title: Factor inhibiting HIF (FIH) recognizes distinct molecular features within hypoxia-inducible factor-α (HIF-α) versus ankyrin repeat substrates.
abstract: Factor Inhibiting HIF (FIH) catalyzes the β-hydroxylation of asparagine residues in HIF-α transcription factors as well as ankyrin repeat domain (ARD) proteins such as Notch and Gankyrin. Although FIH-mediated hydroxylation of HIF-α is well characterized, ARDs were only recently identified as substrates, and less is known about their recognition and hydroxylation by FIH. We investigated the molecular determinants of FIH substrate recognition, with a focus on differences between HIF and ARD substrates. We show that for ARD proteins, structural context is an important determinant of FIH-recognition, but analyses of chimeric substrate proteins indicate that the ankyrin fold alone is not sufficient to explain the distinct substrate properties of the ARDs compared with HIF. For both substrates the kinetic parameters of hydroxylation are influenced by the amino acids proximal to the target asparagine. Although FIH tolerates a variety of chemically disparate residues proximal to the asparagine, we demonstrate that certain combinations of amino acids are not permissive to hydroxylation. Finally, we characterize a conserved RLL motif in HIF and demonstrate that it mediates a high affinity interaction with FIH in the presence of cell lysate or macromolecular crowding agents. Collectively, our data highlight the importance of residues proximal to the asparagine in determining hydroxylation, and identify additional substrate-specific elements that contribute to distinct properties of HIF and ARD proteins as substrates for FIH. These distinct features are likely to influence FIH substrate choice in vivo and, therefore, have important consequences for HIF regulation.
authors.affiliation: School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia 5005, Australia.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Wilkins
authors.forename: Sarah
authors.initials: S
authors.lastname: Karttunen
authors.forename: Rachel J
authors.initials: RJ
authors.lastname: Hampton-Smith
authors.forename: Iain
authors.initials: I
authors.lastname: Murchland
authors.forename: Anne
authors.initials: A
authors.lastname: Chapman-Smith
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Peet
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017089:Ankyrin Repeat; D006801:Humans; D006900:Hydroxylation; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051881:Receptor, Notch1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000074562:Receptor, Notch4; D051880:Receptors, Notch / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D011518:Proto-Oncogene Proteins; D051881:Receptor, Notch1; D000074562:Receptor, Notch4; D051880:Receptors, Notch; D012097:Repressor Proteins; D014157:Transcription Factors; C525549:gankyrin protein, mouse; C073572:Notch4 protein, mouse; D006899:Mixed Function Oxygenases; C551240:factor inhibiting hypoxia-inducible factor 1, mouse; C439790:HIF1AN protein, human