Structural basis for recruitment of CBP/p300 coactivators by STAT1 and STAT2 transactivation domains

Abstract

CBP/p300 transcriptional coactivators mediate gene expression by integrating cellular signals through interactions with multiple transcription factors. To elucidate the molecular and structural basis for CBP-dependent gene expression, we determined structures of the CBP TAZ1 and TAZ2 domains in complex with the transactivation domains (TADs) of signal transducer and activator of transcription 2 (STAT2) and STAT1, respectively. Despite the topological similarity of the TAZ1 and TAZ2 domains, subtle differences in helix packing and surface grooves constitute major determinants of target selectivity. Our results suggest that TAZ1 preferentially binds long TADs capable of contacting multiple surface grooves simultaneously, whereas smaller TADs that are restricted to a single contiguous binding surface form complexes with TAZ2. Complex formation for both STAT TADs involves coupled folding and binding, driven by intermolecular hydrophobic and electrostatic interactions. Phosphorylation of S727, required for maximal transcriptional activity of STAT1, does not enhance binding to any of the CBP domains. Because the different STAT TADs recognize different regions of CBP/p300, there is a potential for multivalent binding by STAT heterodimers that could enhance the recruitment of the coactivators to promoters.

Figure 1

Characterization of STAT-TAD and TAZ domains. (A) Domain structure of mouse CBP showing the positions of the TAZ1 and TAZ2 domains in the mouse sequence. (B) Alignment of the human STAT1, STAT3 and STAT4 TAD amino-acid sequences. Bold-faced type highlights amino acids that are similar between family members. Asterisks indicate residues in the STAT1-TAD that directly contact TAZ2 in the complex structure. The grey bar denotes amino acids that form the helix in the bound STAT1-TAD. The circled ‘P' denotes the position of the phosphorylation site. A dash in the sequence indicates a gap with no corresponding residue. (C) Amino-acid sequences of the human STAT2 C-terminal TAD (ctTAD) sequences and the truncated construct with two point mutations (nmrTAD). Underlined residues indicate an imperfect repeat in the STAT2-ctTAD sequence. A dash in the sequence indicates a gap with no corresponding residue. A dot in the STAT2-nmrTAD sequence indicates a position where the amino acid is identical to STAT2-ctTAD. Asterisks indicate residues in the STAT2-nmrTAD that contact TAZ1 in the complex structure. Grey bars denote the amino acids that comprise the three helical structures in the bound STAT2-nmrTAD.

Figure 2

Interactions of TAZ and STAT domains. (A) SDS–PAGE analysis of mouse TAZ1 and TAZ2 assayed for interactions with the immobilized human STAT sequences shown in Figure 1B and C. GB1 (lanes 3 and 10), GB1–STAT1-TAD (lanes 4 and 11), GB1–STAT2-ctTAD (lanes 5 and 12), GB1–STAT2-nmrTAD (lanes 6 and 13), GB1–STAT3-TAD (lanes 7 and 14) and GB1–STAT4-TAD (lanes 8 and 15) bound to IgG Sepharose were incubated with purified TAZ1 (lanes 3–8) or TAZ2 (lanes 10–15). The bands at ∼25 and ∼50 kDa (lanes 3–8 and 10–15) correspond to IgG light and heavy chains. Lanes 2 and 9 contain free TAZ1 and TAZ2, respectively. (B) Results of pull-down assays showing that phosphorylation of S727 does not enhance the affinity of STAT1 TAD for CBP. TADs of STAT1 and MLL (used as a control for KIX binding) were bound to IgG Sepharose through an N-terminal GB1 fusion tag and binding of CBP domains TAZ1, KIX, TAZ2 and NCBD to STAT1 TAD (710–750), phosphoS727 and S727A STAT1 TADs was measured by pull-down assays monitored by reversed phase HPLC.

Figure 3

NMR spectra of free and bound STAT-TAD. (A) ¹H–¹⁵N HSQC spectrum of STAT1-TAD free (black) and bound to TAZ2 (red). (B) ¹H–¹⁵N HSQC spectrum of STAT2-TAD free (black) and bound to TAZ1 (red). Selected cross-peaks of the bound protein are labelled.

Figure 4

NMR solution structure of the TAZ1:STAT2-TAD. (A) Ensemble of the 20 lowest energy structures superimposed on the backbone atoms of TAZ1:STAT2-TAD (TAZ1, residues Pro347-Ala372 and Cys384-Asn434; STAT2-TAD, residues Asp790-Met816). The backbones of TAZ1 and STAT2-TAD are shown in blue and green, respectively. The boundaries of the well-structured region of STAT2-TAD, and the N and C termini of TAZ1 are labelled. The backbone atoms of the flexible C-terminal region of the STAT2-TAD (residues Pro817-Ser835) are shown in light green, and the flexible loop of TAZ1 (residues 373–383) is shown in light blue. (B) Ribbon diagram showing the backbone of the lowest energy structure with zinc atoms and secondary structure elements of TAZ1 labelled. The ribbon is traced from residues Thr344-Ser436 of TAZ1 (blue) and Glu788-Met816 of the STAT2-TAD (green), with the C-terminal tail shown in light green. (C–E) Close-up views showing TAZ1 (yellow surface) and STAT2-TAD (white backbone), with the side chains of residues that form intermolecular contacts. The colour scheme for the side chains is as follows: acidic residues (Asp and Glu), red; basic residues (Arg, Lys and His), blue; neutral polar residues (Ser, Thr and Gln) and backbone carbonyl atoms, cyan; hydrophobic residues (Ala, Leu, Ile, Phe, Tyr, Val, Met and Pro), green.

Figure 5

NMR solution structure of the TAZ2:STAT1-TAD. (A) Ensemble of the 20 lowest energy structures superimposed on the backbone atoms of TAZ2:STAT1-TAD (TAZ2, residues Gln1766-Arg1852; STAT1-TAD, residues Asp721-Asn748). The backbones of TAZ2 and STAT1-TAD are shown in purple and yellow, respectively. The N and C termini of TAZ2 and STAT1-TAD are labelled. (B) Ribbon diagram showing the backbone of the lowest energy structure with zinc atoms and secondary structure elements of TAZ2 labelled. (C–E) Close-up views showing TAZ2 (yellow surface) and STAT1-TAD (white backbone), with the side chains of residues that form intermolecular contacts. The colour scheme for the side chains is the same as for Figure 4. The position of the side chain of S727, the phosphorylation site characterized in Figure 1, is shown in magenta.

Figure 6

Electrostatic potentials on the surfaces of the TAZ domains. Positively charged surfaces are shown in blue, and negatively charged in red. (A) TAZ2 in complex with the STAT1-TAD (yellow backbone). (B) TAZ1 in complex with STAT2-TAD (green backbone). The left and right images represent a 180° rotation around the vertical axis in the plane of the page. (C) Superposition of the structure of STAT2-TAD (green) on the TAZ2:STAT1-TAD complex (transparent surface plus yellow backbone). (D) Superposition of the structure of STAT1-TAD (yellow) on the TAZ1:STAT2-TAD complex (transparent surface plus green backbone).

Figure 7

Comparison of TAZ1 ligand structures. (A) Surface representation of the structure of TAZ1 in complex with the STAT2-TAD (green), HIF-1α-CTAD (red) and CITED2-TAD (blue). The left and right images represent a 180° rotation around the vertical axis in the plane of the page. The N and C termini of each ligand are labelled. (B) Superposition of the complexes of TAZ1 with STAT2-TAD (green) and CITED2-TAD (blue), showing a region of similar structure in the two complexes, even though the sequences run in opposite directions. (C) Superposition of the complexes of STAT2-TAD (green) and CITED2-TAD (blue), showing different secondary structures bound to the same region of TAZ1. (D) Sequence alignment showing proteins with homology to the helical region of STAT1-TAD. Amino acids are coloured red (acidic), blue (basic), yellow (non-charged polar), green (hydrophobic) and purple (Pro and Gly).