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, HIF␣ and ARNT. Although this complex involves protein-protein interactions mediated by basic helixloop-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-2␣ by NMR spectroscopy. We demonstrate that HIF-2␣ 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-2␣ central ␤-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 ␤-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. C ellular responses to oxygen availability are essential for the development and homeostasis of mammalian cells, demonstrated most critically by the link between the cellular adaptation to reduced tissue oxygenation and disease progression (1, 2). In mammalian cells, these responses are mediated in part by the hypoxia-inducible factor (HIF), a heterodimeric transcription factor composed of HIF␣ and aryl hydrocarbon receptor nuclear translocator (ARNT, also known as HIF␤) (3). HIF activity is tightly controlled under normoxic conditions by multiple O 2 -dependent hydroxylation events of the HIF␣ subunit, which coordinately promote the ubiquitin-mediated destruction of this protein (4) and impair its ability to interact with transcriptional coactivators (5, 6) (Fig. 1a). These controls are relieved during hypoxia, allowing HIF to activate the transcription of genes that facilitate metabolic adaptation to low oxygen levels and increase local oxygen supply by angiogenesis (7).All three isoforms of HIF␣ [HIF-1␣, -2␣ (EPAS1), and -3␣] (8, 9) and ARNT belong to the basic helix-loop-helix (bHLH)-Per-ARNT-Sim (PAS) family of eukaryotic transcription factors, which contain bHLH and PAS domains (Fig. 1). The bHLH domains of these proteins serve as dimerization elements, helping determine the specificity of complex formation while providing a DNA-binding interface composed of the basic regions from each monomer (10). PAS domains are widespread components of signal transduction proteins, currently identified in Ͼ2,000 proteins from organisms in all three kingdoms of life. These domains, shown to be ...

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For several well-documented reasons, it has been challenging to develop artificial small molecule inhibitors of protein/protein complexes. Such reagents are of particular interest for transcription factor complexes given links between their misregulation and disease. Here we report parallel approaches to identify regulators of a hypoxia signaling transcription factor complex, involving the ARNT subunit of the HIF (Hypoxia Inducible Factor) activator and the TACC3 (Transforming Acidic Coiled Coil Containing Protein 3) coactivator. In one route, we used in vitro NMR and biochemical screening to identify small molecules that selectively bind within the ARNT PAS (Per-ARNT-Sim) domain that recruits TACC3, identifying KG-548 as an ARNT/TACC3 disruptor. A parallel, cell-based screening approach previously implicated the small molecule KHS101 as an inhibitor of TACC3 signaling. Here, we show that KHS101 works indirectly on HIF complex formation by destabilizing both TACC3 and the HIF component HIF-1α. Overall, our data identify small molecule regulators for this important complex and highlight the utility of pursuing parallel strategies to develop protein/protein inhibitors.

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a basic helix-loop-helix Period/ARNT/Single-minded (bHLH-PAS) protein that controls various biological pathways as part of dimeric transcriptional regulator complexes with other bHLH-PAS proteins. The two PAS domains within ARNT, PAS-A and PAS-B, are essential for the formation of these complexes because they mediate protein-protein interactions via residues located on their ␤-sheet surfaces. While investigating the importance of residues in ARNT PAS-B involved in these interactions, we uncovered a point mutation (Y456T) on the solvent-exposed ␤-sheet surface that allowed this domain to interconvert with a second, stable conformation. Although both conformations are present in equivalent quantities in the Y456T mutant, this can be shifted almost completely to either end point by additional mutations. A high-resolution solution structure of a mutant ARNT PAS-B domain stabilized in the new conformation revealed a 3-residue slip in register and accompanying inversion of the central I␤-strand. We have demonstrated that the new conformation has >100-fold lower in vitro affinity for its heterodimerization partner, hypoxia-inducible factor 2␣ PAS-B. We speculate that the pliability in ␤-strand register is related to the flexibility required of ARNT to bind to several partners and, more broadly, to the abilities of some PAS domains to regulate their activities in response to small-molecule cofactors.alternative conformations ͉ NMR ͉ Per-ARNT-Sim ͉ proline isomerization ͉ protein folding

The aryl hydrocarbon receptor nuclear translocator (ARNT) serves as the obligate heterodimeric partner for bHLH-PAS proteins involved in sensing and coordinating transcriptional responses to xenobiotics, hypoxia, and developmental pathways. Although its C-terminal transactivation domain is dispensable for transcriptional activation in vivo, ARNT has recently been shown to use its N-terminal bHLH and/or PAS domains to interact with several transcriptional coactivators that are required for transcriptional initiation after xenobiotic or hypoxic cues. Here we show that ARNT uses a single PAS domain to interact with two coiled coil coactivators, TRIP230 and CoCoA. Both coactivators interact with the same interface on the ARNT PAS-B domain, located on the opposite side of the domain used to associate with the analogous PAS domain on its heterodimeric bHLH-PAS partner HIF-2␣. Using NMR and biochemical studies, we identified the ARNT-interacting motif of one coactivator, TRIP230 as an LXXLL-like nuclear receptor box. Mutation of this motif and proximal sequences disrupts the interaction with ARNT PAS-B. Identification of this ARNT-coactivator interface illustrates how ARNT PAS-B is used to form critical interactions with both bHLH-PAS partners and coactivators that are required for transcriptional responses. PAS (Per-ARNT-Sim)3 domains are small, modular domains that mediate interactions with proteins (and often small molecule ligands) to coordinate cellular responses to diverse environmental stimuli (1). The largest class of eukaryotic PAS-containing proteins are transcription factors containing a basic helix-loop-helix (bHLH) DNA-binding domain followed by two PAS domains, known as the bHLH-PAS family (2). As functional heterodimers, bHLH-PAS complexes bind DNA at specific promoter elements and recruit transcriptional coactivators via their C-terminal transactivation domains (TADs) to regulate transcriptional responses to diverse stimuli. In particular, the ubiquitously expressed protein ARNT is of central importance within the bHLH-PAS family, acting as the obligate heterodimeric partner for the aryl hydrocarbon receptor (AHR), hypoxia-inducible factor-␣ (HIF-␣), and single-minded (SIM) to regulate transcriptional responses to xenobiotics, hypoxia, and neurogenesis, respectively (3). ARNT heterodimeric complexes are involved in the etiology and progression of many forms of human cancer through the metabolic activation of dietary and environmental carcinogens (AHR⅐ARNT) and the adaptation of solid tumors to chronic hypoxia (HIF-␣⅐ARNT, also known as HIF) (4, 5).Heterodimerization of bHLH-PAS proteins is mediated by contacts between both the bHLH and the tandem PAS domains, with inter-PAS domain contacts playing a critical role in the specificity and stability of heterodimer formation (Fig. 1A) (6 -9). Structures of the HIF-2␣⅐ARNT PAS-B heterodimer demonstrate that the two PAS-B domains associate via their ␤-sheets (Refs. 10, 11 and Fig. 1B) and disruption of this interface by point mutations significantly attenuate...