Structural basis for PAS domain heterodimerization in the basic helix–loop–helix-PAS transcription factor hypoxia-inducible factor

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, 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 ...

Identification of Cys255 in HIF‐1α as a novel site for development of covalent inhibitors of HIF‐1α/ARNT PasB domain protein–protein interaction

“…1 H- 15 N HSQC experiments were recorded at 30 C on a Bruker Avance 700 MHz spectrometer equipped with a TCI cryo-probe. Mnova 7.0 (Mestrelab Research) was used in data analysis based on the sequential assignments.…”

“…This suggests that direct modulation of the HIF-2a/ARNT PasB interface may reduce hypoxia-driven gene transcription. 14,15 While not as exhaustive as classical alanine scan mutagenesis studies for identification of ''hot-spots'' on protein-protein interfaces, 16 these results suggest that the HIF-2a/ARNT PasB interface could be a potential target for therapeutic intervention.…”

Coactivators necessary for transcriptional output of the hypoxia inducible factor, HIF, are directly recruited by ARNT PAS-B

“…bHLH-PAS heterodimers are dependent on intersubunit contacts between the basic bHLH and tandem PAS domains (2)(3)(4). The second of two PAS domains, PAS-B, plays a critical role in maintaining the stability of this complex, given that mutations in HIF-2α PAS-B disrupt HIF-α/ARNT interactions and decrease transactivation in vivo (3,4).…”

The Structure of Phytochrome: A Picture Is Worth a Thousand Spectra

“…The interactions between the PAS, GAF, and PHY domains have remained opaque, and there is considerable variability among PAS-PAS and GAF-GAF interactions in published protein structures (Ho et al, 2000;Martinez et al, 2002Martinez et al, , 2005Erbel et al, 2003;Kurokawa et al, 2004;Yildiz et al, 2005). It has thus been difficult to predict how the domains in phytochrome might assemble, much less how light absorption results in signal transmission to the C-terminal portion of the protein.…”