title: Structural analysis of the TKB domain of ubiquitin ligase Cbl-b complexed with its small inhibitory peptide, Cblin.
abstract: Cbl-b is a RING-type ubiquitin ligase. Previously, we showed that Cbl-b-mediated ubiquitination and proteosomal degradation of IRS-1 contribute to muscle atrophy caused by unloading stress. The phospho-pentapeptide DGpYMP (Cblin) mimics Tyr612-phosphorylated IRS-1 and inhibits the Cbl-b-mediated ubiquitination and degradation of IRS-1 in vitro and in vivo. In this study, we confirmed the direct interaction between Cblin and the TKB domain of Cbl-b using NMR. Moreover, we showed that the shortened tripeptide GpYM also binds to the TKB domain. To elucidate the inhibitory mechanism of Cblin, we solved the crystal structure of the TKB-Cblin complex at a resolution of 2.5 Å. The pY in Cblin inserts into a positively charged pocket in the TKB domain via hydrogen-bond networks and hydrophobic interactions. Within this complex, the Cblin structure closely resembles the TKB-bound form of another substrate-derived phosphopeptide, Zap-70-derived phosphopeptide. These peptides lack the conserved intrapeptidyl hydrogen bond between pY and a conserved residue involved in TKB-domain binding. Instead of the conserved interaction, these peptides specifically interact with the TKB domain. Based on this binding mode of Cblin to the TKB domain, we can design drugs against unloading-mediated muscle atrophy.
authors.affiliation: Departments of Nutritional Physiology, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, 770-8503, Japan.
authors.forename: Ayako
authors.initials: A
authors.lastname: Ohno
authors.affiliation: Departments of Nutritional Physiology, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, 770-8503, Japan.
authors.forename: Arisa
authors.initials: A
authors.lastname: Ochi
authors.affiliation: Institute for Enzyme Research, University of Tokushima, Tokushima, 770-8503, Japan.
authors.forename: Nobuo
authors.initials: N
authors.lastname: Maita
authors.affiliation: Departments of Nutritional Physiology, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, 770-8503, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Ueji
authors.affiliation: Departments of Nutritional Physiology, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, 770-8503, Japan.
authors.forename: Aki
authors.initials: A
authors.lastname: Bando
authors.affiliation: Departments of Nutritional Physiology, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, 770-8503, Japan; National Institute of Advanced Industrial Science and Technology, Ibaraki, 305-8566, Japan.
authors.forename: Reiko
authors.initials: R
authors.lastname: Nakao
authors.affiliation: Departments of Nutritional Physiology, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, 770-8503, Japan; Graduate School of Fisheries Science and Environmental Studies, Nagasaki University, Nagasaki, 852-8521, Japan.
authors.forename: Katsuya
authors.initials: K
authors.lastname: Hirasaka
authors.affiliation: Departments of Nutritional Physiology, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, 770-8503, Japan.
authors.forename: Tomoki
authors.initials: T
authors.lastname: Abe
authors.affiliation: Departments of Nutritional Physiology, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, 770-8503, Japan.
authors.forename: Shigetada
authors.initials: S
authors.lastname: Teshima-Kondo
authors.affiliation: Departments of Bioorganic Synthetic Chemistry, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, 770-8503, Japan.
authors.forename: Hisao
authors.initials: H
authors.lastname: Nemoto
authors.affiliation: Departments of Nutritional Physiology, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, 770-8503, Japan; Graduate School of Nutritional Science, Sagami Woman's University, Kanagawa, 252-0383, Japan.
authors.forename: Yuushi
authors.initials: Y
authors.lastname: Okumura
authors.affiliation: Japan Aerospace Exploration Agency (JAXA), Tsukuba, 305-8505, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Higashibata
authors.affiliation: Japan Aerospace Exploration Agency (JAXA), Tsukuba, 305-8505, Japan.
authors.forename: Sachiko
authors.initials: S
authors.lastname: Yano
authors.affiliation: Graduate School of Engineering, Kyoto University, Kyoto, 615-8510, Japan.
authors.forename: Hidehito
authors.initials: H
authors.lastname: Tochio
authors.affiliation: Departments of Nutritional Physiology, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, 770-8503, Japan. Electronic address: nikawa@tokushima-u.ac.jp.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Nikawa
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D055504:Insulin Receptor Substrate Proteins / Q000378:metabolism; D008958:Models, Molecular; D009842:Oligopeptides / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D050721:Proto-Oncogene Proteins c-cbl / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; D009842:Oligopeptides; C492211:CBLB protein, human; D050721:Proto-Oncogene Proteins c-cbl
keywords: Cbl-b inhibitor; Muscle atrophy; Pentapeptide; TKB domain; X-ray crystallography