PDBsum entry 3d8c

Transcription regulator, oxidoreductase

PDB id: 3d8c

Contents

Protein chains

341 a.a. *

13 a.a. *

Ligands

SO4 ×2

AKG

GOL

Metals

_ZN

Waters ×223

* Residue conservation analysis

PDB id:

3d8c

Name:

Transcription regulator, oxidoreductase

Title:

Factor inhibiting hif-1 alpha d201g mutant in complex with zn(ii), alpha-ketoglutarate and hif-1 alpha 19mer

Structure:

Hypoxia-inducible factor 1 alpha inhibitor. Chain: a. Synonym: hypoxia-inducible factor asparagine hydroxylase, factor inhibiting hif-1, fih-1. Engineered: yes. Mutation: yes. Hypoxia-inducible factor 1 alpha. Chain: b. Fragment: ctad.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: hif1an, fih1. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes. Other_details: synthetic peptide fragment of hif-1 alpha

Resolution:

2.10Å R-factor: 0.218 R-free: 0.256

Authors:

M.A.Mcdonough,R.Chowdhury,C.J.Schofield

Key ref:

K.S.Hewitson et al. (2008). Evidence that two enzyme-derived histidine ligands are sufficient for iron binding and catalysis by factor inhibiting HIF (FIH). J Biol Chem, 283, 25971-25978. PubMed id: 18611856 DOI: 10.1074/jbc.M804999200

Date:

23-May-08 Release date: 12-Aug-08

Protein chain

Q9NWT6  (HIF1N_HUMAN) -  Hypoxia-inducible factor 1-alpha inhibitor from Homo sapiens

Seq: 349 a.a.

Struc: 341 a.a.*

Protein chain

No UniProt id for this chain

Struc: 13 a.a.

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class 2: Chain A: E.C.1.14.11.30 - hypoxia-inducible factor-asparagine dioxygenase.
• Reaction: L-asparaginyl-[hypoxia-inducible factor alpha subunit] + 2-oxoglutarate + O2 = (3S)-3-hydroxy-L-asparaginyl-[hypoxia-inducible factor alpha subunit] + succinate + CO2

L-asparaginyl-[hypoxia-inducible factor alpha subunit] (Bound ligand (Het Group name = AKG) corresponds exactly) + 2-oxoglutarate + O2 = (3S)-3-hydroxy-L-asparaginyl-[hypoxia-inducible factor alpha subunit] + succinate + CO2

• Cofactor: Fe(2+); L-ascorbate
• Enzyme class 3: Chain A: E.C.1.14.11.n4 - ?????

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1074/jbc.M804999200

J Biol Chem 283:25971-25978 (2008)

PubMed id: 18611856

Evidence that two enzyme-derived histidine ligands are sufficient for iron binding and catalysis by factor inhibiting HIF (FIH).

K.S.Hewitson, S.L.Holmes, D.Ehrismann, A.P.Hardy, R.Chowdhury, C.J.Schofield, M.A.McDonough.

ABSTRACT

A 2-His-1-carboxylate triad of iron binding residues is present in many non-heme iron oxygenases including the Fe(II) and 2-oxoglutarate (2OG)-dependent dioxygenases. Three variants (D201A, D201E, and D201G) of the iron binding Asp-201 residue of an asparaginyl hydroxylase, factor inhibiting HIF (FIH), were made and analyzed. FIH-D201A and FIH-D201E did not catalyze asparaginyl hydroxylation, but in the presence of a reducing agent, they displayed enhanced 2OG turnover when compared with wild-type FIH. Turnover of 2OG by FIH-D201A was significantly stimulated by the addition of HIF-1alpha(786-826) peptide. Like FIH-D201A and D201E, the D201G variant enhanced 2OG turnover but rather unexpectedly catalyzed asparaginyl hydroxylation. Crystal structures of the FIH-D201A and D201G variants in complex with Fe(II)/Zn(II), 2OG, and HIF-1alpha(786-826/788-806) implied that only two FIH-based residues (His-199 and His-279) are required for metal binding. The results indicate that variation of 2OG-dependent dioxygenase iron-ligating residues as a means of functional assignment should be treated with caution. The results are of mechanistic interest in the light of recent biochemical and structural analyses of non-heme iron and 2OG-dependent halogenases that are similar to the FIH-D201A/G variants in that they use only two His-residues to ligate iron.

Selected figure(s)

Figure 1.
The reaction catalyzed by wild-type FIH (and FIH-D201G). hydroxylation of the β-carbon of Asn-803 within the C-terminal transactivation domain of HIF-1α. The reaction requires Fe(II) as a cofactor and dioxygen and 2OG as co-substrates that are converted to succinate and carbon dioxide concomitant with hydroxylation of HIF-1α substrate. For the FIH-D201A/E variants, 2OG decarboxylation is uncoupled from the hydroxylation of HIF-1α substrate and is stimulated by the presence of a reducing agent (ascorbate or DTT).

Figure 2.
Insights from crystal structures of FIH-D201A and FIH-D201G. a, stereo view of the iron binding site of the FIH-D201A·Fe(II)·2OG·HIF-1α[786–826] complex. Experimental electron density OMIT map (F[o] - F[c]) contoured to 5σ represented as blue mesh (electron density is carved out around the residues and ligands displayed for clarity). The unanticipated electron density adjacent to the iron was provisionally modeled as (bi)carbonate (see “Results” for discussion). b, comparison of the wild-type FIH·Fe(II)·2OG·HIF-1α[786–826] complex (PDB ID 1H2L) with the FIH-D201A·Fe(II)·2OG·HIF-1α[786–826] complex (wild-type FIH (blue) in complex with HIF substrate (cyan) and FIH-D201A (yellow) in complex with HIF substrate (magenta)). This figure emphasizes several important interactions between wild-type FIH and HIF-1α that are lost in the FIH-D201A complex: wild-type FIH Asp-201 and HIF-1α Asn-803 main chain nitrogen (yellow dash), wild-type FIH Gln-239 and HIF-1α Asn-803 side chain, and wild-type FIH Trp-296 and HIF-1α Val-802. Note the presence of the assigned sulfate ion (orange and red) in the FIH-D201A structure apparently replacing the carboxylate of the HIF-1α Glu-801 in the wild-type FIH·Fe(II)·2OG·HIF-1α[786–826] complex, which is also observed in uncomplexed wild-type FIH structure (PDB 1H2N, not shown). c, stereo views from the crystal structures of 2OG-dependent halogenase, SyrB2 (pink) superimposed on the FIH-D201A variant (yellow). The FIH-D201A variant shares the same HXA... H motif as SyrB2 (PDB ID 2FCT); however, the FIH-D201A apparently does not provide enough space for a chloride ion to complete octahedral coordination to the Fe(II), which could explain why FIH-D201A does not have halogenase activity toward HIF-1α under our assay conditions. Distances between the FIH-D201A Ala-201 Cβ methyl group and Fe(II) in each of the structures are shown as black dashed lines to emphasize this point. d, stereo view ball-and-stick representation of the FIH-D201G·Zn(II)·2OG·HIF-1α[786–826] complex metal binding site. Experimental electron density 2F[o] - F[c] contoured to 1.0σ represented as blue mesh (electron density is carved out around the residues and ligands displayed for clarity). FIH and 2OG are colored green, HIF-1α[786–826] is colored yellow, Zn(II) is colored as a gray sphere, and the Zn(II) bound water is colored as a red sphere. Wat, water.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2008, 283, 25971-25978) copyright 2008.

Figures were selected by an automated process.