Structural characterization of mammalian bHLH-PAS transcription factors

Abstract

The mammalian basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) transcription factors share common architectural features that include a bHLH DNA-binding domain and tandemly positioned PAS domains. The sixteen members of this family include the hypoxia-inducible factors (HIF-1α and HIF-2α), ARNT (also known as HIF-1β), CLOCK and BMAL1. Most bHLH-PAS proteins have been genetically linked to variety of diseases in humans, including cancers, metabolic syndromes and psychiatric conditions. To function as transcription factors, the bHLH-PAS proteins must form heterodimeric complexes. Recent crystallographic studies of HIF-α-ARNT and CLOCK-BMAL1 complexes have unveiled the organization of their multi-domain bHLH-PAS-A-PAS-B segments, revealing how these architectures can give rise to unique patterns of heterodimerization. As our structural understanding becomes better integrated with ligand-discovery and target gene identification, a more comprehensive picture of their architectural and functional properties will emerge.

Figure 1

The bHLH-PAS family of transcription factors. (a) Domain organization within the bHLH-PAS polypeptides. bHLH, basic helix-loop-helix; PAS, PER-ARNT-SIM; TAD, transactivation domain; TRD, transrepression domain. (b) Members of the mammalian bHLH-PAS family and their selective heterodimerization partners. (c) NMR solution structures of the single PAS-B domains of HIF-2α (magenta, PDB: 1P97) and ARNT (green, PDB: 1x0O). (d) Crystal structure of the PAS-B heterodimer formed by mutated HIF-2α and ARNT (PBD: 3F1P). The two mutations (R247E and E362R) are shown and labeled in the figure, as well as the location of HIF-2α cavity. (e) Crystal structure of the AHR PAS-A domain homodimer (brown, PDB: 4M4X). (f–h) Overall crystal structures of three complexes CLOCK-BMAL1 (PDB: 4F3L) (f), HIF-2α-ARNT (PDB: 4ZP4) (g) and NPAS1-ARNT (h), with each of bHLH, PAS-A and PAS-B domains labeled. The colors used for CLOCK, BMAL1, HIF-2α, ARNT and NPAS1 are orange, lemon, magenta, green and cyan, respectively.

Figure 2

Analysis of domain-domain interfaces. (a) Overall structures of HIF-2α-ARNT and CLOCK-BMAL1 complexes, with each of domains labelled and interfaces numbered. The colors used for HIF-2α, ARNT, CLOCK and BMAL1 are magenta, green, orange and lemon, respectively. (b) A table summarizing the information for each domain interface about the composition and calculated interface area. The numbers in black color are for the heterodimer interfaces; and those in magenta and lemon are for the intramolecular domain interfaces of HIF-2α and BMAL1, respectively. (c) Comparisons of the interfaces between corresponding domains (Interface 1 for bHLH/bHLH, 2 for PAS-A/PAS-A, and 4 for PAS-B/PAS-B) within two heterodimers, by the side-by-side displaying (left) and ARNT/BMAL1 alignment (right). The solid arrow shows position of the ARNT PAS-B C-terminal helix (colored in black), and the dashed one shows that of the CLOCK helix (gray). (d) Comparison of the PAS-A/PAS-B interfaces revealing a conserved position at two β-strands of the PAS-A domain (“patch”) interacting with different positions at the PAS-B domains. ARNT, HIF-2α, BMAL1 and PER2 (PDB: 3GDI) are colored in green, magenta, lemon and blue, respectively. The alignment of interfaces by the PAS-A domains is displayed on the right, with the enlarged figure below showing the four conserved residues on the “patch”. (e) The unique bHLH/PAS-A interface of HIF-2α within the HIF-2α-ARNT complex.

Figure 3

Functional interactions formed between HIF-1α and other macromolecules. (a) The oxygen-dependent regulation of HIF-1α-ARNT transactivation by pVHL-mediated HIF-1α protein degradation. PHD, prolyl hydroxylase domain protein; FIH, Factor-inhibiting HIF; pVHL, the von Hippel-Lindau protein; ub, ubiquitin. (b) A diagram of HIF-1α protein showing the function and interacting macromolecules (DNA or proteins) for each domain. DBD, DNA binding domain; LBD, ligand binding domain; ODD, oxygen-dependent degradation domain; TAD, transactivation domain. (c) Crystal structure of HIF-1α-ARNT-DNA complex showing the binding and recognition of HRE site (5′-TACGTG-3′, colored in cyan) on the DNA by bHLH domains (PDB: 4ZPR). (d) Crystal structure of the catalytic domain of PHD2 complexed with the ODD region of HIF-1α including the P564 residue (DPB: 3HQR). (e) Crystal structure of P564-hydroxylated HIF-1α ODD peptide binding to the pVHL-Elongin B-Elongin C complex (PDB: 1LQB). (f) Crystal structure of FIH in complex with the C-terminal TAD fragment of HIF-1α including the N803 residue (PDB: 1H2M). (g) NMR solution structure of the TAZ1 motif of CBP binding to the C-TAD peptide of HIF-1α including the N803 residue (PDB: 1L8C). Colors used in the labels match those used in figures for the same components.