Contribution of the Per/Arnt/Sim (PAS) Domains to DNA Binding by the Basic Helix-Loop-Helix PAS Transcriptional Regulators

Abstract

The basic helix-loop-helix (bHLH) PAS transcriptional regulators control critical developmental and metabolic processes, including transcriptional responses to stimuli such as hypoxia and environmental pollutants, mediated respectively by hypoxia inducible factors (HIF-␣) and the dioxin (aryl hydrocarbon) receptor (DR). The bHLH proteins contain a basic DNA binding sequence adjacent to a helix-loop-helix dimerization domain. Dimerization among bHLH.PAS proteins is additionally regulated by the PAS region, which controls the specificity of partner choice such that HIF-␣ and DR must dimerize with the aryl hydrocarbon nuclear translocator (Arnt) to form functional DNA binding complexes. The basic helix-loop-helix (bHLH) 1 family of transcriptional regulators is characterized by a highly conserved basic DNA binding sequence adjacent to a helix-loop-helix dimerization domain, which allows homo-or heterodimerization among bHLH proteins to form functional DNA binding complexes. Structural studies indicate that the HLH dimer forms a compact four-helical bundle that positions the basic region to facilitate interaction with the DNA recognition sequence (1). There are two subclasses of bHLH proteins in which formation of protein dimers is additionally regulated by an adjacent secondary dimerization domain, either a leucine zipper (Zip) or a Per/Arnt/Sim (PAS) homology domain (2, 3).The PAS region in bHLH.PAS proteins consists of two adjacent PAS domains, degenerate repeats of ϳ130 amino acids, termed PAS A and PAS B (3). PAS domains are not restricted to bHLH DNA-binding proteins and are a widely observed protein-protein interaction and signaling module forming a highly conserved structure, despite having low primary sequence homology (3-5). The minimal PAS domain structure consists of a 5-stranded ␤-sheet with a central ␣-helical PAS core region, often containing a binding site for small ligands, and a helical connector spanning the two halves of the ␤-scaffold (5, 6). Some PAS domains also appear to include a highly variable N-terminal ␣-helical cap, although the function of this structure is not clear (7,8).In vivo, the aryl hydrocarbon nuclear translocator (Arnt) is the common obligate dimerization partner for a number of bHLH.PAS proteins, including the dioxin (or aryl hydrocarbon) receptor (DR), the hypoxia inducible factors (HIF) 1␣ and 2␣, and the Sim proteins (9). The cytoplasmic DR, on binding an activating ligand such as dioxin or structurally related halogenated hydrocarbons within the PAS B domain (10), translocates to the nucleus and heterodimerizes with Arnt to form a functional DNA binding complex (11). The DR/Arnt dimer recognizes the xenobiotic response element (XRE) in enhancers of target genes and promotes transcription of a battery of xenobiotic-metabolizing enzymes. The HIF␣ proteins mediate cellular responses to oxygen. They are rapidly turned over at normoxia whereas under hypoxic stress, HIF␣ is stabilized in a form competent to recruit coactivators (12). Under hypoxia, HIF␣ heterodimerizes with Ar...

Role of the Per/Arnt/Sim Domains in Ligand-dependent Transformation of the Aryl Hydrocarbon Receptor

“…5c). These findings not only suggest that the PASB domain is capable of initiation of dimerization and that the PASA domain is necessary for successful completion of this process (resulting in Hsp90 displacement), but are also consistent with the previously reported inhibitory effect of the PASB domain on transformation (16,18) and demonstration of PASA as a major contributor to the stability of the AhR:Arnt dimer (14). Moreover, it has been found that a conformational change in the AhR is required for stable dimer formation because geldanamycin-or salt-mediated dissociation of Hsp90 from the AhR resulted in less stable dimer formation (5,13).…”

“…Although the PASA domain is essential for formation of the stable AhR:Arnt dimer, the PASB domain contains a ligand binding site and one of the Hsp90 binding sites (6,14,15). The PASB domain has been suggested to exert an inhibitory effect on AhR transformation and its deletion results in an AhR that is constitutively active with respect to its transformation/DNA binding and transcriptional activation functions (16 -18).…”

ARNT PAS-B has a fragile native state structure with an alternative β-sheet register nearby in sequence space

“…ARNT constitutively dimerizes with other bHLH-PAS family members that themselves are regulated, including AHR and hypoxia-inducible factors (HIFs). PAS domains are essential for stable heterodimer formation, as supported by biochemical studies that show that deletion of PAS domains from ARNT (12,13) or its partners [e.g., HIF-2␣ (14)] impairs the formation and activity of regulatory complexes. Biophysical studies of PAS domains from HIF-2␣ and ARNT have shown that the C-terminal PAS-B domains from these proteins bind each other via an antiparallel ␤-sheet-␤-sheet packing (15,50).…”

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).…”