorylated KID, leading to a significant increase in both basal and induced transcriptional activity of CREB.14 No such increase was observed for c-Myb binding to the mutant KIX. This mutation appears to rearrange the deep hydrophobic pocket in a way that specifically enhances the imperfect binding of KID but not the more optimized binding of c-Myb. Additional c-Myb residues that complement the hydrophobic surface of KIX better than the corresponding KID residues are Leu301, Met303 and Thr305. The latter side-chain is accommodated perfectly in a secondary pocket on KIX (Figure 2). In addition, the ends of the hydrophobic groove of KIX are lined by three charged residues, His602, Arg646 and Glu665, which can make electrostatic interactions with c-Myb residues Glu308, Glu306 and Arg294, but not with KID residues. We suggest that the differences in the interface of the c-Myb and KID complexes are crucial to enable constitutive binding of c-Myb to KIX and at the same time to achieve only low-level basal interactions between CREB and KIX. Thus, the lack of specific electrostatic interactions in the N-terminal part of the bound c-Myb helix, in contrast to those present at the N terminus of pKID helix aB can explain the inducible nature of the CREB:CBP binding reaction and the higher affinity of pKID for KIX. Conversely, the presence of the central leucine residue that binds into the deep pocket, together with additional strong, specific interactions in the C-terminal part of the bound helix, can explain the constitutive binding of c-Myb and its higher affinity for KIX compared to that of unphosphorylated KID. The LXXLL motif in transcriptional activation complexes Leu302 of c-Myb is part of an LXXLL motif, which appears commonly in complexes formed

Solution Structure of KIX:c-Myb Complex

between nuclear receptors and their co-activators. Structures of complexes between nuclear receptors and coactivators from the p160 family15 – 18 resemble the c-Myb:KIX complex in the binding of an amphipathic helix containing the LXXLL sequence in a hydrophobic groove of the second protein. In addition, while the LXXLL motif contributes most of the binding energy, adjacent residues increase the affinity and confer specificity.17 In the complex between the thyroid hormone receptor and the p160 co-activator GRIP1, all three leucine residues of the motif bind in relatively shallow hydrophobic pockets within the hydrophobic groove and mutation of either one is detrimental for binding.15 In contrast, the contribution of the three leucine residues for the c-Myb:KIX interaction varies: Leu302, which binds in the deep hydrophobic pocket, buries 98% of its solvent-accessible surface, Leu298, which binds in a shallow hydrophobic pocket in the hydrophobic groove, buries 80%, and Leu301, which binds in a shallow amphipathic pocket outside the hydrophobic groove, buries only 33%. Consistently, Leu302 has a small tolerance for conservative mutations, Leu298 has a larger tolerance for conservative mutations and Leu301 can be mutated even to alanine without a significant reduction in binding or transcriptional activity.4 Indeed, sequence alignment of c-Myb and CREB according to the structures of their KIX complexes (Figure 5) reveals the identity of only Leu302. There is a conservative change of Leu298 to isoleucine, and a non-conservative change of Leu301 to aspartate. Thus, the c-Myb binding site contains a sequence motif in common with a number of other domains involved in binding interactions in transcriptional activation. Although the specific interactions of the c-Myb LXXLL motif in the KIX complex do not appear to have much in common with such sequences in other complexes, the presence of this binding “signature” in c-Myb but not in KID may be correlated with the constitutive binding of c-Myb versus the low basal binding of unphosphorylated KID. pH-Dependence of pKID and c-Myb affinities Binding of c-Myb at the more acidic pH of 5.5 is

529

accompanied by a significantly more favorable enthalpy, while for the pKID –KIX interaction the change of enthalpy is in the opposite direction (modestly more favorable at neutral pH) (Figure 3A; Table 2). The significant change of thermodynamics in the pH range 5.5 – 7.0 suggests that a histidine residue is mediating the effect via an electrostatic interaction that differs for each complex. Since there is no histidine in the c-Myb and pKID sequences, the putative histidine residue must reside in KIX. An alternative explanation for the pH effect in the pKID – KIX interaction could be the ionization of the phosphoserine residue, resulting in a stronger electrostatic interaction with Lys662. This possibility is consistent with 31 P-NMR experiments showing titration of the phosphoserine residue in the same pH range.19 However, we have observed a similar increase in affinity of KIX for unphosphorylated pKID at pH 7.0 compared to pH 5.5 (data not shown), suggesting that the pH effect is unlikely to be associated with the phosphoserine residue. The buffer-effect data (Figure 3B) indicate that at neutral pH there is uptake of almost a complete proton by a KIX residue, probably histidine, upon binding to c-Myb. This binding reaction is significantly more favorable at acidic pH, since the pHsensitive electrostatic interaction can take place without the cost of proton transfer. On the other hand, the proton release required to generate an uncharged histidine residue upon binding of KIX to pKID causes this binding reaction to be more favorable as the pH increases from acidic to neutral pH, where the energy cost of proton transfer is avoided. There are five histidine residues in the KIX construct used in the structural studies of the pKID5 and the c-Myb complexes. His592 and His594 form part of the G1 310 helix at the N terminus of KIX; their side-chains are rather disordered, and are far from the bound c-Myb. His602 and His605 are in the middle of the a1 helix, and His651 is towards the N terminus of a3. The positions of these side-chains are shown in the family of c-Myb: KIX structures in Figure 6. The solvent-accessible area of His592, His594 and His605 is not changed upon complex formation with either c-Myb or pKID. His651 also appears far from the c-Myb

Figure 5. Sequence alignment of pKID and c-Myb. Sequences of various KID constructs referred to here, aligned with the sequence of the 25-mer c-Myb peptide according to the similarity in the three-dimensional structures of their complexes with the KIX domain of CBP. Sequence KID60 corresponds to the construct used to obtain the solution structure of the pKID:KIX complex.5 Sequence pKID34 was used for the buffer sensitivity ITC measurements, and sequence pKID29 was used for the pH-dependence measurements and for all previous thermodynamic measurements.6 Asterisks ( p ) indicate sites of contact with KIX in the three-dimensional structure of each complex. Helices observed in the complexes are outlined in blue (pKID complex) and red (c-Myb complex). Ser133, which is phosphorylated in the pKID proteins, is outlined in orange. Leu302 of c-Myb, and the analogous Leu141 of pKID, which is buried in the same hydrophobic pocket of KIX, is outlined in yellow. The LXXLL sequence of c-Myb is shown in green letters.

530

helix, but the side-chain interacts in half of the pKID:KIX structures with Arg125 from the aA helix of pKID. This interaction is electrostatically unfavorable, and therefore the affinity of KIX for pKID should be higher when His651 is deprotonated and uncharged. The only intermolecular interaction of His651 in the c-Myb:KIX complex is between its Ha and the H1 of Met303, which would be pH-independent. The most likely histidine residue to mediate the pH-dependence of c-Myb:KIX complex formation is His602, which makes an electrostatic interaction with Glu308 of c-Myb. Consistent with the ITC results, proton uptake would be required for this interaction to occur at neutral pH; binding would be more favorable at acidic pH. The residue equivalent to Glu308 of c-Myb in the pKID – KIX complex is Gly147, which is uncharged and therefore unable to make such an electrostatic interaction. (It is possible that an electrostatic interaction occurs for one of the pKID constructs, where Gly147 forms the C-terminal residue; in this case, the C-terminal carboxyl group could possibly substitute for the sidechain of Glu308.) The structures5 show that His602 is close to Ala145 and Asp144 of pKID, but the distance and angle between the His602 side-chain and the latter residue do not support an electrostatic interaction or a hydrogen bond. The non-polar interactions of His602 in the pKID – KIX complex predict that binding would be stronger in the uncharged state, possibly contributing to the increased affinity of KIX for pKID as the pH is increased from acidic to neutral. Physiological rationale for observed binding affinities The intermolecular interactions of the phosphoserine residue of pKID increase its affinity for KIX at neutral pH by 20-fold over that of the con-

Solution Structure of KIX:c-Myb Complex

stitutive c-Myb. Lower affinity may be an advantage for c-Myb, since it is regulated mainly at the level of its expression and degradation8,20 and is required to maintain the proliferative state of immature hematopoietic cells.21,22 Therefore, the lower binding affinity of the c-Myb activation domain is consistent with the kinetic profile of its constitutive activity, which is turned on and off slowly and has a cumulative effect over time. In contrast, CREB is turned on more rapidly by an extra-cellular signal leading to PKA-mediated phosphorylation on Ser133,23 and it is turned off rapidly following dephosphorylation by the Ser/ Thr phosphatase PP-1.24 Activation of the inducible pathway mediated by CREB may require highaffinity binding to CBP in order to compete effectively with constitutive pathways converging at CBP. The fact that c-Myb binds CBP with a lower relative affinity might also be an advantage for regulation by other transcription factors, to either increase (C/EBPb, Ets-1 and AML-1) or attenuate (GATA-1) its activity in a synergistic fashion.1,9,25 The distinct physiological requirements of the two transcriptional activators correlate well with the pH optima of co-activator recruitment, physiological pH for pKID and lower pH for c-Myb. Inducible versus constitutive: a common binding pattern? In addition to the KIX domain of CBP, serveral other systems in the cell utilize ligand binding that may be inducible or constitutive. For example, the 14-3-3 family of proteins mediates various signal transduction pathways by binding to a number of phosphoserine-containing proteins.26 The structure of a 14-3-3 protein bound to its target phosphopeptide27 reveals many similarities to that of the KIX:pKID complex. The serine-phosphorylated protein binds in an amphipathic groove on

Figure 6. Positions of KIX histidine side-chains. Stereoview of the family of 20 structures of the KIX:c-Myb complex, showing the positions of the five histidine residues of KIX, His592 (orange), His594 (yellow), His602 (green), His605 (blue) and His651 (purple). The backbone of KIX is shown in light blue, and that of c-Myb in red.

531

Solution Structure of KIX:c-Myb Complex

the 14-3-3 surface, with an electrostatic interaction between the phosphoserine and three basic residues, and a hydrogen bond between the phosphoserine and a tyrosine residue. Other residues in addition to the phosphoserine, both hydrophobic and polar, are essential for binding. In spite of the significant contribution of non-phosphate interactions, dephosphorylation results in loss of binding.27 In addition, several non-phosphorylated proteins are able to bind with high affinity to the amphipathic groove of 14-3-3 proteins.28,29 The 14-3-3 proteins are therefore similar to KIX in their ability to bind the inducible protein only after phosphorylation and yet to bind constitutive nonphosphorylated proteins with high affinity, using the same surface. As it is in the KIX:c-Myb complex, binding of a constitutive non-phosphorylated peptide to 14-3-3 depends on good complementarity of both hydrophobic and polar residues. However, in contrast to c-Myb, the constitutive 14-3-3 binding peptide has two acidic residues that substitute for the doubly charged phosphoserine by making electrostatic interactions in the same basic pocket that accommodates the phosphoserine residue, thereby bringing the affinity of the constitutive peptide for 14-3-3 to the level of inducible peptides in the phosphorylated state.30,31 By contrast, the absence of such acidic residues in c-Myb keeps its affinity for KIX lower than that of pKID in spite of the excellent interface complementarity. The functional similarity between KIX and 14-3-3 proteins suggests a general pattern of recognition for proteins that competitively bind both phosphorylated and non-phosphorylated targets. The binding of an inducible protein would involve strong interaction with the phosphate moiety by electrostatic and hydrogen bonds and imperfect but essential additional interactions along the groove. Those interactions would not be sufficient for binding in the absence of the phosphate but ensure specificity and higher affinity of binding following the phosphorylation event. The binding of a constitutive partner would involve optimal complementarity along the groove to ensure specificity and reasonable affinity and, if higher affinity is required, it may dictate the presence of acidic residues substituting for the phosphate interactions.

Conclusions The structure of the KIX:c-Myb complex and our thermodynamic analysis provide new insights into the mechanism of recognition of various transcriptional activator domains by the highly conserved KIX domain of CBP and p300, and illustrate the structural and thermodynamic basis for constitutive activation by c-Myb and inducible activation by CREB. We suggest that the minimal requirements for low-affinity interaction with the hydrophobic groove of KIX are an amphipathic a

helix that binds along the groove and a critical leucine residue that is inserted into the deep hydrophobic pocket of KIX. Optimal complementarity of the c-Myb:KIX interface, in particular within the deep hydrophobic pocket, results from the sequence of c-Myb and from a bend in the helix of bound c-Myb. Modulation of that complementarity enables the KIX domain to distinguish between the constitutive c-Myb that activates transcription and the unphosphorylated form of CREB that should not activate transcription. Induction of CREB by phosphorylation converts the low-affinity complex into a specific high-affinity complex, mainly via formation of additional inter-molecular interactions involving the phosphate moiety, which compensate for the imperfect complementarity of the pKID –KIX interface.

Materials and Methods Protein expression and purification The KIX domain (residues 586– 672) of mouse CBP (amino acid sequence identical with that of human) was expressed in either unlabeled or uniformly 15N or 13 C,15N-enriched forms, in BL21(DE3) Escherichia coli and purified to homogeneity as described.5 Uniformly 15N or 13 C,15N-labeled c-Myb (residues 291– 315 from mouse) were overexpressed in E. coli using a ubiquitin fusion protein system (a generous gift from Dr Toshiyuki Kohno) and purified to homogeneity as described.6 Unlabeled c-Myb and phosphorylated KID peptides (residues 119 – 147 (pKID29) or residues 116 – 149 (pKID34) of mouse CREB, amino acid sequence identical with that of human) were synthesized chemically using a Perseptive Biosystems peptide synthesizer (Perkin – Elmer) and purified to homogeneity by reverse-phase HPLC. The identity and integrity of all proteins and peptides were confirmed by mass spectrometry. NMR spectroscopy NMR samples were prepared in the concentration range of 0.5– 1.0 mM in 90% (v/v) H2O/10% (v/v) 2H2O buffer (20 mM Tris d11-acetate d4 (pH 5.5), 50 mM NaCl, 2 mM NaN3). NMR spectra were recorded at 27 8C on Bruker AMX500, DRX600 and DMX750 spectrometers, equipped with triple axis gradient probes. Titration of labeled KIX with unlabeled c-Myb or labeled c-Myb with unlabeled KIX was monitored by 2D 1H – 15N HSQC spectra. The unlabeled component was added in 10 – 20% excess. NMR data processing and analysis were performed using Felix97 (Molecular Simulations Inc. San Diego) or NMRPipe32 and NMRView.33 Nearly complete backbone assignments for c-Myb-bound KIX were accomplished using 3D HNCACB,34,35 CBCA(CO)NH,36 HNCO,37 HCACO,38 15N-edited NOESY-HSQC and 15Nedited TOCSY-HSQC39 ðtm ¼ 51 msÞ spectra. Only the backbone amides of Arg623 and Lys667 were not assignable due to exchange broadening. Aliphatic side-chain resonances were assigned using 3D 15N-edited TOCSY, HCCH-TOCSY, and HCCH-COSY40 spectra. Aromatic side-chain resonances were assigned from 3D CB(CG)CD and CB(CG)CE41 spectra. Although backbone resonances could be assigned, only partial assignments could be made for the side-chain protons of Arg600, Lys606,

532

Lys621 and the histidine residues. Complete backbone assignments for KIX-bound c-Myb were accomplished using 3D HNCACB and CBCA(CO)NH spectra. 3D 15 N-edited TOCSY-HSQC ðtm ¼ 43:2 msÞ and 13C-edited HCCH-TOCSY, HCCH-COSY, CCH-COSY, and CCHTOCSY42 spectra were acquired for side-chain assignments. Several exchange-broadened resonances could be assigned only by using sequential NOE connectivities observed in 3D 15N and 13C-edited NOESY spectra. The 13 C chemical shifts were referenced relative to 2,2dimethyl-2-silapentane-5-sulphonate (DSS). Generation of restraints Intra-molecular distance constraints for KIX and c-Myb protons were obtained from 15N-edited NOESY (tm ¼ 80 ms and 100 ms, respectively) and 13C-edited NOESY (tm ¼ 120 ms and 80 ms, respectively). Intensities were calibrated against known inter-proton distances in regular structural elements (a helices) or between protons of fixed separation. Upper limits of distance con˚, 4A ˚ and 5 A ˚ , while all lower limits straints were 3 A ˚ ). were set to the van der Waals contact distance (1.8 A Due to the longer mixing time, the upper limits for the constraints derived from 15N-edited NOESY, were ˚ . Appropriate pseudo-atom corrections increased by 0.5 A to the upper bounds were applied to constraints involving methyl and methylene groups, and aromatic ring protons. No explicit hydrogen bonding constraints were imposed. Inter-molecular distance constraints were derived from a 3D 13C(v2)-edited, 13C(v3)-filtered NOESY (tm ¼ 120 ms) experiment.43 A scaling factor was determined by comparing the intensities of wellresolved peaks with those of the corresponding peaks in the 13C-edited NOESY spectrum acquired for KIX. On ˚, 4A ˚ and 5 A ˚ were that basis, upper bounds of 3 A assigned. Backbone f and w angles in helical regions, as indicated by the chemical-shift index,44 were restrained to 2 50(^30)8 and 2 40(^30)8, respectively. Structure calculations and analysis Initial structures were calculated separately with the program DYANA45 for KIX and c-Myb, using intra-molecular distance and torsion angle constraints determined in the bound state. Intermolecular restraints were then added to dock the c-Myb:KIX complex, and 200 DYANA structures were generated. The 30 structures with the lowest target functions were refined by simulated annealing using the AMBER 6 software package.46 The final structures were calculated by one cycle of DYANA, generating 100 structures with lowest target functions (out of 200) for refinement by two cycles of AMBER. The 20 structures with the lowest constraint violation energies were selected for analysis. The structures show excellent convergence, low AMBER energies (mean of 2 1700 kcal mol21) and no NOE violation ˚ . Statistics are given in Table 1. greater than 0.3 A Analysis of the structures was performed using PROCHECK,47 and in-house programs. Graphics images were generated and interface area was calculated using MOLMOL.48 Isothermal titration calorimetry (ITC) The titration of a pKID or c-Myb peptide into a KIX protein was performed at 27 8C using an MCS titration calorimeter from MicroCal Inc. Simultaneous dialysis of

Solution Structure of KIX:c-Myb Complex

all peptides and proteins against the ITC buffer, followed by filtration and degassing was performed to minimize background noise. The ITC buffer was prepared from 50 mM Tris titrated to pH 7.0 with HCl or to pH 5.5 with acetic acid or 50 mM sodium phosphate buffer at pH 7.0. All ITC buffers included 50 mM NaCl. KIX and pKID concentrations were determined by measuring absorbance at 280 nm. The stoichiometric ratio obtained from the curve fit was consistently 1:1, within 5% error. The concentration of c-Myb was determined according to the stoichiometric ratio obtained from the curve fit. The concentration of KIX in the ITC cell was 40 – 225 mM with higher concentrations used for the lower affinity and/or lower enthalpy complexes, while the concentration of the peptide in the syringe was 12-fold over that of KIX. Typically, two injections of 5 ml were followed by 28 injections of 10 ml until a molar ratio of 2.5 was obtained. Integration of the thermogram and subtraction of the blanks yielded a binding isotherm that was fit to a model of one-site interaction (ITC data analysis software in Origin 2.3 of MicroCal Inc.). Data bank accession numbers The assigned chemical shift list has been deposited in the BioMagResBank with accession number 6095 and the coordinates in the Protein Data Bank with accession number 1SB0.

Acknowledgements This work was supported by grant CA96865 from the National Institutes of Health and by the Skaggs Institute for Chemical Biology. T.Z. is an EMBO fellow and R.N.D. is supported by the Leukemia and Lymphoma Society. We thank Dr Toshiyuki Kohno for providing us with the ubiquitin fusion construct. We are grateful to Linda Tennant and Dr Maria Martinez-Yamout for help with sample preparation, and Drs Eduardo Zaborowski, Gerard Kroon and John Chung for expert advice regarding NMR experiments. We are particularly grateful to Dr Natalie Goto for critical reading of the manuscript and we thank Dr Ishwar Radhakrishnan and Gabriela PerezAlvarado for helpful discussions.

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Edited by M. F. Summers (Received 21 January 2004; accepted 23 January 2004)