5JWP

Crystal structure of human FIH D201E variant in complex with Zn, alpha-ketoglutarate, and HIF1 alpha peptide.

Summary for 5JWP

• Entry DOI: 10.2210/pdb5jwp/pdb
• Related: 5JWK 5JWL
• Descriptor: Hypoxia-inducible factor 1-alpha inhibitor, Hypoxia-inducible factor 1-alpha, 2-OXOGLUTARIC ACID, ... (7 entities in total)
• Functional Keywords: hypoxia sensing, factor inhibiting hif, alpha-ketoglutarate dependent oxygenase, hypoxia inducible factor, hydroxylase, oxidoreductase
• Biological source: Homo sapiens (Human); More
• Total number of polymer chains: 2
• Total formula weight: 43331.56

Authors

Taabazuing, C.Y.,Garman, S.C.,Eron, S.,Knapp, M.J. (deposition date: 2016-05-12, release date: 2016-11-23, Last modification date: 2023-09-27)

Primary citation

Hangasky, J.A.,Taabazuing, C.Y.,Martin, C.B.,Eron, S.J.,Knapp, M.J.
The facial triad in the alpha-ketoglutarate dependent oxygenase FIH: A role for sterics in linking substrate binding to O2 activation.
J.Inorg.Biochem., 166:26-33, 2016

PubMed Abstract: The factor inhibiting hypoxia inducible factor-1α (FIH) is a nonheme Fe(II)/αKG oxygenase using a 2-His-1-Asp facial triad. FIH activates O via oxidative decarboxylation of α-ketoglutarate (αKG) to generate an enzyme-based oxidant which hydroxylates the Asn residue within the C-terminal transactivation domain (CTAD) of HIF-1α. Tight coupling of these two sequential reactions requires a structural linkage between the Fe(II) and the substrate binding site to ensure that O activation occurs after substrate binds. We tested the hypothesis that the facial triad carboxylate (Asp) of FIH linked substrate binding and O binding sites. Asp variants of FIH were constructed and thoroughly characterized in vitro using steady-state kinetics, crystallography, autohydroxylation, and coupling measurements. Our studies revealed each variant activated O with a catalytic efficiency similar to that of wild-type (WT) FIH (kaK=0.17μMmin), but led to defects in the coupling of O activation to substrate hydroxylation. Steady-state kinetics showed similar catalytic efficiencies for hydroxylation by WT-FIH (k/K=0.42μMmin) and D201G (k/K=0.34μMmin); hydroxylation by D201E was greatly impaired, while hydroxylation by D201A was undetectable. Analysis of the crystal structure of the D201E variant revealed steric crowding near the diffusible ligand site supporting a role for sterics from the facial triad carboxylate in the O binding order. Our data support a model in which the facial triad carboxylate Asp provides both steric and polar contacts to favor O access to the Fe(II) only after substrate binds, leading to coupled turnover in FIH and other αKG oxygenases.

PubMed: 27815979
DOI: 10.1016/j.jinorgbio.2016.10.007

Experimental method

X-RAY DIFFRACTION (2.1 Å)