Ligand-induced perturbation of the HIF-2α:ARNT dimer dynamics

Abstract

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.