Computational Repacking of HIF-2α Cavity Replaces Water-Based Stabilized Core  Fernando Corrêa, Jason Key, Brian Kuhlman, Kevin H. Gardner  Structure  Volume 24, Issue 11, Pages 1918-1927 (November 2016) DOI: 10.1016/j.str.2016.08.014 Copyright © 2016 Elsevier Ltd Terms and Conditions

Structure 2016 24, 1918-1927DOI: (10.1016/j.str.2016.08.014) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 HIF-2α PAS-B Contains a Fully Hydrated Cavity (A) Crystal structure of HIF-2α PAS-B (PDB: 3F1P; Scheuermann et al., 2009) demonstrates the presence of a fully hydrated 290 Å3 cavity within the protein core. (B) The HIF-2α cavity is filled with eight ordered water molecules, which form an extensive hydrogen bond network with several backbone and polar side chain atoms, with distances between donor and acceptor groups labeled in the figure. See also Table S1. Structure 2016 24, 1918-1927DOI: (10.1016/j.str.2016.08.014) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Redesigning HIF-2α Protein Core (A) Overview of the Rosetta protocol, starting with energy minimization of HIF-2α PAS-B backbone coordinates (PDB: 3F1P; Scheuermann et al., 2009) using Foldit (Eiben et al., 2012), followed by two distinct routes of sequence optimization. In route A, all residues lining the cavity were allowed to mutate, while in route B, residues participating in the internal native hydrogen bond network were held fixed. Rosetta energy values of designed molecules were compared with the value for the native protein. Of the top six designs with best energy scores and other practical advantages (i.e., numbers of mutated residues), we selected three for experimental testing (one soluble [S], two insoluble [I]) while the remaining three were not tested (NT). Column RS provides information on Rosetta energy score, while SOL gives information for solubility. (B) Overlap of HIF-2α D1 NMR structure ensemble with the initial Rosetta model and X-ray structure of the WT HIF-2α PAS-B (PDB: 3F1P; Scheuermann et al., 2009) indicate that protein repacking caused no distinctive changes in the overall protein topology. (C) The NMR ensemble demonstrates a high degree of precision in side-chain orientations, which in turn agree with the predicted Rosetta models. (D) Core repacking generates a newly designed HIF-2α PAS-B protein with an extensively reduced internal cavity, approximately 25% of the volume of the WT protein. Residue substitutions are highlighted in the right-hand panel. (E) NMR data indicate that several residues lining internal cavity (labeled) maintain contacts with water molecules, suggesting the presence of residual solvent within the protein core. Mutated residues are highlighted in red, while those with the WT sequence are labeled in black. See also Figures S1–S5. Structure 2016 24, 1918-1927DOI: (10.1016/j.str.2016.08.014) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 HIF-2α D1 Binds to ARNT (A) Comparison of 15N-1H HSQC spectra of isolated U-15N labeled ARNT (100 μM, black) with HIF-2α PAS-B D1 (HIF-2α D1, 200 μM, red) or WT HIF-2α PAS-B (HIF-2α WT, 200 μM, purple). Addition of native or designed HIF-2α PAS-B led to the same peaks broadening, suggesting that similar heterodimer complexes are formed in solution. (B) SEC demonstrates the formation of a stabilized heterodimer between single mutants HIF-2α PAS-B D1∗ (R247E) and ARNT PAS-B∗ (E362R). Dashed lines correspond to the elution volumes of molecular weight standards: aprotinin (6.5 kDa), RNase A (13.7 kDa), carbonic anhydrase (29 kDa), ovalbumin (43 kDa), conalbumin (75 kDa), and Blue Dextran 2000 (void). See also Figure S5. Structure 2016 24, 1918-1927DOI: (10.1016/j.str.2016.08.014) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Cavity Repacking Disrupts HIF-2α PAS-B Binding to an Artificial Small-Molecule Ligand Isothermal titration calorimetry (ITC) data demonstrate that the binding of a well-characterized HIF-2α PAS-B (A) high-affinity ligand (compound 2, KD = 78 nM; Scheuermann et al., 2013) is disrupted upon core repacking (HIF-2α D1, B). See also Figure S4. Structure 2016 24, 1918-1927DOI: (10.1016/j.str.2016.08.014) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 Microscale Thermophoresis Shows Cavity Repacking Disrupts Binding to Small Molecules Titration assays monitoring binding of red fluorescent-labeled designed (D1, black) or WT (red) HIF-2α PAS-B to compound 2 through thermophoretic mobility (error bars at ± 1SD). Lack of detectable changes in fluorescence signal clearly showed that protein repacking disrupted binding to small molecules in comparison with native protein (KD = 140 ± 48 nM). See also Figure S4. Structure 2016 24, 1918-1927DOI: (10.1016/j.str.2016.08.014) Copyright © 2016 Elsevier Ltd Terms and Conditions