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. 2002 Nov 26;99(24):15351-6.
doi: 10.1073/pnas.202614999. Epub 2002 Nov 13.

Structure of factor-inhibiting hypoxia-inducible factor 1: An asparaginyl hydroxylase involved in the hypoxic response pathway

Charles E Dann 3rd  1 , Richard K Bruick, Johann Deisenhofer
Affiliations

Structure of factor-inhibiting hypoxia-inducible factor 1: An asparaginyl hydroxylase involved in the hypoxic response pathway

Charles E Dann 3rd et al. Proc Natl Acad Sci U S A. .

Abstract

Precise regulation of the evolutionarily conserved hypoxia-inducible transcription factor (HIF) ensures proper adaptation to variations in oxygen availability throughout development and into adulthood. Oxygen-dependent regulation of HIF stability and activity are mediated by hydroxylation of conserved proline and asparagine residues, respectively. Because the relevant prolyl and asparginyl hydroxylases use O(2) to effect these posttranslational modifications, these enzymes are implicated as direct oxygen sensors in the mammalian hypoxic response pathway. Here we present the structure of factor-inhibiting HIF-1 (FIH-1), the pertinent asparaginyl hydroxylase involved in hypoxic signaling. Hydroxylation of the C-terminal transactivation domain (CTAD) of HIF by FIH-1 prevents CTAD association with transcriptional coactivators under normoxic conditions. Consistent with other structurally known hydroxylases, FIH-1 is comprised of a beta-strand jellyroll core with both Fe(II) and the cosubstrate 2-oxoglutarate bound in the active site. Details of the molecular contacts at the active site of FIH-1 have been elucidated and provide a platform for future drug design. Furthermore, the structure reveals the presence of a FIH-1 homodimer that forms in solution and is essential for FIH activity.

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Figures

Fig 1.
Fig 1.
FIH-1 hydroxylates HIF-1α at Asn-803. The FIH-1 active site contains Fe(II) coordinated by three protein side chains: His-199, Asp-201, and His-279. The enzyme binds 2-OG, HIF peptide substrate, and molecular oxygen to facilitate hydroxylation of the β-carbon on Asn-803 of HIF-1α. In the course of the reaction, molecular oxygen is consumed and 2-OG is converted into succinate and carbon dioxide.
Fig 2.
Fig 2.
The primary structure of FIH-1 is labeled with secondary structure elements taken from the x-ray crystallographic model. β-strands and helices are depicted as red arrows and yellow boxes, respectively. Residues responsible for Fe(II) binding are highlighted in red, whereas residues in close contact with 2-OG are highlighted in green.
Fig 3.
Fig 3.
FIH-1 structure contains a β-jellyroll core marked by an extension of one of the β-sheets away from the core and helices dotting the periphery. (A) A ribbon model of the FIH-1 monomer is positioned looking between the β-sheets comprising the jellyroll and into the active site cavity. The active site metal is shown as a red sphere. Structural elements are colored as in Fig. 2. (B) A secondary structure topology diagram shows the arrangement of the 14 β-strands (triangles) and 8 helices (circles) in FIH-1. The core jellyroll motif, structurally homologous to the cupin protein family, is colored in red. (C) FIH-1 exists as a functionally relevant dimer in the crystal. The first monomer of the dimer is colored as in A, whereas the second monomer is blue. N and C termini are marked as black circles. The figure was generated by using RIBBONS (41).
Fig 4.
Fig 4.
Multiple contacts are present in the FIH-1 active site. (A) A 2Fo − Fc simulated annealing omit map contoured at 1.1 σ reveals the presence of 2-OG in the active site. (B) 2-OG makes several hydrophilic contacts with residues of FIH-1, most notably hydrogen bonds to Lys-214 and Thr-196. Additional contacts include coordination to the Fe (red sphere) and hydrophobic interactions with Leu-188, Phe-207, and Ile-281. Ball-and-stick model in B is colored with 2-OG in yellow, Cα positions in green, carbon in black, nitrogen in blue, and oxygen in red. The figure was generated by using SETOR (42) and RIBBONS (41) modeling programs.
Fig 5.
Fig 5.
The C terminus of FIH-1 is required for activity. (A) In vitro hydroxylation of HIF-2α 774–874 by wild-type FIH-1 inhibits interaction with p300. 35S-labeled HIF-2α 774–874 was incubated in the absence (lane 1) or presence of various recombinant MBP-FIH-1 enzymes (lanes 2–6) followed by incubation with immobilized GST-p300 CH1. 35S-labeled HIF-2α 774–874 bound to the GST-p300 CH1 domain was visualized by phosphorimaging after SDS/PAGE. Mutations that interfere with Fe(II) binding (D201A) or delete residues 303–349 compromise FIH-1 ability to block p300 association with the HIF-2α CTAD. (B) Deletion of FIH-1 residues 303–349 prevents interaction with the CTAD. 35S-labeled HIF-2α 774–874 bound to immobilized wild-type or truncated (1–302) MBP-FIH-1 was visualized after SDS/PAGE. The right lane indicates 10% of the input 35S-labeled protein in the pull-down experiments.
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