title: Mapping of the leptin binding sites and design of a leptin antagonist.
abstract: The leptin/leptin receptor system shows strong similarities to the long-chain cytokine interleukin-6 (IL-6) and granulocyte colony-stimulating factor cytokine/receptor systems. The IL-6 family cytokines interact with their receptors through three different binding sites I-III. The leptin structure was superposed on the crystal structures of several long-chain cytokines, and a series of leptin mutants was generated focusing on binding sites I-III. The effect of the mutations on leptin receptor (LR) signaling and on binding to the membrane proximal cytokine receptor homology domain (CRH2) of the LR was determined. Mutations in binding site I at the C terminus of helix D show a modest effect on signaling and do not affect binding to CRH2. Binding site II is composed of residues at the surface of helices A and C. Mutations in this site impair binding to CRH2 but have only limited effect on signaling. Site III mutations around the N terminus of helix D impair receptor activation without affecting binding to CRH2. We identified an S120A/T121A mutant in binding site III, which lacks any signaling capacity, but which still binds to CRH2 with wild type affinity. This leptin mutant behaves as a potent leptin antagonist both in vitro and in vivo.
authors.affiliation: Department of Medical Protein Research, Faculty of Medicine and Health Sciences, Flanders Interuniversity Institute for Biotechnology, VIB09, Ghent University, Albert Baertsoenkaai 3, Belgium.
authors.forename: Frank
authors.initials: F
authors.lastname: Peelman
authors.forename: Katrien
authors.initials: K
authors.lastname: Van Beneden
authors.forename: Lennart
authors.initials: L
authors.lastname: Zabeau
authors.forename: Hannes
authors.initials: H
authors.lastname: Iserentant
authors.forename: Peter
authors.initials: P
authors.lastname: Ulrichts
authors.forename: Delphine
authors.initials: D
authors.lastname: Defeau
authors.forename: Annick
authors.initials: A
authors.lastname: Verhee
authors.forename: Dominiek
authors.initials: D
authors.lastname: Catteeuw
authors.forename: Dirk
authors.initials: D
authors.lastname: Elewaut
authors.forename: Jan
authors.initials: J
authors.lastname: Tavernier
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D019556:COS Cells; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D017930:Genes, Reporter; D006801:Humans; D020738:Leptin / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008811:Mice, Inbred DBA; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection
chemical_list: D020738:Leptin; D011994:Recombinant Proteins; D008156:Luciferases