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

Stefano Motta,Claudia Minici,Dario Corrada,Laura Bonati,Alessandro Pandini,Ozlem Keskin

doi:10.1371/journal.pcbi.1006021

Abstract

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

Highlights

Cells in a tumor react to low oxygen levels with a metabolism modification induced by the activation of hypoxia inducible factors (HIFs) through dimerization with a partner protein and binding to a DNA target
We exploit molecular dynamics to identify the crucial interfaces in the Hypoxia inducible factors (HIFs) dimer stabilization and, by comparing the results obtained in the bound and unbound forms, we reveal the mechanism of ligand inhibition at atomic detail
Hypoxia inducible factors (HIFs) are obligate heterodimers belonging to the basic helix−loop −helix superfamily of transcription factors that mediate the physiological responses to hypoxia

Summary

Introduction

Hypoxia inducible factors (HIFs) are obligate heterodimers belonging to the basic helix−loop −helix (bHLH) superfamily of transcription factors that mediate the physiological responses to hypoxia. This extensive protein family is characterized by a 4–6 basic amino acids next to a HLH dimerization domain, both required to properly bind DNA targets. Within the bHLH superfamily HIFs belong to the subfamily containing the PER/aryl hydrocarbon receptor nuclear translocator (ARNT)/single minded (SIM) (PAS) homology domain (bHLH-PAS) [1,2,3] Based on their heterodimerization behavior, bHLH-PAS proteins can be further divided into two classes: class I members only form heterodimers with a member of class II, which, by contrast, can promiscuously homo- and heterodimerize. HIF-α subunit dimerizes with the constitutive ARNT ( known as HIF-β) subunit, the best characterized class II protein; other members of this class include the tissue restricted ARNT2, and the circadian rhythm proteins BMAL1 and BMAL2 [2,3]

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Discussion

Conclusion