||2ILM deposited: 2006-10-03 modified: 2011-07-13
||Factor Inhibiting HIF-1 Alpha D201A Mutant in Complex with FE(II), Alpha-Ketoglutarate and HIF-1 Alpha 35mer
||Chowdhury, R., Ehrismann, D., Hardy, A.P., Hewitson, K.S., Holmes, S.L., McDonough, M.A., Schofield, C.J.
||resolution None rfactor None rfree None
|Related PDB Entries
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.
J.Biol.Chem. 2008 Sep; 283(38):25971-25978 doi:10.1074/jbc.M804999200
A set of four non-heme iron(II) and 2-oxoglutarate-dependent enzymes catalyze the post-translational modification of a transcription factor, hypoxia inducible factor (HIF), that mediates the hypoxic response in animals. Hydroxylation of HIF both causes its degradation and limits its activity. We describe how the use of structural data coupled to solid-phase synthesis led to the discovery of a selective inhibitor of one of the HIF hydroxylases. The inhibitor N-oxalyl-d-phenylalanine was shown to inhibit the HIF asparaginyl hydroxylase (FIH) but not a HIF prolyl hydroxylase. A crystal structure of the inhibitor complexed to FIH reveals that it binds in the 2OG and, likely, in the dioxygen binding site. The results will help to enable the modulation of the hypoxic response for the up-regulation of specific genes of biomedical importance, such as erythropoietin and vascular endothelial growth factor.
J.Am.Chem.Soc. 2005 Jun; 127(21):7680-7681 doi:10.1021/ja050841b
The activity of the transcription factor hypoxia-inducible factor (HIF) is regulated by oxygen-dependent hydroxylation. Under normoxic conditions, hydroxylation of proline residues triggers destruction of its alpha-subunit while hydroxylation of Asn(803) in the C-terminal transactivation domain of HIF-1 alpha (CAD) prevents its interaction with p300. Here we report crystal structures of the asparagine hydroxylase (factor-inhibiting HIF, FIH) complexed with Fe((II)), 2-oxoglutarate cosubstrate, and CAD fragments, which reveal the structural basis of HIF modification. CAD binding to FIH occurs via an induced fit process at two distinct interaction sites. At the hydroxylation site CAD adopts a loop conformation, contrasting with a helical conformation for the same residues when bound to p300. Asn(803) of CAD is buried and precisely orientated in the active site such that hydroxylation occurs at its beta-carbon. Together with structures with the inhibitors Zn((II)) and N-oxaloylglycine, analysis of the FIH-CAD complexes will assist design of hydroxylase inhibitors with proangiogenic properties. Conserved structural motifs within FIH imply it is one of an extended family of Fe((II)) oxygenases involved in gene regulation.
J.Biol.Chem. 2003 Jan; 278(3):1802- doi:10.1074/JBC.C200644200