title: The ubiquitin binding region of the Smurf HECT domain facilitates polyubiquitylation and binding of ubiquitylated substrates.
abstract: Mono- and polyubiquitylation of proteins are key steps in a wide range of biological processes. However, the molecular mechanisms that mediate these different events are poorly understood. Here, we employed NMR spectroscopy to map a non-covalent ubiquitin binding surface (UBS) on the Smurf ubiquitin ligase HECT domain. Analysis of mutants of the HECT UBS reveal that interfering with the UBS surface blocked Smurf-dependent degradation of its substrate RhoA in cells. In vitro analysis revealed that the UBS was not required for UbcH7-dependent charging of the HECT catalytic cysteine. Surprisingly, although the UBS was required for polyubiquitylation of both Smurf itself and the Smurf substrate RhoA, it was not required for monoubiquitylation. Furthermore, we show that mutating the UBS interfered with efficient binding of a monoubiquitylated form of RhoA to the Smurf HECT domain. Our findings suggest the UBS promotes polyubiquitylation by stabilizing ubiquitylated substrate binding to the HECT domain.
authors.affiliation: Center for Systems Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada.
authors.forename: Abiodun A
authors.initials: AA
authors.lastname: Ogunjimi
authors.forename: Silke
authors.initials: S
authors.lastname: Wiesner
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Briant
authors.forename: Xaralabos
authors.initials: X
authors.lastname: Varelas
authors.forename: Frank
authors.initials: F
authors.lastname: Sicheri
authors.forename: Julie
authors.initials: J
authors.lastname: Forman-Kay
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Wrana
mesh_terms: D001665:Binding Sites; D002460:Cell Line; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D025821:Polyubiquitin / Q000378:metabolism*; D011485:Protein Binding; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D025801:Ubiquitin; D025821:Polyubiquitin; C498342:SMURF1 protein, human; D044767:Ubiquitin-Protein Ligases; D020742:rhoA GTP-Binding Protein