br Acknowledgement The work was supported by the NSFC throug
6. Acknowledgement The work was supported by the NSFC through Grant Nos. 11535016 and 11475232. It was also supported by CAS. The authors thank their collaborators for beneficial discussions and enthusiastic supports in the simulations and calculations.
Introduction Hypoxia-inducible factors (HIFs) are responsible for cellular adaptations to low oxygen stress through the activation of transcriptional processes, such as erythropoiesis and angiogenesis , , , , , . Because these processes are related to tumor growth and progression, HIFs have become attractive targets for cancer therapy , , , , , , , , , . To execute their transcriptional function, HIFs must form a heterodimer between an oxygen-dependent α subunit (HIF-1α or HIF-2α) and an oxygen-independent β subunit (HIF-1β), which is also referred to as aryl hydrocarbon receptor nuclear translocator (ARNT). All HIF-1α, HIF-2α, and ARNT proteins have a basic-helix-loop-helix (bHLH) domain for DNA reading at their amino termini, followed by two tandem Per-ARNT-Sim (PAS) domains, namely PAS-A and PAS-B, for HIF-α–ARNT dimerization and one or two transactivation domains (TAD) at their carboxyl termini (Fig. 1(A)). An oxygen-dependent degradation domain (ODDD), which is required for oxygen-dependent hydroxylation and degradation, lies between the PAS-B and TAD domains of HIF-1α and HIF-2α. Under normoxia, two proline residues in the ODDD of HIF-α (P402 and P564 in HIF-1α; P405 and P531 in HIF-2α) are hydroxylated by prolyl hydroxylase domain (PHD) enzymes. The proline modifications cause the degradation of HIF-1α and HIF-2α through the ubiquitin-proteasome (26S) pathway , , , . In addition, an asparagine residue, N803 in HIF-1α and N851 in HIF-2α, is hydroxylated to block the interaction between HIF-α and the p300 coactivator. Under hypoxia, PHD Nexturastat A are inactivated, facilitating the entry of accumulated HIF-α into the nucleus to dimerize with ARNT. The HIF-α–ARNT heterodimer binds to hypoxia response element (HRE) sequences in the promoter region of HIF target genes. Furthermore, the HIF-α–ARNT heterodimer binds to transcriptional coactivator proteins, p300 and cyclic AMP response element-binding protein, to promote the context-dependent expression of multiple genes that offset hypoxia at the cellular level , , . Wu et al.  demonstrated the structures of the HRE gene-bound HIF-1α–ARNT (PDB code: 4ZPR), HRE gene-bound HIF-2α–ARNT (PDB code: 4ZPK), and apo HIF-2α–ARNT (PDB code: 4ZP4), characterizing their bHLH, PAS-A, and PAS-B domains through X-ray crystallography. Fig. 1(B) presents the structure of the HRE gene-bound HIF-2α–ARNT heterodimer where HIF-2α and ARNT intertwine through their PAS domains, and two adjacent bHLH domains to form a DNA-reading head for recognizing the HRE gene. Notably, loops linking PAS-A to PAS-B as well as PAS-A to bHLH in ARNT are long and flexible, and therefore were not defined in X-ray crystallography. By contrast, linkages among the bHLH, PAS-A, and PAS-B domains in HIF-2α are relatively short, making HIF-2α compact compared with ARNT. However, the loop linking the bHLH and PAS-A domains in HIF-2α was defined in the HRE gene-bound HIF-2α–ARNT–DNA complex but not in the apo HIF-2α–ARNT complex. The missing coordinates of the loop between the bHLH and PAS-A domains of HIF-2α indicate the extent of flexibility required for the intertwined association in the DNA-reading head of the HIF-2α–ARNT heterodimer. The overall heterodimer structure reveals that the bHLH, PAS-A, and PAS-B domains of HIF-2α are in close contact, whereas they are spread out, encompassing HIF-2α, in ARNT because of its long flexible loops. Moreover, because long flexible loops link the bHLH, PAS-A, and PAS-B domains, ARNT can form heterodimers with various bHLH-PAS family members, such as HIF-1α, HIF-2α, HIF-3α, AhR, AHR repressor, and neuronal PAS 1 , , , , , , , , .