Hypoxia inducible factors (HIFs) are transcription factors belonging to the basic helix−loop−helix
28 PER-ARNT-SIM (bHLH-PAS) protein family with a role in sensing oxygen levels in the cell.
29 Under hypoxia, the HIF- degradation pathway is blocked and dimerization with the aryl
30 hydrocarbon receptor nuclear translocator (ARNT) makes HIF- transcriptionally active. Due to the
31 common hypoxic environment of tumors, inhibition of this mechanism by destabilization of HIF-
32 α:ARNT dimerization has been proposed as a promising therapeutic strategy. Following the
33 discovery of a druggable cavity within the PAS-B domain of HIF-2α, research efforts have been
34 directed to identify artificial ligands that can impair heterodimerization. Although the
35 crystallographic structures of the HIF-2α:ARNT complex have elucidated the dimer architecture
36 and the 0X3-inhibitor placement within the HIF-2α PAS-B, unveiling the inhibition mechanism
37 requires investigation of how ligand-induced perturbations could dynamically propagate through the
38 structure and affect dimerization. To this end, we compared evolutionary features, intrinsic
39 dynamics and energetic properties of the dimerization interfaces of HIF-2α:ARNT in both the apo
40 and holo forms. Residue conservation analysis highlighted inter-domain connecting elements that
41 have a role in dimerization. Analysis of domain contributions to the dimerization energy
42 demonstrated the importance of bHLH and PAS-A of both partners and of HIF-2α PAS-B domain
43 in dimer stabilization. Among quaternary structure oscillations revealed by Molecular Dynamics
44 simulations, the hinge-bending motion of the ARNT PAS-B domain around the flexible PAS45
A/PAS-B linker supports a general model for ARNT dimerization in different heterodimers.
46 Comparison of the HIF-2α:ARNT dynamics in the apo and 0X3-bound forms indicated a model of
47 inhibition where the HIF-2α-PAS-B interfaces are destabilised as a result of water-bridged ligand48
protein interactions and these local effects allosterically propagate to perturb the correlated motions
49 of the domains and inter-domain communication. These findings will guide the design of improved
50 inhibitors to contrast cell survival in tumor masses