The ubiquitin binding region of the Smurf HECT domain facilitates polyubiquitylation and binding of ubiquitylated substrates.
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.
Ogunjimi
Abiodun A
AA
Center for Systems Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada.
Wiesner
Silke
S
Briant
Douglas J
DJ
Varelas
Xaralabos
X
Sicheri
Frank
F
Forman-Kay
Julie
J
Wrana
Jeffrey L
JL
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
D025801:Ubiquitin; D025821:Polyubiquitin; C498342:SMURF1 protein, human; D044767:Ubiquitin-Protein Ligases; D020742:rhoA GTP-Binding Protein