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Structural basis for Hif-1 alpha /CBP recognition in the cellular hypoxic response - PubMed

  • ️Tue Jan 01 2002

Structural basis for Hif-1 alpha /CBP recognition in the cellular hypoxic response

Sonja A Dames et al. Proc Natl Acad Sci U S A. 2002.

Abstract

The cellular response to low tissue oxygen concentrations is mediated by the hypoxia-inducible transcription factor HIF-1. Under hypoxic conditions, HIF-1 activates transcription of critical adaptive genes by recruitment of the general coactivators CBP/p300 through interactions with its alpha-subunit (Hif-1 alpha). Disruption of the Hif-1 alpha/p300 interaction has been linked to attenuation of tumor growth. To delineate the structural basis for this interaction, we have determined the solution structure of the complex between the carboxy-terminal activation domain (CAD) of Hif-1 alpha and the zinc-binding TAZ1 (CH1) motif of cyclic-AMP response element binding protein (CREB) binding protein (CBP). Despite the overall similarity of the TAZ1 structure to that of the TAZ2 (part of the CH3) domain of CBP, differences occur in the packing of helices that can account for differences in specificity. The unbound CAD is intrinsically disordered and remains relatively extended upon binding, wrapping almost entirely around the TAZ1 domain in a groove through much of its surface. Three short helices are formed upon binding, stabilized by intermolecular interactions. The Asn-803 side chain, which functions as a hypoxic switch, is located on the second of these helices and is buried in the molecular interface. The third helix of the Hif-1 alpha CAD docks in a deep hydrophobic groove in TAZ1, providing extensive intermolecular hydrophobic interactions that contribute to the stability of the complex. The structure of this complex provides new insights into the mechanism through which Hif-1 alpha recruits CBP/p300 in response to hypoxia.

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Figures

Figure 1
Figure 1

1H-15N heteronuclear single quantum coherence spectra (600 MHz) of Hif-1α (776–826) free (red) and bound to unlabeled TAZ1 (black).

Figure 2
Figure 2

NMR structure of the Hif-1α:TAZ1 complex. (A) Stereo view of the best 20 structures superposed on backbone heavy atoms in ordered regions. The TAZ1 backbone is shown in blue, Hif-1α in pink, and the N and C termini of each chain are labeled in the corresponding colors. Bound zinc ions are shown as yellow spheres. (B) Ribbon representation of a single structure in a similar orientation to A. Helices α1–α4 of TAZ1 and αA–αC of the Hif-1α CAD are labeled. The zinc ions are represented as white spheres, and the side chains of the cysteine and histidine ligands are shown in yellow and blue, respectively.

Figure 3
Figure 3

(A) Amino acid sequences of the zinc-binding sites (Zn1, Zn2, and Zn3) in the TAZ1 and TAZ2 domains of mouse CBP. The histidine (blue) and cysteine (yellow) ligands are highlighted. The sequence of part of helix α3 preceding the Zn3 site is shown; note the deletion in TAZ1 immediately preceding the histidine ligand. (B) Superposition of TAZ1 (blue) and TAZ2 (yellow) structures, represented as backbone ribbons. The proteins are aligned on the backbone heavy atoms of helices α1 and α2. The structures are rotated ≈180° relative to the view of TAZ1 in Fig. 2 to reveal the similar packing arrangements of helices α1, α2, and α3, and the similar locations of the Zn1 and Zn2 sites. The arrows indicate the location of the Zn3 site in the two structures. (C) Close-up stereoview of the Zn3 site, showing the different position of the α4-helix in the TAZ1 (blue) and TAZ2 (yellow) structures and the different locations of Zn3 (blue and yellow spheres) and the zinc-binding loops. The backbone of the αC-helix of Hif-1α in the complex with TAZ1 is shown as a pink tube to illustrate how binding to TAZ2 would be occluded by the different arrangement of Zn3 and α4.

Figure 4
Figure 4

(A) Alignment of Hif-1α and Hif-2α CAD sequences showing conserved residues. The asterisks indicate sites where mutations abrogate CBP/p300 binding. The arrow indicates Asn-803, which functions as a hypoxic switch. (B) Binding site for αB-helix and the preceding extended region of Hif-1α CAD (pink tube) in a groove on the surface of TAZ1 (blue). The side chain of Asn-803 is shown in magenta. The side chains of Cys-800 (yellow) and other critical residues that form the molecular interface are shown and labeled in black. The surfaces of interacting positively charged side chains from TAZ1 are labeled in blue. (C) Interactions involving Asn-803 of Hif-1α. Hydrogen bonds to the Hif-1α Asp-799 carbonyl and the TAZ1 Asp-346 backbone NH are drawn as black lines. The helix N-cap hydrogen bond formed by the Thr-796 Oγ is also shown. (D) Binding site for the amphipathic αC-helix and the extended region linking αC and αB. The hydrophobic side chains that form the molecular interface are shown in green. The position of the conserved Glu-817 and Asp-823 side chains and the carboxy terminus (labeled C) of Hif-1α are shown in red; the conserved basic residues on TAZ1 with which they interact are labeled in blue.

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