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 (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 (k_cat/K_M = 830 ± 40 M^-1 s^-1) in hydroxylating a peptide mimic of hypoxia inducible transcription factor HIF-1α, as compared to WT PHD2 (k_cat/K_M = 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.

Keywords: cellular signaling; gas tunnels; non-heme iron; oxygen sensing; protein design.

Figure 1.

A) Range of catalytic efficiencies with respect to O₂ (k_cat/K_M(O₂)) for O₂ sensing non-heme iron hydroxylases in H. sapiens. B) Protein phylogeny of PHD2 (also referred to as EGLN1) across 1844 organisms. PHD2 is well conserved throughout evolution with the protein being observed in simple animals such as T. adhaerens up to complex mammals such as H. sapiens. C) Schematic describing the HIF-based O₂ sensing and signaling system in mammals. Cells sense adequate O₂ levels (physoxia, shaded blue) via O₂-dependent proline hydroxylation of HIF-1α leading to subsequent degradation. Under hypoxia (shaded pink), HIF-1⍺ translocates to the nucleus and binds to HIF-1β. This transcription complex binds to DNA and upregulates the transcription of hypoxia response elements (HRE) to restore O₂ homeostasis.

Figure 2.

A) Full-view and B) zoomed-in crystal structure of PHD2 and HIF peptide (PDB: 5L9B) with CAVER computed tunnel. Tunnel is shown in blue with lining residues from PHD2 and HIF peptide shown in gray and teal, respectively. Bottleneck of the tunnel is represented as a circle and labelled BN. C) Heat map depicting the frequency and bottleneck radius of the primary tunnel in PHD2 variants from three concatenated 100 ns MD trajectories. The width of lane gives a time-localized estimate of the duration for which the primary tunnel stays open and the color of each block represents the bottleneck radius of each observed tunnel. D) Impact of mutations on bottleneck radius (BR) from three concatenated 100 ns MD trajectories. Box plot reveals BR distribution (0, 25, 50, 75 and 100% quantile) for each variant. Trend line connects the average BR across PHD2 variants. E) Representative structures from MD simulations of PHD2 variants. The CAVER computed tunnels are shown in blue. WT tunnel forming residues that were targets of our rational design studies are shown in yellow in each panel. Residues that were mutated are shown in dark blue. Residues that potentially constricted the tunnel but were not targets of mutagenesis are shown in gray.

Figure 3.

A) UV-Vis difference spectra of WT PHD2 active-site assembly and MLCT perturbation. 2OG-bound PHD2 exhibits a broad MLCT centered at 521 nm (red). Binding of the HIF-1α peptide (HIF) forces the loss of an aqua ligand resulting in a 10 nm blue-shift of the MLCT (blue). B) UV-Vis spectra of iron nitrosyl formation in WT PHD2 and determination of k_on(NO) for PHD2 variants (inset). Upon exposure to NO, PHD2 forms an iron nitrosyl complex which exhibits a distinct UV-Vis signature at 445 nm (green). Monitoring the kinetics of nitrosyl formation (k_obs) at varying NO concentrations leads to the determination of k_on(NO) for PHD2 variants (inset).

Figure 4

A) Hydroxylation activity screen for PHD variants at ambient O₂ (21% or 256 μM O₂). Hydroxylation activity was determined by monitoring the conversion of a HIF-1α peptide mimic via MALDI-TOF MS. Averages and S.D. are from three independent experiments. B) Steady-state kinetics as a function of varied O₂ concentrations for PHD variants. Averages and S.D. for each data point are from two independent experiments.

Figure 5.

A) Schematic of hypoxia signaling experiments. HEK-293T cells were transfected with WT and PHD2 variants and incubated under hypoxia (1% O₂). HIF-1α levels were determined using western blot. Downstream transcripts were quantified using RT-qPCR. B) HIF-1α protein levels from western blot. Values were quantified in ImageJ and normalized by WT PHD2 using the equation: normalized HIF-1α = HIF-1α(PHD2 variant/WT PHD2). Averages and S.D. are from three independent experiments. Asterisks denote statistical significance compared to WT PHD2 (*p < 0.05). C) RT-qPCR results for the quantification of CA9, HK2, LDHA, and ADM transcripts after transfection with PHD2 variants. Averages and S.D. are from three independent experiments. Asterisks denote statistical significance compared to WT PHD2 (*p < 0.05).