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, 100 (26), 15504-9

Structural Basis for PAS Domain Heterodimerization in the Basic helix--loop--helix-PAS Transcription Factor Hypoxia-Inducible Factor

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Structural Basis for PAS Domain Heterodimerization in the Basic helix--loop--helix-PAS Transcription Factor Hypoxia-Inducible Factor

Paul J A Erbel et al. Proc Natl Acad Sci U S A.

Abstract

Biological responses to oxygen availability play important roles in development, physiological homeostasis, and many disease processes. In mammalian cells, this adaptation is mediated in part by a conserved pathway centered on the hypoxia-inducible factor (HIF). HIF is a heterodimeric protein complex composed of two members of the basic helix-loop-helix Per-ARNT-Sim (PAS) (ARNT, aryl hydrocarbon receptor nuclear translocator) domain family of transcriptional activators, HIFalpha and ARNT. Although this complex involves protein-protein interactions mediated by basic helix-loop-helix and PAS domains in both proteins, the role played by the PAS domains is poorly understood. To address this issue, we have studied the structure and interactions of the C-terminal PAS domain of human HIF-2alpha by NMR spectroscopy. We demonstrate that HIF-2alpha PAS-B binds the analogous ARNT domain in vitro, showing that residues involved in this interaction are located on the solvent-exposed side of the HIF-2alpha central beta-sheet. Mutating residues at this surface not only disrupts the interaction between isolated PAS domains in vitro but also interferes with the ability of full-length HIF to respond to hypoxia in living cells. Extending our findings to other PAS domains, we find that this beta-sheet interface is widely used for both intra- and intermolecular interactions, suggesting a basis of specificity and regulation of many types of PAS-containing signaling proteins.

Figures

Fig. 1.
Fig. 1.
Oxygen-dependent regulation and domain architecture of HIF proteins. (a) HIF regulation is tightly linked to intracellular oxygen levels. Under normoxic conditions, HIFα is posttranslationally hydroxylated, promoting its degradation [modification of the oxygen-dependent degradation domain (ODD)] and interfering with its ability to interact with CBP/p300 coactivators (modification of the transcriptional activation domains NTAD and CTAD). These modifications are not made under hypoxic conditions, allowing HIFα to accumulate and enter the nucleus where it associates with ARNT and binds to HREs upstream of hypoxia-activated genes. The red box highlights the HIFα and ARNT PAS-B domains. (b) Domain topology of HIFα subunits, including a bHLH domain, two PAS domains, and C-terminal regulatory domains. A sequence alignment of the HIFα PAS-B orthologs is shown, with bold letters indicating the mutated residues described in the text. HIF-2α PAS-B secondary structure elements are indicated with a gray background.
Fig. 2.
Fig. 2.
Solution structure of HIF-2α PAS-B. (a) Superimposition of 20 lowestenergy structures for HIF-2α PAS-B, calculated as indicated in the text. (b) Ribbon diagram of the structure closest to the mean of the ensemble shown in a. Circles indicate the approximate locations of the ligand-binding sites of several PAS domains (, –36).
Fig. 3.
Fig. 3.
Characterization of the HIF-2α/ARNT PAS-B-binding interaction. (a) Titration of unlabeled ARNT PAS-B (black, 0 μM; light blue, 200 μM; blue, 600 μM; red, 800 μM ARNT) into a 200 μM 15N-labeled HIF-2α PAS-B solution. Arrow shows direction of peak shifts with increasing amounts of ARNT. Residues with peak broadening beyond detection during the titration are indicated with *.(b) Normalized peak heights of HIF-2α PAS-B (38 resonances) plotted against increasing amounts of ARNT PAS-B. The concentration dependence of the observed reduction in peak heights can be fit to a 1:1 binding event with a Kd of ≈30 μM (dotted line).
Fig. 4.
Fig. 4.
Identification of the ARNT PAS-B-binding site on the surface of HIF-2α PAS-B. (a) Chemical-shift changes from 15N/1H HSQC spectra of HIF-2α PAS-B in the presence of 800 μM ARNT are plotted as a function of residue number. The red line indicates chemical-shift changes >0.16 ppm. Residues with peak broadening beyond detection are shown as an arbitrary chemical-shift change of 0.5 ppm. Secondary structure elements are indicated with a gray background. (b) Surface representations of HIF-2α PAS-B showing the location of the ARNT PAS-B-binding site. Colors indicate residues with large chemical-shift changes (>0.4 ppm) or broadening beyond detection (red), residues with a significant chemical-shift changes (>0.16 ppm) (orange), and the site of the mutated residues that disrupt the HIF/ARNT PAS-B interactions (yellow). Figs. 4 and 6 were made with pymol (www.pymol.org).
Fig. 5.
Fig. 5.
Point mutations in the HIFα PAS-B central β-sheet disrupt the binding of ARNT PAS-B. (a) Superimposed 15N/1H HSQC spectra of 250 μM 15N labeled HIF-2α PAS-B (Left) or triple mutant (Q322E/M338E/Y342T) (Right). Spectra in the presence of 900 μM unlabeled ARNT PAS-B are shown with red contours; those without ARNT are shown in black contours. Similar data for HIF-1α PAS-B are provided in Supporting Methods. (b) PAS-B domain interaction is important to form a biologically active HIF/ARNT complex. A construct expressing a luciferase reporter under the control of an HRE promoter was transfected into Ka-13 (columns 1–5) or CHO (column 6) cells along with various HIFα constructs. Values represent the average luciferase activity of three samples, with bars indicating standard error. Luciferase expression was induced by cotransfection of HIF-1α (column 2) or HIF-2α (column 4), particularly under hypoxic conditions. Cotransfection of trHIF-1α (column 3) or trHIF-2α (column 5), full-length HIFα proteins containing the three PAS-B mutations, shows a significant drop in luciferase activity compared with wild-type HIFα.
Fig. 6.
Fig. 6.
Versatility of protein interactions involving PAS domain β-sheets. HIF2α is shown in the same orientation as Fig. 4b and colored by residues experiencing significant 15N/1H chemical shifts on complex formation (red) and those used to generate the complex-disrupting trHIF-2α (blue). Phototropin (AsLOV2) (36) and photoactive yellow protein (33) highlight the α-helices external to the PAS core (magenta) and any atoms located within 5 Å of those helices (pink). HERG (42) shows functionally important, solvent-exposed residues (dark blue) and residues present in a surface hydrophobic patch suggested to be important for channel function (light blue) (42).

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