Structural basis for binding of the renal carcinoma target hypoxia‐inducible factor 2α to prolyl hydroxylase domain 2

Abstract The hypoxia‐inducible factor (HIF) prolyl‐hydroxylases (human PHD1‐3) catalyze prolyl hydroxylation in oxygen‐dependent degradation (ODD) domains of HIFα isoforms, modifications that signal for HIFα proteasomal degradation in an oxygen‐dependent manner. PHD inhibitors are used for treatment of anemia in kidney disease. Increased erythropoietin (EPO) in patients with familial/idiopathic erythrocytosis and pulmonary hypertension is associated with mutations in EGLN1 (PHD2) and EPAS1 (HIF2α); a drug inhibiting HIF2α activity is used for clear cell renal cell carcinoma (ccRCC) treatment. We report crystal structures of PHD2 complexed with the C‐terminal HIF2α‐ODD in the presence of its 2‐oxoglutarate cosubstrate or N‐oxalylglycine inhibitor. Combined with the reported PHD2.HIFα‐ODD structures and biochemical studies, the results inform on the different PHD.HIFα‐ODD binding modes and the potential effects of clinically observed mutations in HIFα and PHD2 genes. They may help enable new therapeutic avenues, including PHD isoform‐selective inhibitors and sequestration of HIF2α by the PHDs for ccRCC treatment.


| INTRODUCTION
In humans and other animals, the hypoxia-inducible factor (HIF) transcription factors play key roles in responses to limiting O 2 availability by promoting context-dependent expression of genes working to alleviate the effects of hypoxia.HIF is an α,β-heterodimeric protein; the levels of HIFβ, also known as the aryl hydrocarbon receptor nuclear translocator protein, are not regulated directly by O 2 concentrations. 1 contrast, as a consequence of catalysis by the HIF prolyl hydroxylase domain enzymes (human PHD1-3), HIFα levels are strongly regulated by O 2 availability. 2,3PHD1-3 catalyze trans-4-prolyl hydroxylation of the N-and C-terminal oxygen-dependent degradation (NODD and CODD, respectively) domains in HIF1-3α isoforms (note, HIF3α only contains a CODD) (Figure 1A).5][6][7] A second HIFα hydroxylase, factor-inhibiting HIF (FIH) catalyzes the C3 hydroxylation of an asparagine-residue in the C-terminal transcriptional activation domains of HIF1α and HIF2α (but not HIF3α), a modification that reduces the interaction of HIF with histone acetyltransferases (CREB binding protein and p300). 810][11] In hypoxia, PHD1-3 activity decreases and HIF1-3α levels rise (Figure S1). 2,3HIF1-3α translocate to the nucleus and dimerize with F I G U R E 1 Overview of HIFα prolyl hydroxylase catalysis and view of the conserved double-stranded β-helix fold of the 2OG oxygenases.(A) PHD1-3 catalyze 2OG-dependent trans-4-prolyl hydroxylation of HIFα isoforms.Sequence alignment of the five human HIFα N-/C-terminal oxygen-degradation domains.The secondary structure (α-helix: red and β-strand: orange-NODD/green-CODD) assignments are as observed in crystal structures of HIF1α 394-413 -NODD and HIF1α 556-574 -CODD in complex with the PHD2 catalytic domain (PDB: 5L9V and 5L9B).The hydroxylated proline is marked with a yellow star.(B, C) The distorted double-stranded β-helix core fold (teal βI-βVIIΙ refer to the eight DSBH strands), the β2-β3 finger loop (red cartoon), and the N-/C-terminal extensions of the DSBH (N: α1-α3 and C: α4 helices-blue) are labeled.(B) The PHD2 181-407 .Mn(II).NOG.HIF1α-CODD.3C(6YW3) complex is depicted as a cartoon showing the N-terminal binding site of the cocrystallized 3C cyclic peptide (yellow sticks with Connolly surface).(C) A view of the DSBH of the PHD2 181-407 .Mn(II).2OG.HIF2α-CODD (7Q5X) complex, the main focus of this work.Key active site residues (teal), target proline (orange), and NOG (lemon) are represented as sticks.The HIF1α-CODD (olive) and HIF2α-CODD (orange) substrates are displayed as cartoons.Waters (red) and Mn (violet) are shown as spheres.CODD, C-terminal oxygen-dependent degradation; HIF, hypoxia-inducible factor; NODD, N-terminal oxygen-dependent degradation.
HIFβ to form transcriptionally active α,β-HIF heterodimers (Figure S1). 2,3,1213 The PHDs and FIH are both Fe(II) and 2-oxoglutarate (2OG)dependent oxygenases that couple hydroxylation with the conversion of 2OG to succinate and CO 2 (Figures 1A and S1). 3,8,11,14,150][21][22] PHD2 also forms a relatively stable complex with Fe and 2OG, even after exposure to O 2 .0][21][22][23] The PHDs are more sensitive than FIH to limiting O 2 levels 24 and HIFα-NODD hydroxylation is reported to be more sensitive than CODD hydroxylation to O 2 levels. 24,25The PHDs also show different selectivity toward the various HIFα-ODDs, with PHD3 being reported to be particularly selective for the HIF1αand HIF2α-CODD domains (Figures 1A and S1). 12,22,26ystal structures of PHD2.HIFα-ODD complexes and kinetic studies have revealed the importance of a conformationally mobile loop (the β2-β3 loop) that links β2 and β3 of the catalytic domain of PHD2 and which is involved in HIFα-ODD binding and selectivity; in the PHD2.
8][29][30] Overall, these observations support the proposal that β2-β3 loop dynamics are important both in catalysis and determining PHD/HIFα-ODD substrate selectivity, though the precise molecular details are undefined (Figure 1B,C). 27,29,30L gene mutation is common in clear cell renal cell carcinoma (ccRCC) patients causing upregulation of HIFα isoforms, so increasing the expression of the HIF2α target VEGF, in a manner apparently promoting tumorigenesis and cancer progression. 31,32Belzutifan (MK-6482 or PT-2977) inhibits HIF2α-mediated expression and is used for ccRCC treatment (Figure S1). 33Mutations in EPAS1 (encoding for HIF2α), EGLN1 (encoding for PHD2), are also linked to disease, including familial/idiopathic erythrocytosis and ccRCC (Figure S2A). 34us, structural information of how the PHDs bind HIFα-ODDs, and in particular HIF2α, may inform on the clinically observed pathologies of the mutant EPAS1-related diseases.
Although structures of HIF1α-CODD and NODD in complex with the catalytic domain of PHD2 are available, analogous structures with HIF2α-CODD have not been reported. 11,27,28The PHD.
Here, we report high-resolution crystal structures of the truncated catalytic domain of PHD2 (residues 181-407) in complex with HIF2α-CODD (residues 523-542), a manganese ion, and 2OG or its close isostere N-oxalylglycine (NOG) (Figures 1C and 2).The structures inform on differences in HIFα-ODD binding that may influence the different selectivity of the PHDs.
HIF2α-CODD complexes, clear electron density for 3C was not observed in the crystal structures (Figure S3B), that is there was insufficient electron density to model in 3C as reported in the PHD2 181-426 .HIF1α-CODD complex structure (PDB: 6YW3).
Comparison of the overall PHD2 181-426/407 .Mn.NOG/2OG.HIF2α-CODD structures reveals conservation of the distorted double stranded β-helix (DSBH) and associated HIFα-ODD substrate binding elements (Figures 1B,C and 3). 11,27,28,36,37The NOG and 2OG structures are very similar to each other (backbone RMSD: 0.078 Å) and, to a somewhat lesser extent in terms of details, with other PHD2.HIFα-ODD structures (Figure 3). 11,27,28In particular, variations in the conformations of α1, the β2-β3 loop, and the C-terminal α4 regions are observed.Note that the constructs used vary in the length of their C-terminus and in our case reversible binding of 3C may promote formation of, or stabilize, specific conformations that promote crystallization (Figure 3). 28e previously reported PHD2 active site chemistry is also conserved in the PHD2 181-407 .Mn.NOG/2OG.HIF2α-CODD structures (Figure 3), 37 with a single manganese ion (substituting for iron) being coordinated by the side chains of His313, Asp315, and His374, as well as a well-defined water molecule/hydroxide ion. 11,27,28The use of Mn(II) in PHD2 crystallization/inhibition is of interest given links between disease associated with Mn metabolism and erythropoiesis. 382OG and NOG bind the manganese ion in a bidentate manner via their C1 carboxylate and C2 carboxylate oxygens.The 2OG and NOG C5 carboxylates are positioned to interact with the guanidino group of Arg383 (Figures 2 and 3).The 2OG C1 carboxylate coordinates the manganese ion in the position adjacent to Pro531 HIF2α , i.e., 2OG coordination is in an off-line mode, suggesting that at some stage a metal-centered rearrangement may be required to present the reactive ferryl adjacent to the oxidized Pro531 HIF2α C-H bond.The pyrrolidine ring of Pro531 HIF2α is clearly observed in the C4-endo conformation, as observed in previous PHD2-substrate complex structures. 11,27,39However, the C4 of Pro564 HIF1α-CODD is $0.5 Å closer to the metal than the Pro531 HIF2α-CODD, though whether this has any kinetic relevance is unclear (Figure 3A). 11llectively, these observations reveal a conserved mode of binding for HIFα-ODD substrate proline-residues at the active site, including with respect to the substrate proline-ring conformation and off-line 2OG binding.The overall binding mode is also conserved in PHD type prolyl hydroxylases in Trichoplax adhaerens (TaPHD), 40 including bacteria (Pseudomonas putida PHD (PPHD) and Bacillus anthracis prolyl-4-hydroxylase (BaP4H)), which catalyze prolyl hydroxylation of elongation factor-thermally unstable (EF-Tu) (Figure S4). 41,42This conservation is important because these features T A B L E 1 Data collection and crystallographic processing statistics of the PHD2 181-407.HIF2α complex structures.are proposed to be involved in the HIF/PHD/VHL "O 2 -sensing" mechanism.Thus, off-line 2OG binding may help enable the slow reaction of the PHDs with O 2 , a property proposed to be important in their "O 2 -sensing" role. 43C4 proline hydroxylation is proposed to enable a stereoelectronic preference for the C4-exo over the C4-endo proline ring conformation, with the former being observed in PHD.
HIFα-ODD complexes and the latter in VHL.hydroxylated-HIFα-ODD complexes. 11,39,44mparison of reported PHD2 181-426 .NOG.HIF1α-ODD complex structures (PDB: 3HQR and 5L9V) with the new PHD2 181-407 .NOG.Note: Single crystal diffraction data were collected from samples at 100K with conventional, rotation-based methods.Statistics for the highest-resolution shell are in parentheses.R factor is equal to P hkl jjFobs( hkl )j À jFcalc( hkl )jj/ P hkl jFobs( hkl )j and was calculated for the working set of reflections (R work ). 1 Å for the Lys402 Cα (Figure 3).These differences in α4 might, in part, reflect variations in the HIFα-ODD substrate binding modes at the C-terminal region of PHD2 and their impact on catalysis. 27,29,30wever, it cannot be ruled out if these are caused by variations in crystal lattice packing, possibly relating to 3C binding. 28e β2-β3 loops of the PHDs are important in catalysis and in determining HIFα-ODD substrate selectivity. 27,30,37In the absence of HIFα-ODD substrates, the β2-β3 loop is likely conformationally mobile/disordered and principally adopts conformations that are not near the active site, including those observed by crystallography. 11,27- 29,45In all reported PHD2.HIFα-ODD structures, the β2-β3 loop folds to enclose the substrate proline residue in the active site, as is observed in our PHD2 181-407 .HIF2α-CODD structures (Figure 2).
Although the overall PHD2 181-407 .HIF2α-CODD structures with NOG and 2OG are very similar (RMSD: 0.078 Å), there are some differences in the conformations of the β2-β3 loop involving PHD2 residues Gln243-Asp246 (Figure 2).In the 2OG.HIF2α-CODD complex, the side chain amide NΗ 2 group of PHD2 Gln243 β2-β3 is positioned to form a hydrogen bond (2.74 Å) with the main chain carbonyl O atom of PHD2 Asp246 (Figure 2B).This hydrogen bond is, however, not observed in the NOG.HIF2α-CODD complex, where Gln243 β2-β3 is oriented away from the loop and adopts a more solvent-exposed position (Figure 2A).
Although further work is required, given the 2OG and isostreric NOG structures have the same space group and similar crystal packing, the differences in the β2-β3 loop between them suggests that small differences at the active site region may influence the conformation of relatively distant structural elements within PHD2 181-407 .This observation is interesting in part because recent studies on the mechanism of isopenicillin N synthase, which is structurally and mechanistically related to the 2OG-dependent oxygenases, imply that conformational changes distant from the active site are involved in catalysis. 46It is also of interest because it supports the previous proposal that inhibition by 2OG mimetics involves effects on structural dynamics in addition to simple blockade of 2OG binding in the active site. 30,47,48Modeling studies on 2OG oxygenases, including demethylases, also imply the relevance of conformational changes both at and relatively distant from the active site during catalysis. 49,50However, defining the precise effects of Fe-binding inhibitors on the overall structural dynamics (and in some cases including complexed substrate) is technically challenging, requiring room temperature solution as well as low-temperature biophysical crystallographic studies. 48Hence, in addition to studies with isolated PHDs, empirical optimization of inhibitors in a cellular context is desirable.
We compared the β2-β3 loop conformations in In our PHD2 181-407 .HIF2α 523-542 -CODD structures, interactions of the HIFα-ODD residues with residues both on the N-terminal and C-terminal sides of the target proline peptide with PHD2 are conserved, including the interaction with α4 (Arg396 PHD2 -Asp539 HIF2α- CODD ) (Figure 4C).The β2-β3 loop residues (Val241, Ser242, Lys244, and Ile251) interact with Glu527/Thr528 HIF2α-CODD ("XX" residues of the LXXLAP motif of HIF2α-CODD) in a similar fashion to the previously reported PHD2 structures with HIF1α-NODD/-CODD. 27ese combined observations further support a role for the β2-β exposed.In conformation-B, Glu538 HIF2α-CODD projects towards a symmetry-related chain forming a hydrogen bond with Glu538 HIF2α- CODD in a symmetry-related molecule (Figure S3A).It is possible that the conformational flexibility of Gly537 HIF2α /Glu538 HIF2α unit relates to the presence of the additional Gly537 in HIF2α-CODD on the C-terminal side of the hydroxylated proline, compared to HIF1α/3α-CODD and HIF1/2α-NODD (Figure 1A).When compared with HIF1/2α-NODD, hydrophobic Ile-residues are in the same position as the polar Gly/Glu unit of HIF2α-CODD; these may alter the dynamics of the enzyme-substrate interaction (Figure 1A). 27e above-described differences may, at least to some extent, influence PHD2 HIFα-isoform selectivity.To investigate the preference of PHD2 towards HIFα-CODD substrates, we carried out assays comparing the PHD2 catalyzed hydroxylation of HIF1-3α CODD peptides, both individually and as a mixture (Figure 5).The results with PHD2 and individual peptides showed no clear preference for the HIF1-3α-CODD.However, when conducting the reaction with a 1:1:1 mixture of HIF1-3α-CODD peptides, PHD2 showed a clear preference for HIF1α-over the HIF3α-and HIF2α-CODDs (Figure 5A).This result supports the proposal that the presence of the additional Gly537/Glu538 unit in HIF2α-CODD on the C-terminal side of the hydroxylated proline, compared to HIF1α/3α-CODD causes PHD2 to preferentially catalyze hydroxylation of HIF1α peptide over HIF2-3α-CODDs peptides. 51The Gly537/Glu538 unit in HIF2α-CODD may also reflect differences in crystallization conditions required for the various PHD2.HIFα-ODD complexes (Table S1).interactions between PHD3 and HIF1α-NODD might, in part, rationalize the preference of PHD3 for HIF1-2α-CODD over NODD. 26mparison of the HIFα-ODD binding modes of PHD1-3 with respect to the N-terminal sides of the target substrate prolines (Figure S6) also implies differences in the PHD.HIFα-ODD interactions between PHD1/PHD3 models and the PHD2 crystal structures.In Ser242 PHD2 (β2-β3 loop) is substituted by Pro64 PHD3 , the latter of which cannot make the same polar interactions (Figure S6).Pro64 PHD3 may also alter the dynamics of the β2-β3 loop during catalysis compared with PHD1-2.

| Structural comparison of PHD1-3.HIF1-3α between crystallographic and AlphaFold predicted structures
The predicted weaker interactions of PHD3.NODD residues both on the N-and C-terminal sides of the proline substrate residue may explain the low level of PHD3.HIF1-2α-NODD turnover observed from these substrates. 26,27Due to the preliminary nature of the models and given the multitude of interactions in the PHD.HIFα-ODD complexes, to what extent these structural conformational changes/ induced fit processes directly influence catalysis and PHD isoform selectivity remains unclear.

| DISCUSSION
Interactions between the PHDs and the HIFα-ODDs play a central role in the hypoxic response in humans and other animals.PHD-like prolyl-hydroxylases are also present in certain non-animal eukaryotes and prokaryotes, though to date these identified substrates are not HIF (like) transcription factors.In early metazoan PHD/HIF-containing organisms, there is typically only one PHD and one HIFα, as exemplified by studies on T. adhaerens (Figure S4). 40,54However, in humans and other complex HIF containing animals, there are commonly more than one PHD isoform and more than one HIFα isoform, though (typically) likely only one von Hippel-Lindau protein and one FIH. 54There are some subsequent bioinformatic studies that have revealed multiple PHDs and HIFα-ODDs present in complex animals, at least in part, a reflection of the need for context-dependent regulation of the hypoxic response. 54,55This increased complexity may introduce vulnerabilities with respect to mutations enabling specific disease states including cancer, for example, by using modulation of one HIFα isoform in tumor progression whilst maintaining an ability to execute a robust hypoxic response, with another HIFα isoform. 31,56,57In this regard, the link between HIF2α upregulation in ccRCC (most commonly associated with VHL gene mutation) and diseases related to erythrocytosis is of interest.
Reduction of HIF2α mediated expression is the mode of action of Belzutifan which is used to treat ccRCC. 31,33However, there is a need for new treatments for ccRCC and other diseases associated with VHL/HIFα/PHD gene mutations.Such treatments could, in principle, involve modulation of PHD.HIFα-ODD interactions, for example, by sequestering a HIF2α-CODD in complex with PHD, possibly in a manner that signals for a non-VHL mediated protein degradation process, by using a small-molecule and/or metal ion that promotes the PHD.HIF2α-CODD interaction.The structures presented here may help in the design of such small molecules.
At least some of the observed EPAS1/HIF2α mutations will likely impact on PHD catalysis as they involve residues that interact with the PHD2 active site as shown by our PHD2 181-407 .NOG/2OG.
HIF2α-CODD structures (PDB: 7Q5V and 7Q5X) and inferred by models of PHD1-3.HIFα-ODD complexes (Figure S2).Strikingly, some of the clinically observed mutations (M535V, M535T, G537W, and G537R) affect the Gly537/Glu538 unit, thus likely altering PHD.HIFα-ODD binding potentially in a manner affecting catalytic efficiency and/or HIFα-ODD selectivity in a disease-relevant manner.Differences in PHD1-3.HIF1-3α-ODD related interactions are also important in the normal hypoxic response and knowledge of them may help enable treatments including modulation of specific sets of HIF target genes.
The combined crystallographic and NMR studies, further highlight the importance of the conformationally mobile β2-β3 loop and the C-terminal PHD region in HIFα-ODD hydroxylation and selectivity (Figures 2 and 4). 11,27,29,30,36,45However, the available evidence also supports the dynamic nature of PHD.HIFα-ODD interactions, at least in certain stages of the catalytic cycle.This means that structurebased attempts to modulate PHD.HIFα-ODD interactions, for example, to alter PHD isoform selectivity, should be coupled with empirical approaches in cells (note PHD.HIFα interactions likely involve other components and regions beyond the immediate PHD catalytic domain and HIFα-ODD reactions).
The presence of an additional residue (Gly537/Glu538 unit) in HIF2α-CODD compared to other HIFα-ODDs, likely results in increased flexibility of HIF2α-CODD, possibly weakening its binding to PHD2 (Figure S3A).This may, at least partially, explain the preference of PHD2 for HIF1α > HIF3α > HIF2α-CODDs as observed in our biochemical studies (Figure 5).However, it is important to note that multiple interactions occur between the PHDs and the HIFα-ODDs and given the dynamic nature of at least some of these interactions, it is difficult to predict the effects of individual residue changes with confidence.
By contrast, the dynamic and multivalent interaction of the PHDs with the overall HIFα-ODDs, the chemistry in the immediate active site vicinity appears to be highly conserved in the PHDs, an observation which even extends, at least substantially, to PHD-like enzymes with non-HIFα substrates (Figure S4C). 41,42The conservation includes with respect to the nature of Fe(II) and 2OG binding, including the positioning of the 2OG C1 carboxylate adjacent to the methylene of the proline-residue that undergoes hydroxylation, an arrangement that is likely partially responsible for the unusually slow reaction of the PHDs with O 2 , though other factors also likely impact this aspect of the mechanism. 43Another chemically relevant conservation is the conformation of the unhydroxylated substrate proline ring at the active site, which to date has always been observed in the C4-endo formation, at least in the PHD2 substrate complexes.PHD catalyzed trans-4-hydroxylation results in a bias of the proline ring to the C4-exo protein conformation, due to operation of a stereoelectronic effect, as observed in pVHL-hydroxylated-HIFα-ODD complex structures. 39mparison of AlphaFold models of PHD1 and PHD3 with

| Expression and protein purification
The PHD2 181-407 -pET-28a(+) plasmid was expressed in Escherichia coli BL21(DE3) cells (New England Biolab Inc.). 27Expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (OD 600 nm 0.6-1.2) at 28 C for 3-4 h. 27Cells were harvested and stored at À80 C until purification.Cells were freeze-thawed at 4 C in the lysis buffer (20 mM Tris-HCl pH 7.5 RT, 0.5 M NaCl, 5 mM imidazole, and 5% (vol/vol) glycerol). 27 glycerol), finally with 5 CV of lysis buffer. 30The loaded column was washed with 30 CV of wash buffer (   ). 65 PHENIX.Xtriage was used to assess the data quality of the reflections. 66Phaser-Molecular replacement (PHASER-MR) was used to phase the processed diffraction data for the structures (PDB: 7Q5V and 7Q5X). 67A previously determined structure of PHD2 (PDB: 3HQR) was used as a search model for MR-phasing. 11COOT (version 0.9.5, CCP4) was used to semi-manual model build based on the overlaid 2mF o -DF c and difference mF o -DF c electron density maps from the phased structures (PDB: 7Q5V and 7Q5X). 68,69e geometry of the model was adjusted based on calculated electron density maps and was improved in COOT with subsequent refinement cycles using PHENIX.Refine. 66,70Three cycles were typically run for each refinement round before manual fitting.PHENIX.Refine was used to modify and improve the model in iterative cycles. 70

F I G U R E 2
Structural basis for HIF2α-CODD binding to PHD2.(A, B) Views from structures of PHD2 181-407 .Mn.2OG/NOG.HIF2α 523-542 -CODD displayed as cartoons (PHD2 181-407 -blue; HIF2α-orange) (PDB: 7Q5V-A and 7Q5X-B).PHD2 residues (blue/teal), β2-β3 loop (red), 2OG/NOG (lime), and HIF2α (orange) residues are shown as sticks.The electron density map (contoured at 1.0 σ) is depicted as a mesh (blue).Key polar interactions are represented by black dashes.Waters (red) and Mn (violet) are displayed as spheres.Differences in the β2-β3 loop conformation in the two structures are highlighted by a black circle and a red arrow.Note the C4-endo conformation of the substrate proline ring in both structures.CODD, C-terminal oxygen-dependent degradation; HIF, hypoxia-inducible factor.

3
loop in positioning the HIFα-ODD substrates at the PHD active site, notably via interactions with the conserved LXXLAP motif in HIFα-ODDs.Despite most of the interactions appearing to be conserved in the different HIFα-ODDs, a striking conformational feature is observed at the C-terminal site of HIF2α 523-542 -CODD in both the 2OG and NOG PHD2 181-407 .HIF2α-CODD complex structures (PDB: 7Q5V and 7Q5X).Glu538 HIF2α-CODD is observed to adopt two conformations in both structures, one of which, conformation-A, is less solvent exposed and one of which, conformation-B, is more solvent F I G U R E 4 Comparison of PHD.HIFα-ODD binding interactions.(A-C) Views showing the conformations of HIF1-2α (HIF1α-NODD-green, HIF1α-CODD-olive, and HIF2α-CODD-orange) as observed by crystallography in complex with truncated PHD2 displayed as sticks with solventexcluded surface representation (Connolly) (PDB: 5L9V-gray, 7Q5V-blue, and 3HQR-dark gray).HIF1-2α-ODDs are displayed as cartoons and sticks.Hydrogen bonding and electrostatic interactions are represented by black dashes.(A) Disulfide cross-linked residues in (A), produced to enable stable complex formation, are shown as yellow sticks.CODD, C-terminal oxygen-dependent degradation; HIF, hypoxia-inducible factor; NODD, N-terminal oxygen-dependent degradation.

4. 5 |
Solid phase extraction-MS based enzymatic activity assaysActivity assays were conducted using a RapidFire ® RF360 sampling robot (Agilent Technologies).Samples were loaded onto a C4 SPE cartridge (Agilent Technologies) and peptides were eluted with 85% (vol/vol) acetonitrile and 15% (vol/vol) water mixture added with 0.1% (v/v) formic acid.Real-time activity assays were performed in reaction buffer containing 50 mM Tris-HCl pH 7.8 and 50 mM NaCl.Stock solutions of each component were made freshly.100 mM stock solution of sodium-L-ascorbate and 50 mM stock solution of 2OG were made in water (LC-MS Grade, LiChrosolv ® ). 10 mM peptides stock solution were made in dimethyl sulfoxide (DMSO).To limit oxidation of Fe(II) to Fe(III), a 100 mM stock solution of (NH 4 ) 2 Fe(II)(SO 4 ) was made in HCl (20 mM), then diluted to 10 mM with water (LC-MS Grade, LiChrosolv ® ). 1 mL final volume solutions containing 200 μM sodium-L-ascorbate, 20 μM 2OG, 20 μM (NH 4 ) 2 Fe(II)(SO 4 ) and 10 μM peptide (HIF1α 556-574 -CODD, HIF2α 523-542 -CODD, or HIF3α 484-505 -CODD) were prepared as control reactions.1 mL solutions containing 200 μM sodium-L-ascorbate, 20 μM 2OG, 20 μM (NH 4 ) 2 Fe(II)(SO 4 ), and 10 μM of a 1:1:1 mixture of peptides (HIF1α 556-574 -CODD, HIF2α 523-542 -CODD, and HIF3α 484-505 -CODD) were prepared for the competition reactions.About 500 μL of the substrate mixture was transferred into 96-well polypropylene plates (Agilent Technologies).After a first injection onto the C4 SPE cartridge (Agilent Technologies), data acquisition was paused, then 500 μL of 300 nM PHD2 181-426 in reaction buffer was added into the well to initiate the reaction.The control reactions were monitored for 32 min (one injection every 2.5 min).The competition reactions were monitored for 67 min (one injection every 5 min).The positive ion mode was used to monitor peptide charge states.RapidFire Integrator software (Agilent Technologies) was used to integrate the area of the peaks extracted from the chromatogram.Excel was used to calculate percent (%) hydroxylation of the CODD peptide substrates using the formula: % hydroxylated substrate = 100 Â hydroxylated/(hydroxylated + nonhydroxylated peptide).Oxidation of the methionine residues in the CODD sequences was 4%-6% in the no enzyme control.Every data set was normalized to a no enzyme buffer control.

4. 6 |
X-ray data analysis and software X-ray diffraction data were collected at Diamond Light Source synchrotron at I24 MX beamline and autoprocessed with Xia2 (DIALS, Diamond Light Source Ltd. Model improvement was assessed by the decrease in, and convergence of R work /R Free values between cycles of refinement.MolProbity was used to assess the geometric quality of the refined model and to guide re-building in COOT.68,71Resolution was defined depending on the completeness of the resolution bin (>95% in all resolution bins).PDB extract online tool (version 3.24, Research Collaboratory for Structural Bioinformatics PDB) was used to prepare coordinate and structure factors files in macromolecular CIF format (mmCIF) to be uploaded to Onedep for PDB deposition.72 PyMOL™ (Schrodinger) was used for graphical representation and structure alignment.