Fig. 5: Structural analysis of the BMAL1–HIF2A heterodimer in a complex with DNA.

a, Cryo-EM map of the BMAL1–HIF2A–DNA complex. The map was sharpened using DeepEMhancer. HIF2A and BMAL1 are coloured in purple and red, respectively, with two HRE DNA strands in green and yellow. b, The overall structure of the BMAL1–HIF2A–DNA complex. c, Individual structures of HIF2A and BMAL1 within the complex. d, The four major interfaces (I to IV) between HIF2A and BMAL1. Interaction residues in BMAL1 (red) and HIF2A (purple) are shown; the BMAL1 residues mutated for pull-down analysis in e are shown in magenta. e, Pull-down analysis showing impaired interaction between GST–HIF2A and Flag-tagged BMAL1 mutants. Mutations in the bHLH, PAS-A and PAS-B domains of the BMAL1 are indicated. n = 3 independent experiments. f, The relative binding of BMAL1 mutants compared with WT BMAL1, with WT BMAL1 binding to HIF2A set to 1. n = 3 independent experiments. g, HEK293 cells overexpressing WT or mutated Flag-tagged BMAL1 were exposed to ambient hypoxia (1% O₂) for 4 h, followed by IP with Flag and blotted with the indicated antibodies. n = 3 independent experiments. h, Quantification of the relative binding in g. n = 3 independent experiments. i, HEK293 cells were transfected with either WT or mutant BMAL1 along with a HIF2A vector. A luciferase reporter assay was performed to evaluate transcriptional activation by the BMAL1–HIF2A complex of the AREG promoter, which contains a shared binding site. n = 4 independent experiments. For f,h and i, data are mean ± s.e.m. j, Schematic illustrating the substantial structural rearrangement of BMAL1 (red) when accommodating various partners to be involved in different pathways. The PAS domains of BMAL1 (red) bend towards nearly opposite direction and position separately when intertwining with HIF2A (purple). Statistical analysis was performed using one-way ANOVA (f, h and i). The diagram in j was created using BioRender.

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