Asparaginyl hydroxylation of the Notch ankyrin repeat domain by factor inhibiting hypoxia-inducible factor

Mathew L Coleman, Michael A McDonough, Kirsty S Hewitson, Charlotte Coles, Jasmin Mecinovic, Mariola Edelmann, Kristina M Cook, Matthew E Cockman, David E Lancaster, Benedikt M Kessler, Neil J Oldham, Peter J Ratcliffe, Christopher J Schofield

Research output: Contribution to journalArticlepeer-review

165 Citations (Scopus)


The stability and activity of hypoxia-inducible factor (HIF) are regulated by the post-translational hydroxylation of specific prolyl and asparaginyl residues. We show that the HIF asparaginyl hydroxylase, factor inhibiting HIF (FIH), also catalyzes hydroxylation of highly conserved asparaginyl residues within ankyrin repeat (AR) domains (ARDs) of endogenous Notch receptors. AR hydroxylation decreases the extent of ARD binding to FIH while not affecting signaling through the canonical Notch pathway. ARD proteins were found to efficiently compete with HIF for FIH-dependent hydroxylation. Crystallographic analyses of the hydroxylated Notch ARD (2.35A) and of Notch peptides bound to FIH (2.4-2.6A) reveal the stereochemistry of hydroxylation on the AR and imply that significant conformational changes are required in the ARD fold in order to enable hydroxylation at the FIH active site. We propose that ARD proteins function as natural inhibitors of FIH and that the hydroxylation status of these proteins provides another oxygen-dependent interface that modulates HIF signaling.

Original languageEnglish
Pages (from-to)24027-38
Number of pages12
JournalJournal of Biological Chemistry
Issue number33
Publication statusPublished - 17 Aug 2007


  • Ankyrin Repeat
  • Asparagine
  • Crystallography, X-Ray
  • Humans
  • Hydroxylation
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Mixed Function Oxygenases
  • Protein Structure, Tertiary
  • Receptor, Notch1
  • Receptor, Notch2
  • Receptors, Notch
  • Repressor Proteins
  • Transcription Factors


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