Figures and data
Analysis of disease causing PHD2 mutants.
(A) Distribution of mutation type calculated from clinical case reports. Case reports have been compiled and summarized in the supplementary information (B) Linear map of mutant location and frequency on PHD2. The zinc finger is comprised of residues 21-58 while the catalytic core ranges from 181-426. Each dot represents a clinical report of a disease-causing mutation at the given residue. Mutations selected for analysis have been highlighted in red. (C) Structure of PHD2 HIF1αCODD complex (PDB:5L9B) with PHD2 mutant locations highlighted (yellow). PHD2 (grey) and HIF1αCODD (red) are depicted as ribbons. PHD2 mutants selected for analysis are highlighted in green and labelled. The structure was turned 260° on the y-axis to highlight all mutant locations.
Methods in HEK293A cells detect defects in some PHD2 mutants but not all.
(A) Dual luciferase reporter assays were performed to measure HIF1α transcriptional activity in the presence of PHD2 mutants in PHD2 -/- HEK293A cells. Individual data points are plotted. Loading accuracy was evaluated via immunoblotting for FLAG-tagged PHD2 and vinculin. (B) PHD2 mutant stability was measured via cycloheximide chase assay. HEK293A cells were transfected with wild type or mutant PHD2 constructs, with the amount of transfected adjusted to ensure equal expression at 0 hours. After 24 hours, the transfected cells were treated with CHX to halt protein production and monitor the stability of PHD2. Cells were harvested at various time points up to 24 hours and lysates were immunoblotted to measure PHD2 levels. (C) FLAG immunoblot density was quantified at each time point and normalized with vinculin density to yield a relative density. 24-hour time points were compared to determine significance. For A and C, bars represent mean values, and standard error is represented by error bars. * indicates P < 0.0332, ** indicates P < 0.0021, *** indicates P < 0.0002, and **** indicates P < 0.0001 (two-tailed t-test).
PHD2 mutants display instability through aggregation and decreased thermostability.
(A) Chromatograms of PHD2 catalytic cores were acquired via SEC on a Superdex200 column. Curves have been normalized according to molecular concentrations. Dashed red lines indicate mutants that were not purified. n = 2 (B) CD was performed on PHD2 mutants to predict secondary structure variations. The far-UV spectra (190-260 nm) of the purified catalytic cores was measured and converted to molar ellipticity. (C) Molar ellipticity of PHD2 mutants was monitored at 220 nm from 25 to 95 °C to evaluate thermal stability. Melting curves were generated from the CD melt. Molar ellipticity values were normalized and transformed into a fraction of folded protein and fitted with a sigmoidal curve. Melting temperatures (Tm) were determined using the EC50 of each curve. Tm and R2 are listed in Table 1.
PHD2 mutant melting temperatures calculated via CD.
PHD2 mutants have minor binding defects to HIF1α peptides.
Microscale thermophoresis was performed on fluorescently labelled PHD2 and HIF1α 555-574 CODD (A) or HIF2α 522-542 CODD (B) peptides. PHD2 P317R displayed a severe binding defect whereas the other three mutants had minor binding defects. It is suspected that amine reactive fluorescent labelling induced a binding defect on PHD2 P317R. Kd values with standard deviation are listed in Table 2.
MST determined dissociation constants between PHD2 mutants and HIFa CODD peptides.
PHD2 P317R does not hydroxylate HIFαNODD, while PHD2 A228S has very minor enzymatic defects.
An assay measuring hydroxylation of HIF1αODD (394-574) by PHD2 via NMR was performed. (A) Resonance shifting was monitored in real time to compare hydroxylation rates of A228S (blue), P317R (red) and WT (black) PHD2. PHD2 A228S displayed minorly impaired hydroxylation of both ODDs. PHD2 P317R displayed no activity on the P402 (NODD), while retaining near WT activity on P564 (CODD). (B) HSQC spectra display the resonance shifting pattern of HIF1a ODD upon prolyl hydroxylation catalyzed by PHD2 (WT, P317R, A228S) over the course of 20.2 h. Neighboring residues, A403 and I566, were used to monitor hydroxylation of P402 and P564 respectively. Spectra recorded at 0 h is shown in black while spectra recorded at the endpoint (20.2 h) is shown in red.
BLI determined binding constants between PHD2 WT and P317R and HIF1a NODD and CODD peptides.
Biolayer interferometry was performed to confirm the binding defect of PHD2 P317R observed via MST.
Measurements with HIF1αCODD and HIF1αNODD peptides indicated that PHD2 P317R did not display a severe binding defect compared to PHD2 WT. While a minor defect was observed with HIF1αCODD and PHD2 P317R, no binding defect was observed with HIF1αNODD. Binding measurements were performed in technical triplicates.
Biolayer interferometry was performed with hydroxylated peptides as a negative control.
As expected, no binding is observed with the hydroxylated peptides compared to unhydroxylated. Hydroxylated peptides are colored in red while unhydroxylated are black. The concentrations used for each experiment are as follows: PHD2 WT + HIF1αCODD: 14.6 μM, PHD2 WT + HIF1αNODD: 21.9 μM, PHD2 P317R + HIF1αCODD: 21.9 μM, PHD2 P317R + HIF1αNODD: 7.3 μM.
Purification of PHD2 181-426.
His6-PHD2 (181-426) was expressed in BL21 (DE3) E. coli cells and purified using a Ni-NTA agarose and size exclusion column (SEC). 10 μl samples were taken throughout the purification and analyzed via Coomassie stained SDS-PAGE gel. The expected final size of His6-PHD2 (181-426) is 27.76 kDa. (1) BLUelf prestained protein ladder, 15 μl (2) Lysate from BL21 (DE3) (3) Ni-NTA agarose flowthrough (4) Ni-NTA agarose 5mM imidazole wash (5) Ni-NTA agarose 30 mM imidazole wash 1 (6) Ni-NTA agarose 30 mM imidazole wash 2 (7) PHD2 elution from Ni-NTA agarose (8) Thrombin cleavage to remove His6 tag (9) Reverse Ni-NTA agarose flowthrough (10) Reverse Ni-NTA agarose wash (11) Reverse Ni-NTA agarose elution (12) Pooled SEC fractions containing PHD2 (13) BLUelf prestained protein ladder, 5 μl.
Validation of PHD2 CRISPR KO in HEK293A cells.
Homozygous PHD2 knockout (KO) HEK293A cells were generated via CRISPR. PHD2 KO was confirmed through immunoblotting. pX330 represents an empty plasmid without EGLN1 gRNA and is shown here as a positive control. Each lane represents single cell clones that were selected for PHD2 KO screening. HIF1α levels were also monitored. F4, D8, and E8 displayed complete loss of PHD2 along with HIF1α stabilization. F4, as indicated by asterisk, was chosen for use in further experiments.
Tracking proline hydroxylation using CON versus HSQC NMR.
HSQC NMR was performed to monitor resonance shifting of ODD neighboring residues, I566 and A403, which were used as reporters for P564 and P402 hydroxylation, respectively. This method yields similar results as directly tracking proline hydroxylation using CON NMR, while requiring only HIF1α-ODD to be isotopically labelled. CON results are red, while HSQC results are black. Residues associated with CODD are represented with a circle and those associated with NODD are represented with a triangle.
Sequences of the HIF1α and HIF2α peptides used in the study.
Peptides used for MST and BLI were N-terminally biotinylated.
