Born to sense: biophysical analyses of the oxygen sensing prolyl hydroxylase from the simplest animal Trichoplax adhaerens

Figures & data

Figure 1 Overview of the HIF system.

Notes: (A) Comparison of domain architectures of HIFα and HIFβ in T. adhaerens and humans. In contrast to the multiple PHD/HIFα-isoforms and ODDs present in vertebrates, the prime components of the HIF system in T. adhaerens involve only one PHD and one HIFα, which has a single ODD.¹⁶ Arrows indicate assigned prolyl and asparaginyl hydroxylation sites. (B) Domain structures of HIF prolyl hydroxylases (PHDs) in T. adhaerens and humans. Domain acronyms: 2OG dioxygenase domain (oxygenase), MYeloid, Nervy, and DEAF-1 (MYND)-type zinc finger domain (MYND). (C) Outline of the conserved mechanism of the response to chronic hypoxia in animals. The HIF transcription factors are regulated by PHD catalyzed hydroxylation of prolyl residues in the ODD(s) of HIFα under normoxia. Recognition of the hydroxylated prolyl residues by pVHL is followed by ubiquitination by the E3 ubiquitin ligase, which tags HIFα for proteasomal degradation. In humans, FIH, which is only sporadically present in non-vertebrate animals,¹⁶ constitutes an additional oxygen dependent regulatory element of HIF activity. FIH catalyzes the hydroxylation of an asparagine residue in the CTAD of HIFα, thus disrupting its interaction with the CBP/p300 transcriptional co-activator, and hindering transcriptional activation.¹¹
Abbreviations: PHD, Prolyl hydroxylases; HIF, hypoxia-inducible transcription factor; ODD, oxygen-dependent degradation domain; bHLH, basic helix-loop-helix motif; PAS, Per-ARNT-Sim domain; CTAD, C-terminal transactivation domain; PAC, PAS-associated C-terminal domain; FIH, factor inhibiting HIF.

Figure 2 Evidence for conservation of biochemical properties between TaPHD and HsPHD2.

Notes: (A) Sequence alignment of TaHIFa, HsHIFa, and substrates of PHD-like enzymes in Dictyostelium discoideum (DdSkp1) and Pseudomonas putida (PpEF-Tu). (B) Kinetic parameters determined for TaPHD and HsPHD2 by MALDI-TOF-MS based assays, conditions: HsPHD2 or TaPHD (3.5 µM–7.0 µM), HsHIF1α CODD 19mer peptide (DLDLEMLAPYIPMDDDFQL-NH₂, 100 µM) or TaHIFa ODD 25mer peptide (PINEKEDYDDLAPFVPPPSFDNRLY-NH₂, 100 µM), (NH₄)₂Fe(II)(SO₄)2 (50 μM), sodium L-ascorbate (4 mM) and 2OG disodium salt (300 µM) in Tris (50 mM), pH 7.5. Initial rates were determined by varying the concentrations of the respective peptide or 2OG. Peptide hydroxylation was analyzed by MALDI-MS; the apparent non-enzymatic Met oxidation was subtracted. Data were fitted with the Michaelis-Menten equation using GraphPad Prism^® (errors are indicated as standard deviations, n=3). (C) Results of 1D CLIP HSQC [¹³C]-HsHIF1α CODD and [¹³C]-HsHIF1α NODD displacement experiments with a [¹³C]-prolyl-labelled reporter HsHIF1α CODD/NODD peptide reveal a higher binding affinity of TaPHD for HsHIF1α CODD over NODD (errors are indicated as standard deviations, n=3); conditions: [¹³C]-proline HsHIF1α CODD/NODD (DLDLEMLAPYIPMDDDFQL-NH₂/DALTLLAPAAGDTIISLDF-NH₂, 50 µM), TaPHD (50 µM), 2OG disodium salt (50 µM) buffered with Tris-D₁₁ (50 mM), pH 7.5, in 10% D₂O and 90% H₂O. (D) Comparison of coupled and uncoupled (ie, in the absence of substrate) 2OG turnover by TaPHD and (E) HsPHD2. 2OG turnover was monitored by ¹H CPMG NMR, conditions: TaPHD or HsPHD2 (20 μM), (NH₄)₂Fe(II)(SO₄)₂ (125 µM), sodium L-ascorbate (1 mM), HsHIF1α CODD (500 µM) or TaHIFa ODD substrate (500 µM) (where necessary), and 2OG disodium salt (400 µM), in 10% D₂O and 90% H₂O, Tris-D11 (50 mM), pH 7.5; [Succ]=Succinate. (F) Steady-state O2-dependence of TaPHD and (G) HsPHD2 (published in;²⁹ conditions: 4 µM TaPHD/HsPHD2, 100 µM TaHIFa ODD/HsHIF1α CODD, (NH₄)₂Fe(II)(SO₄)2 (50 µM), 2OG disodium salt (300 µM) and sodium L-ascorbate (4 mM) in Tris (50 mM), pH 7.5 were incubated at 37°C under different % O₂. The extent of hydroxylation was analyzed by MALDI–ToF-MS (errors are indicated as standard deviations, n=3).

Table 1 Data collection and refinement statistics

	TaPHD.Mn (PDB: 6EYI)	TaPHD.TaODD (PDB: 6F0W)
Wavelength (Å)	0.97930	0.97950
Resolution range (Å)^a	38.39-1.20 (1.24-1.20)	30.49-1.30 (1.35-1.30)
Space group	P2^1	P1
Unit cell (a b c, α β γ)	40.58 59.41 51.20, 90 100.74 90	40.84 41.32 42.08, 114.06 95.67 103.69
Total reflections	640,269	377,792
Unique reflections^a	74,236 (6949)	55,424 (5243)
Multiplicity^3	8.6 (4.9)	6.8 (4.1)
Completeness (%)^a	99.24 (93.39)	94.83 (89.72)
Mean 1/σ(1)^a	25.9 (2.0)	14.5 (2.6)
Wilson B-factor	13.06	13.89
Rmerge^b	0.084	0.112
Reflections used in refinement^a	74,191 (6940)	55,413 (5244)
Reflections used for R-free^a	3,738 (333)	2,775 (264)
Rwork^Table Footnotec,Table Footnotea	0.1331 (0.2088)	0.1475 (0.2140)
Rfree^Table Footnotec,Table Footnotea	0.1483 (0.2396)	0.1687 (0.2430)
Number of non-hydrogen atoms	2,026	2,146
Macromolecules	1,808	1,933
Ligands	19	11
Solvent	199	202
Protein residues	211	239
RMS (bonds) (Å)^e	0.011	0.012
RMS (angles) (°)^e	1.09	1.09
Ramachandran favored (%)	97.56	98.24
Ramachandran allowed (%)	2.44	1.76
Ramachandran outliers (%)	0.00	0.00
Rotamer outliers (%)	0.98	0.48
Clashscore	1.37	2.36
Average B-factor (Å^2)	22.75	20.67
Macromolecules (Å^2)	21.24	19.36
Ligands (Å^2)	31.98	12.99
Solvent (Å^2)	35.59	33.67

Notes: ^aParentheses indicate high resolution shell.

^bR_merge =∑_j∑_h| I_hj – < I_h> | /∑_j∑_h < I_h> ×100.

^cR_work =∑||Fobs| – |Fcalc||/|Fobs|×100.

^dR_free, based on 2%–5% of the total reflections.

^eRMS deviation from ideality.

Figure 3 Views from crystal structures of the Trichoplax adhaerens prolyl hydroxylases (PHD) without substrate bound (TaPHD) and in complex with a fragment of its substrate (TaPHD.TaODD).

Notes: (A) Secondary structural elements in TaPHD comprise four α helices and ten β strands, eight of which form the double-stranded β-helix core fold (DSBH, gray, Roman numerals I–VIII). (B) Overall binding mode of TaODD to TaPHD in the TaPHD.TaODD complex structure showing the 2F_o-F_c electron density map for the peptidic substrate (gray mesh, contoured to 1.0 σ). (C) Active site close-up of TaPHD.TaODD reveals that the P486 TaODD C-4 methylene adopts an endo-conformation (2F_o-F_c density, gray mesh, contoured to 1.0 σ). The metal ion (manganese substituting for iron, purple sphere) is octahedrally coordinated by a triad of residues (H209TaPHD, D211TaPHD, and H270TaPHD), N-oxalylglycine (NOG), and a water molecule (W1, red sphere). The stable metal-water coordination observed here is conserved in HsPHD2,^32,34 where it is proposed to enable the oxygen sensing ability in HsPHD2.

Figure 4 Comparison of ODD binding modes by the T. adhaerens and human HIFα PHD for the TaPHD.TaODD, HsPHD2.CODD and HsPHD2.NODD substrate structures.

Notes: (A) Superimposition of structural views of TaPHD.TaODD with HsPHD2.CODD (PDB: 3HQR) and (B) TaPHD.TaODD with HsPHD2.NODD (PDB: 5L9V) reveals major differences between the structures in the PHD flexible β2/β3 finger-loop and PHD C-terminal substrate binding interfaces. Notably, the Pro-Pro-Pro motif in TaODD (PPP motif, P489-491TaODD) adopts a helical bend, which aligns poorly with HsHIF1α CODD and, particularly, with HsHIF1α NODD.
Abbreviations: T. adhaerens, Trichoplax adhaerens; ODD, oxygen dependent degradation domain.

Figure 5 Comparison of substrate binding modes by TaPHD, CrP4H and Pseudomonas putida PPHD.

Notes: Overall superimposition and active site close-up of the TaPHD.TaODD complex with (A) P. putida PPHD (PDB: 4IW3) and (B) CrP4H (PDB: 3GZE) in complex with a proline rich peptidic substrate reveals the conservation of the substrate-binding mode involving the conformationally flexbile β2/β3-finger-loop (in all cases) and the C-terminal α4-helix (in case of TaPHD and PPHD).

Figure 6 Active site metal region details and comparison of the conformations of the target prolyl-residues in the enzyme-substrate complex structures of TaPHD, HsPHD2, PPHD, and CrP4H.

Notes: (A) TaPHD.TaODD, (B) HsPHD2.CODD (PDB: 3HQR,³⁴), (C) HsPHD2.NODD (PDB: 5L9V,³⁴), (D) PPHD.EF-Tu, (PDB: 4IW3,³⁸) and (E) CrP4H.proline-rich substrate (PDB: 3GZE,⁶⁶) complex structures. Note that, in all the enzyme-substrate complexes, the proline C-4- methylene, that is hydroxylated, adopts the endo-conformation. (F) By contrast, Hyp564 in HsHIF1α CODD adopts the C-4 exo-conformation when bound to the VCB complex (PDB: 1LM8,^55–57). In (A and D), N-oxalylglycine (NOG) acts as a 2OG analog. Note that the Zn (substituting for Fe) in the CrP4H active site is tetrahedrally coordinated, with an acetate binding instead of the 2OG co-substrate. The structures reveal conserved orientations of the “target” proline residues, which in each case, adopt a C-4 endo-conformation.⁵⁵ Note that the metal bound water present in the TaPHD (and HsPHD2/PPHD) substrate complex structures is not observed in the CrP4H.(Ser-Pro)₅ structure.

Loenarz C, Coleman ML, Boleininger A, et al. The hypoxia-inducible transcription factor pathway regulates oxygen sensing in the simplest animal, Trichoplax adhaerens. EMBO Rep. 2011;12(1):63–70.

 PubMed Web of Science ®Google Scholar

Lando D, Peet DJ, Whelan DA, Gorman JJ, Whitelaw ML. Asparagine hydroxylation of the HIF transactivation domain a hypoxic switch. Science. 2002;295(5556):858–861.

 PubMed Web of Science ®Google Scholar

Tarhonskaya H, Chowdhury R, Leung IK, et al. Investigating the contribution of the active site environment to the slow reaction of hypoxia-inducible factor prolyl hydroxylase domain 2 with oxygen. Biochem J. 2014;463(3):363–372.

 PubMedGoogle Scholar

McDonough MA, Li V, Flashman E, et al. Cellular oxygen sensing: Crystal structure of hypoxia-inducible factor prolyl hydroxylase (PHD2). Proc Natl Acad Sci U S A. 2006;103(26):9814–9819.

 PubMed Web of Science ®Google Scholar

Chowdhury R, McDonough MA, Mecinović J, et al. Structural basis for binding of hypoxia-inducible factor to the oxygen-sensing prolyl hydroxylases. Structure. 2009;17(7):981–989.

 PubMed Web of Science ®Google Scholar

Scotti JS, Leung IK, Ge W, et al. Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation. Proc Natl Acad Sci U S A. 2014;111(37):13331–13336.

 PubMed Web of Science ®Google Scholar

Koski MK, Hieta R, Hirsilä M, Rönkä A, Myllyharju J, Wierenga RK. The crystal structure of an algal prolyl 4-hydroxylase complexed with a proline-rich peptide reveals a novel buried tripeptide binding motif. J Biol Chem. 2009;284(37):25290–25301.

 PubMed Web of Science ®Google Scholar

Loenarz C, Mecinović J, Chowdhury R, McNeill LA, Flashman E, Schofield CJ. Evidence for a stereoelectronic effect in human oxygen sensing. Angew Chem Int Ed Engl. 2009;48(10):1784–1787.

 PubMed Web of Science ®Google Scholar

Hon WC, Wilson MI, Harlos K, et al. Structural basis for the recognition of hydroxyproline in HIF-1α by pVHL. Nature. 2002;417(6892):975–978.

 PubMed Web of Science ®Google Scholar