Windsor Peter, Ouyang Haiping, da Costa Joseph A G, Damodaran Anoop Rama, Chen Yue, Bhagi-Damodaran Ambika
Department of Chemistry University of Minnesota, Twin Cities Minneapolis, MN, 55455, United States.
Department of Biochemistry and Molecular Biology University of Minnesota, Twin Cities Minneapolis, MN, 55455, United States.
bioRxiv. 2024 May 15:2023.08.07.552357. doi: 10.1101/2023.08.07.552357.
Cells have evolved intricate mechanisms for recognizing and responding to changes in oxygen (O) concentrations. Here, we have reprogrammed cellular hypoxia (low O) signaling via gas tunnel engineering of prolyl hydroxylase 2 (PHD2), a non-heme iron dependent O sensor. Using computational modeling and protein engineering techniques, we identify a gas tunnel and critical residues therein that limit the flow of O 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 (/ = 830 ± 40 M s) in hydroxylating a peptide mimic of hypoxia inducible transcription factor HIF-1α, as compared to WT PHD2 (/ = 90 ± 9 M s). 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% O. Overall, these studies highlight activation of PHD2 as a new pathway to reprogram hypoxia responses and HIF signaling in cells.
细胞已经进化出复杂的机制来识别和响应氧气（O₂）浓度的变化。在此，我们通过对脯氨酰羟化酶2（PHD2，一种非血红素铁依赖性O₂传感器）进行气体通道工程，对细胞缺氧（低O₂）信号进行了重编程。利用计算建模和蛋白质工程技术，我们确定了一个气体通道及其内的关键残基，这些残基限制了O₂流向PHD2的催化核心。我们表明，对这些残基进行系统性修饰可以打开PHD2气体通道的收缩拓扑结构。使用动力学停流测量，以NO作为替代双原子气体，我们证明其与通道工程突变体的铁中心的结合速率提高了3.5倍。与野生型PHD2（kcat = 90 ± 9 M⁻¹ s⁻¹）相比，我们设计最有效的突变体在羟基化缺氧诱导转录因子HIF-1α的肽模拟物时，催化效率提高了9倍（kcat = 830 ± 40 M⁻¹ s⁻¹）。此外，在HEK-293T哺乳动物细胞中表达设计的PHD2突变体的质粒转染显示，在1% O₂的缺氧条件下，HIF-1α和下游缺氧反应转录本显著减少。总体而言，这些研究突出了激活PHD2作为重编程细胞中缺氧反应和HIF信号传导的新途径。
