Principles of ligand binding within a completely buried cavity in HIF2alpha PAS-B

Abstract

Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors responsible for the metazoan hypoxia response and promote tumor growth, metastasis, and resistance to cancer treatment. The C-terminal Per-ARNT-Sim (PAS) domain of HIF2alpha (HIF2alpha PAS-B) contains a preformed solvent-inaccessible cavity that binds artificial ligands that allosterically perturb the formation of the HIF heterodimer. To better understand how small molecules bind within this domain, we examined the structures and equilibrium and transition-state thermodynamics of HIF2alpha PAS-B with several artificial ligands using isothermal titration calorimetry, NMR exchange spectroscopy, and X-ray crystallography. Rapid association rates reveal that ligand binding is not dependent upon a slow conformational change in the protein to permit ligand access, despite the closed conformation observed in the NMR and crystal structures. Compensating enthalpic and entropic contributions to the thermodynamic barrier for ligand binding suggest a binding-competent transition state characterized by increased structural disorder. Finally, molecular dynamics simulations reveal conversion between open and closed conformations of the protein and pathways of ligand entry into the binding pocket.

Figure 1. Crystal structures of HIF2α PAS-B reveal and internal solvent-filled cavity which binds artificial ligands

(a). Crystal structure of the PAS B domain of HIF2α in the apo form showing the eight bound solvent atoms within the core of the domain (red spheres). (b). Cutaway of the HIF-2α surface revealing the 290 ų internal cavity, depicted as a surface (cyan).

Figure 2. HIF2α PAS-B equilibrium ligand-binding thermodynamics

(a). Typical isothermal titration calorimetry results from compound THS-017 binding to HIF2α PAS-B. (b). Compounds evaluated in this study, shown in wireframe with binding and thermodynamic parameters derived from ITC. (c). The ligand binding pocket of HIF2α PAS-B of the THS-017 (green) and THS-020 (cyan) bound structures. All panels are shown in the same orientation and the ARNT PAS-B* domain heterodimer has been omitted for clarity.

Figure 3. NMR ZZ-exchange data from HIF-2α PAS B and compound

Shown are fits to NMR peak intensities as a function of exchange mixing time, with a subset corresponding crosspeaks shown in inset. Arrows indicating the directionality of autopeak to crosspeak are shown in the first inset.

Figure 4. Eyring plots for HIF-2α PAS B ligands KG-721, THS-002 and THS-020

(a). Temperature dependence of k_off. (b). Temperature dependence of k_on.

Figure 5. Molecular dynamics suggest routes of ligand entry and egress

Heat map of HIF2α PAS-B derived from Cα-RMSD values over the course of 35 ns molecular dynamics simulations. Water passage pathways are superimposed on the crystal structure and noted with pathway numbers and percentages of total observed water entry/exit events.