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

Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 01/2004; 100(26):15504-9. DOI: 10.1073/pnas.2533374100
Source: PubMed


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.

Download full-text


Available from: Ozgur Karakuzu, May 20, 2015
  • Source
    • "The NMR structures of hypoxia-inducible factor 2a (HIF-2a) and the aryl hydrocarbon receptor nuclear translocator (ARNT) PAS-B domains were chosen as the templates for homology modeling. Their coordinate files were obtained from the Protein Data Bank: entries 1P97 for HIF-2a (Erbel et al., 2003) and 1X0O for ARNT (Card et al., 2005). A three-dimensional model of the AaMet PAS-B domain was generated using MODELLER version 9v7 (Sali and Blundell, 1993). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Methoprene-tolerant (Met) protein is a juvenile hormone (JH) receptor in insects. JH-bound Met forms a complex with the βFtz-F1-interacting steroid receptor coactivator (FISC) and together they regulate JH response genes in mosquitoes. Both proteins contain basic-helix-loop-helix (bHLH) and PAS motifs. Here we demonstrated that FISC is the obligatory partner of Met for binding to JH-response elements (JHREs). Met or FISC alone could not bind a previously characterized JHRE, while formation of the Met-FISC complex was necessary and sufficient to bind to the JHRE. This binding required participation of the DNA-binding domains of both Met and FISC. The optimal DNA sequence recognized by Met and FISC contained a core consensus sequence GCACGTG. While formation of the Met-FISC complex in mosquito cells was induced by JH, heterodimerization and DNA binding of bacterially expressed Met and FISC were JH-independent, implying that additional mosquito proteins were required to modulate formation of the receptor complex.
    Full-text · Article · Jul 2014 · Molecular and Cellular Endocrinology
  • Source
    • "However, the availability of crystal and NMR structures of homologous proteins of the PAS superfamily allowed development of theoretical models for the LBD of the mouse AhR by applying homology modeling techniques (Pandini et al., 2007). The most recent homology model of the mouse AhR LBD (Fig. 3; Pandini et al., 2009) was built using the NMR-determined structures of the PAS B domains of HIF-2a (Erbel et al., 2003) and ARNT (Card et al., 2005) proteins as templates, given their higher degree of sequence identity and similarity with the AhR PAS B domain and their functional relationship to the AhR (Kewley et al., 2004). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The Ah receptor (AhR) is a ligand-dependent transcription factor that mediates a wide range of biological and toxicological effects that result from exposure to a structurally diverse variety of synthetic and naturally occurring chemicals. Although the overall mechanism of action of the AhR has been extensively studied and involves a classical nuclear receptor mechanism of action (i.e., ligand-dependent nuclear localization, protein heterodimerization, binding of liganded receptor as a protein complex to its specific DNA recognition sequence and activation of gene expression), details of the exact molecular events that result in most AhR-dependent biochemical, physiological, and toxicological effects are generally lacking. Ongoing research efforts continue to describe an ever-expanding list of ligand-, species-, and tissue-specific spectrum of AhR-dependent biological and toxicological effects that seemingly add even more complexity to the mechanism. However, at the same time, these studies are also identifying and characterizing new pathways and molecular mechanisms by which the AhR exerts its actions and plays key modulatory roles in both endogenous developmental and physiological pathways and response to exogenous chemicals. Here we provide an overview of the classical and nonclassical mechanisms that can contribute to the differential sensitivity and diversity in responses observed in humans and other species following ligand-dependent activation of the AhR signal transduction pathway.
    Preview · Article · Sep 2011 · Toxicological Sciences
  • Source
    • "When the modelled structures were superimposed on the crystal structure of the HIF-2α/Arnt PAS.B dimer, where interaction is through the β-sheet surfaces (49), it was clear that the most severe of our identified mutations in both Arnt and AhR cluster on faces away from the HIF-2α/Arnt PAS.B β-scaffold dimerization interface (Figure 6b). This suggests the PAS.A interactions use a face of the domain, encompassing α-C, β-C and β-D, that is different from the HIF-2α/Arnt PAS.B interface, as well as other examples of PAS domain interactions (9,17–21). Further, AhR and Arnt appear to interact through equivalent regions of PAS.A (Figure 6a). Partner specific Arnt D217 is centrally located in the putative dimerization interface in this model, while the S190P substitution on the opposite face may alter interaction with AhR through effects on packing. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The basic helix–loop–helix (bHLH).PAS dimeric transcription factors have crucial roles in development, stress response, oxygen homeostasis and neurogenesis. Their target gene specificity depends in part on partner protein choices, where dimerization with common partner Aryl hydrocarbon receptor nuclear translocator (Arnt) is an essential step towards forming active, DNA binding complexes. Using a new bacterial two-hybrid system that selects for loss of protein interactions, we have identified 22 amino acids in the N-terminal PAS domain of Arnt that are involved in heterodimerization with aryl hydrocarbon receptor (AhR). Of these, Arnt E163 and Arnt S190 were selective for the AhR/Arnt interaction, since mutations at these positions had little effect on Arnt dimerization with other bHLH.PAS partners, while substitution of Arnt D217 affected the interaction with both AhR and hypoxia inducible factor-1α but not with single minded 1 and 2 or neuronal PAS4. Arnt uses the same face of the N-terminal PAS domain for homo- and heterodimerization and mutational analysis of AhR demonstrated that the equivalent region is used by AhR when dimerizing with Arnt. These interfaces differ from the PAS β-scaffold surfaces used for dimerization between the C-terminal PAS domains of hypoxia inducible factor-2α and Arnt, commonly used for PAS domain interactions.
    Full-text · Article · May 2011 · Nucleic Acids Research
Show more