title: Substrate preference of the HIF-prolyl hydroxylase-2 (PHD2) and substrate-induced conformational change.
abstract: HIF prolyl-4-hydroxylase 2 (PHD2) is a non-heme Fe, 2-oxoglutarate (2OG) dependent dioxygenase that regulates the hypoxia inducible transcription factor (HIF) by hydroxylating two conserved prolyl residues in N-terminal oxygen degradation domain (NODD) and C-terminal oxygen degradation domain (CODD) of HIF-1α. Prior studies have suggested that the substrate preference of PHD2 arises from binding contacts with the β2β3 loop of PHD2. In this study we tested the substrate selectivity of PHD2 by kinetic competition assays, varied ionic strength, and global protein flexibility using amide H/D exchange (HDX). Our results revealed that PHD2 preferred CODD by 20-fold over NODD and that electrostatics influenced this effect. Global HDX monitored by mass spectrometry indicated that binding of Fe(II) and 2OG stabilized the overall protein structure but the saturating concentrations of either NODD or CODD caused an identical change in protein flexibility. These observations imply that both substrates stabilize the β2β3 loop to the same extent. Under unsaturated substrate conditions NODD led to a higher HDX rate than CODD due to its lower binding affinity to PHD2. Our results suggest that loop closure is the dominant contributor to substrate selectivity in PHD2.
authors.affiliation: Department of Chemistry, University of Massachusetts, Amherst, MA 01003, USA.
authors.forename: Serap
authors.initials: S
authors.lastname: Pektas
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Knapp
mesh_terms: D001665:Binding Sites; D001667:Binding, Competitive; D002413:Cations, Divalent; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006900:Hydroxylation; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000737:chemistry* / Q000235:genetics; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases / Q000737:chemistry* / Q000235:genetics; D007501:Iron / Q000737:chemistry*; D007656:Ketoglutaric Acids / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D055672:Static Electricity; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D002413:Cations, Divalent; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007656:Ketoglutaric Acids; D011994:Recombinant Proteins; D007501:Iron; C490190:EGLN1 protein, human; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases
keywords: HIF; HIF-prolyl hydroxylase; Hypoxia; Non-heme iron; α-Ketoglutarate