title: Structural analysis of Siah1 and its interactions with Siah-interacting protein (SIP).
abstract: Seven in absentia homologue (Siah) family proteins bind ubiquitin-conjugating enzymes and target proteins for proteasome-mediated degradation. Recently we identified a novel Siah-interacting protein (SIP) that is a Sgt1-related molecule that provides a physical link between Siah family proteins and the Skp1-Cullin-F-box ubiquitin ligase component Skp1. In the present study, a structure-based approach was used to identify interacting residues in Siah that are required for association with SIP. In Siah1 a large concave surface is formed across the dimer interface. Analysis of the electrostatic surface potential of the Siah1 dimer reveals that the beta-sheet concavity is predominately electronegative, suggesting that the protein-protein interactions between Siah1 and SIP are mediated by ionic contacts. The structural prediction was confirmed by site-directed mutagenesis of these electronegative residues, resulting in loss of binding of Siah1 to SIP in vitro and in cells. The results also provide a structural basis for understanding the mechanism by which Siah family proteins interact with partner proteins such as SIP.
authors.affiliation: Burnham Institute, La Jolla, California 92037,USA.
authors.forename: Shu-ichi
authors.initials: S
authors.lastname: Matsuzawa
authors.forename: Chenglong
authors.initials: C
authors.lastname: Li
authors.forename: Chao-Zhou
authors.initials: CZ
authors.lastname: Ni
authors.forename: Shinichi
authors.initials: S
authors.lastname: Takayama
authors.forename: John C
authors.initials: JC
authors.lastname: Reed
authors.forename: Kathryn R
authors.initials: KR
authors.lastname: Ely
mesh_terms: D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D019281:Dimerization; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D044767:Ubiquitin-Protein Ligases; D000098563:Seven in Absentia Proteins
chemical_list: C491778:CACYBP protein, human; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D006360:Heat-Shock Proteins; D009687:Nuclear Proteins; D044767:Ubiquitin-Protein Ligases; D000098563:Seven in Absentia Proteins