Computational Repacking of HIF-2α Cavity Replaces Water-Based Stabilized Core

Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors central to hypoxia response and cancer development. Within the HIF-2 complex, one domain (HIF-2α PAS-B) contains a large (290 Å³) buried cavity filled with water molecules within its hydrophobic core. Such cavities are uncommon except in the case of ligand-binding proteins, leading to the hypothesis that HIF-2α can be regulated by small molecules. The development of artificial HIF-2α inhibitors validates this hypothesis but raises questions about the impact of this cavity on HIF-2α PAS-B structure and function. To answer these points, we used computational methods to construct a repacked protein containing a smaller cavity within the native fold. Experimental validation of a five-mutation variant confirms achieving these objectives and stabilizing the folded structure. Complementary functional data establish that ligands cannot bind this variant although heterodimerization remains unchanged. Altogether, our strategy innovatively addresses the roles of solvated cavities in maintaining protein stability and function.