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

<div><p>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.</p></div

Sequence and structure of the HIF-2α:ARNT dimer.

<p>a. Domains and secondary structures of ARNT and HIF-2α in the bHLH-PAS region. Secondary structures were provided by DSSP on the PDB file 4ZP4: helices are displayed as squiggles and β-strands as arrows, and labelled according to the PAS domain nomenclature [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006021#pcbi.1006021.ref018" target="_blank">18</a>]. b. Cartoon representation of the unbound dimer structure (4ZP4) with the modelled loops and linkers: ARNT in cyan, HIF-2α in orange. Modelled segments are represented in darker colours.</p

Distance cross correlation matrices for apo and holo simulations.

<p>The correlation matrices are shown on the top (upper triangular for apo–lower for holo) with domains (circle = bHLH, vertical lines = PAS-A, light filled = PAS-B) and secondary structure profiles (black = helix, light-grey = sheet) on the sides. A 3D representation of the network derived from the DCCM is represented at the bottom with a close-up of the ARNT PAS-A A’ helix region.</p

Cα RMSF plots for each PAS domain in the dimer.

<p>Structured regions with higher fluctuations are highlighted in green and discussed in text. The long and highly flexible PAS-A loops were excluded from calculation and are indicated by dash lines. Helices and β-strands are represented as black and grey bars, respectively, and labelled according to <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006021#pcbi.1006021.g001" target="_blank">Fig 1A</a>.</p

Perturbed dimerization energy at the holo PAS-B:PAS-B interface.

<p>3D representation of the PAS-B:PAS-B interface: ARNT in cyan, HIF-2α in orange. Residues that mostly affect the dimerization energy at this interface in presence of the 0X3 ligand (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006021#pcbi.1006021.s014" target="_blank">S3 Table</a>) are shown in sticks.</p

Water mediated hydrogen-bond network between S304 and the 0X3 ligand.

<p>In the unbound form (top left panel) the S304 sidechain interacts with the T321 backbone, maintaining this region of the G-strand unstructured. In most of the representative structures of the inhibitor-bound form (remaining panels) the S304 sidechain is involved in a water-mediated hydrogen-bond with the ligand, and the H-bonds between the S304 and T321 backbones facilitate a complete structuring of the strand. H-bond were detected using GROMACS default parameters.</p

Optimal and suboptimal path analysis for the unbound (apo) and 0X3-bound (holo) HIF-2α:ARNT dimer structures.

<p>Left and right panels: paths are shown as red lines connecting residues in the three interface domains (HIF-2α PAS-B and PAS-A in orange cartoon, ARNT PAS-A in cyan cartoon). Central panel: path length distributions between the HIF-2α S304 (in PAS-B) and ARNT A171 (in the A’ helix of PAS-A) residues.</p

Molecular Dynamics for the Optimal Design of Functionalized Nanodevices to Target Folate Receptors on Tumor Cells

Atomistic details on the mechanism of targeting activity by biomedical nanodevices of specific receptors are still scarce in the literature, where mostly ligand/receptor pairs are modeled. Here, we use atomistic molecular dynamics (MD) simulations, free energy calculations, and machine learning approaches on the case study of spherical TiO2 nanoparticles (NPs) functionalized with folic acid (FA) as the targeting ligand of the folate receptor (FR). We consider different FA densities on the surface and different anchoring approaches, i.e., direct covalent bonding of FA γ-carboxylate or through polyethylene glycol spacers. By molecular docking, we first identify the lowest energy conformation of one FA inside the FR binding pocket from the X-ray crystal structure, which becomes the starting point of classical MD simulations in a realistic physiological environment. We estimate the binding free energy to be compared with the existing experimental data. Then, we increase complexity and go from the isolated FA to a nanosystem decorated with several FAs. Within the simulation time framework, we confirm the stability of the ligand–receptor interaction, even in the presence of the NP (with or without a spacer), and no significant modification of the protein secondary structure is observed. Our study highlights the crucial role played by the spacer, FA protonation state, and density, which are parameters that can be controlled during the nanodevice preparation step