title: An Extended Polyanion Activation Surface in Insulin Degrading Enzyme.
abstract: Insulin degrading enzyme (IDE) is believed to be the major enzyme that metabolizes insulin and has been implicated in the degradation of a number of other bioactive peptides, including amyloid beta peptide (Aβ), glucagon, amylin, and atrial natriuretic peptide. IDE is activated toward some substrates by both peptides and polyanions/anions, possibly representing an important control mechanism and a potential therapeutic target. A binding site for the polyanion ATP has previously been defined crystallographically, but mutagenesis studies suggest that other polyanion binding modes likely exist on the same extended surface that forms one wall of the substrate-binding chamber. Here we use a computational approach to define three potential ATP binding sites and mutagenesis and kinetic studies to confirm the relevance of these sites. Mutations were made at four positively charged residues (Arg 429, Arg 431, Arg 847, Lys 898) within the polyanion-binding region, converting them to polar or hydrophobic residues. We find that mutations in all three ATP binding sites strongly decrease the degree of activation by ATP and can lower basal activity and cooperativity. Computational analysis suggests conformational changes that result from polyanion binding as well as from mutating residues involved in polyanion binding. These findings indicate the presence of multiple polyanion binding modes and suggest the anion-binding surface plays an important conformational role in controlling IDE activity.
authors.affiliation: Department of Molecular and Cellular Biochemistry and the Center for Structural Biology, University of Kentucky, Lexington, Kentucky, United States of America.
authors.forename: Eun Suk
authors.initials: ES
authors.lastname: Song
authors.affiliation: Department of Chemistry, University of Miami, Miami, Florida, United States of America.
authors.forename: Mehmet
authors.initials: M
authors.lastname: Ozbil
authors.affiliation: Department of Chemistry, University of Miami, Miami, Florida, United States of America.
authors.forename: Tingting
authors.initials: T
authors.lastname: Zhang
authors.affiliation: Center for Computational Sciences, University of Kentucky, Lexington, Kentucky, United States of America.
authors.forename: Michael
authors.initials: M
authors.lastname: Sheetz
authors.affiliation: Department of Medicine, University of California, San Diego, La Jolla, California, United States of America.
authors.forename: David
authors.initials: D
authors.lastname: Lee
authors.affiliation: Department of Medicine, University of California, San Diego, La Jolla, California, United States of America.
authors.forename: Danny
authors.initials: D
authors.lastname: Tran
authors.affiliation: Department of Medicine, University of California, San Diego, La Jolla, California, United States of America.
authors.forename: Sheng
authors.initials: S
authors.lastname: Li
authors.affiliation: Department of Chemistry, University of Miami, Miami, Florida, United States of America.
authors.forename: Rajeev
authors.initials: R
authors.lastname: Prabhakar
authors.affiliation: Department of Molecular and Cellular Biochemistry and the Center for Structural Biology, University of Kentucky, Lexington, Kentucky, United States of America.
authors.forename: Louis B
authors.initials: LB
authors.lastname: Hersh
authors.affiliation: Department of Molecular and Cellular Biochemistry and the Center for Structural Biology, University of Kentucky, Lexington, Kentucky, United States of America.
authors.forename: David W
authors.initials: DW
authors.lastname: Rodgers
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D003903:Deuterium / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry; D007339:Insulysin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008968:Molecular Conformation; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009154:Mutation; D000071228:Polyelectrolytes; D011108:Polymers / Q000737:chemistry*; D011485:Protein Binding
chemical_list: D000255:Adenosine Triphosphate; D003903:Deuterium; D006859:Hydrogen; D007339:Insulysin; D000071228:Polyelectrolytes; D011108:Polymers