Gas tunnel engineering of prolyl hydroxylase reprograms hypoxia signaling in cells

Abstract

Cells have evolved intricate mechanisms for recognizing and responding to changes in oxygen concentrations. Here, we have manipulated cellular hypoxia (low oxygen) signaling via gas tunnel engineering of prolyl hydroxylase 2 (PHD2), an iron-dependent oxygen sensor. Using computational modeling and protein engineering techniques, we identify a gas tunnel and critical residues therein that limit the flow of oxygen to PHD2's catalytic core. We demonstrate that systematic modification of these residues can open the constriction topology of PHD2's gas tunnel. Our most effectively designed mutant displays 11-fold enhanced hydroxylation efficiency in a biochemical assay that employs a peptide mimic of hypoxia-inducible transcription factor HIF-1α. Furthermore, transfection of plasmids that express these engineered PHD2 mutants in HEK-293T mammalian cells reveal 2-fold reduction in HIF-1α levels under extreme hypoxia. Overall, our studies highlight activation of PHD2 as a new modality in reprogramming HIF signaling pathways to assume physoxia-like conditions despite extreme hypoxia.