Gas Tunnel Engineering of Prolyl Hydroxylase Reprograms Hypoxia Signaling in Cells

• Published in: Angewandte Chemie 2024-11, Vol.136 (48), p.n/a
• Main Authors: Windsor, Peter, Ouyang, Haiping, G. da Costa, Joseph A., Rama Damodaran, Anoop, Chen, Yue, Bhagi‐Damodaran, Ambika
• Format: Article
• Language: English
• Subjects: Cell activation; cellular signaling; Diatomic gases; Flow measurement; gas tunnels; Hypoxia; Iron; Mammalian cells; Mutants; non-heme iron; oxygen sensing; Plasmids; Prolyl hydroxylase; protein design; Protein engineering; Protein folding; Residues; Topology; Transfection
• ISSN: 0044-8249; 1521-3757
• DOI: 10.1002/ange.202409234

Cells have evolved intricate mechanisms for recognizing and responding to changes in oxygen (O2) concentrations. Here, we have reprogrammed cellular hypoxia (low O2) signaling via gas tunnel engineering of prolyl hydroxylase 2 (PHD2), a non‐heme iron dependent O2 sensor. Using computational modeling and protein engineering techniques, we identify a gas tunnel and critical residues therein that limit the flow of O2 to PHD2’s catalytic core. We show that systematic modification of these residues can open the constriction topology of PHD2’s gas tunnel. Using kinetic stopped‐flow measurements with NO as a surrogate diatomic gas, we demonstrate up to 3.5‐fold enhancement in its association rate to the iron center of tunnel‐engineered mutants. Our most effectively designed mutant displays 9‐fold enhanced catalytic efficiency (kcat/KM=830±40 M−1 s−1) in hydroxylating a peptide mimic of hypoxia inducible transcription factor HIF‐1α, as compared to WT PHD2 (kcat/KM=90±9 M−1 s−1). Furthermore, transfection of plasmids that express designed PHD2 mutants in HEK‐293T mammalian cells reveal significant reduction of HIF‐1α and downstream hypoxia response transcripts under hypoxic conditions of 1 % O2. Overall, these studies highlight activation of PHD2 as a new pathway to reprogram hypoxia responses and HIF signaling in cells. The gas tunnel of PHD2, an iron‐based O2 sensing enzyme, is engineered using a computation‐guided rational design approach. The engineered mutants exhibit an increased rate of gas transport to the active site, along with increased catalytic activity in vitro. These mutants also display enhanced intracellular activity demonstrating the ability to reprogram hypoxia signaling by activating PHD2.