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225 a.a.
17 a.a.
_MN
Key reference
DOI no: 10.1016/j.str.2009.06.002 Structure 17:981-989 (2009) PubMed id: 19604478
Structural basis for binding of hypoxia-inducible factor to the oxygen-sensing prolyl hydroxylases. R.Chowdhury, M.A.McDonough, J.Mecinović, C.Loenarz, E.Flashman, K.S.Hewitson, C.Domene, C.J.Schofield. ABSTRACT
The oxygen-dependent hydroxylation of proline residues in the alpha subunit of hypoxia-inducible transcription factor (HIFalpha) is central to the hypoxic response in animals. Prolyl hydroxylation of HIFalpha increases its binding to the von Hippel-Lindau protein (pVHL), so signaling for degradation via the ubiquitin-proteasome system. The HIF prolyl hydroxylases (PHDs, prolyl hydroxylase domain enzymes) are related to the collagen prolyl hydroxylases, but form unusually stable complexes with their Fe(II) cofactor and 2-oxoglutarate cosubstrate. We report crystal structures of the catalytic domain of PHD2, the most important of the human PHDs, in complex with the C-terminal oxygen-dependent degradation domain of HIF-1alpha. Together with biochemical analyses, the results reveal that PHD catalysis involves a mobile region that isolates the hydroxylation site and stabilizes the PHD2.Fe(II).2OG complex. The results will be of use in the design of PHD inhibitors aimed at treating anemia and ischemic disease.
Selected figure(s)
Figure 2.
Figure 2. Conformational Changes in PHD2 Catalysis and Effect of PHD2 Variations on Activity and Selectivity
(A) Stereo view ribbons representation of the tPHD2.CODD complex structure. The tPHD2 fold comprises four α helices and ten β strands of which eight form a double-stranded β helix (DSBH, dark blue) (McDonough et al., 2006). Three of the four α helices (α1, α2, and α3) pack along the major β sheet and stabilize the DSBH.
(B) Stereo view ribbons representation of the tPHD2.CODD complex structure (green) superimposed with tPHD2.Fe(II).A structure (cyan) showing structural differences in the β2β3/loop (PHD2[237-254]) and C-terminal α4-helix conformations.
(C) Bicyclic inhibitors, such as A (salmon) disrupt the Arg-252:Asp-254 salt-bridge observed in the closed substrate binding conformation (P2[1]2[1]2[1]); they also apparently cause rotation of Tyr-310 C[β]-C[γ] (byvert, similar 45°) relative to that observed in the P6[3] form (not shown). Distances for selected salt bridges are given in angstroms.
(D) The tPHD2.CODD complex showing mutation sites (highlighted in white).
(E) 2OG turnover activity of wild-type (wt) and variant tPHD2 using NODD (blue) and CODD (red) substrates. 2OG turnover in absence of substrate was subtracted. Errors are standard deviations (n ≥ 3).
(F) Substrate hydroxylation and selectivity of wt and variant tPHD2 as determined by MALDI-TOF MS using CODD and NODD substrates.Figure 3.
Figure 3. The Binding of Proline/Hydroxyproline Residues to PHD2 and VCB
(A and B) Comparisons of the Pro-564[CODD] conformations when bound to tPHD2 (A, green) and VCB (B, wheat). Superimposition of CODD (yellow)/ CODD[Hyp564] (purple) in complex with tPHD2 /VCB yielded rmsd of 2.6 Å for 14 residues (561–574, Cα atoms). The Pro-564[CODD] C^4-methylene is in the endo conformation when bound to tPHD2 and the exo conformation when Hyp-564[CODD] is bound to the VCB complex. The C^4 Pro-564 hydrogen(s) are modeled in black.
(C) Stereo view of coordination at the PHD2 (green) and the FIH (blue) active site; note the alternative position of the NOG 1-carboxylate. In the PHD2 structure, NOG (orange) chelates the metal via one of its carboxylate oxygens (O-Mn[II]; 2.4 Å; trans to His-374 Nepsilon 2 (N2-Mn[II]; 1.9 Å) and its amide α-carbonyl oxygen (Oα-Mn(II); 2.1 Å;
The above figures are reprinted by permission from Cell Press: Structure (2009, 17, 981-989) copyright 2009. Figures were selected by the author.
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