title: Regulation of ubiquitin transfer by XIAP, a dimeric RING E3 ligase.
abstract: RING domains of E3 ligases promote transfer of Ub (ubiquitin) from the E2~Ub conjugate to target proteins. In many cases interaction of the E2~Ub conjugate with the RING domain requires its prior dimerization. Using cross-linking experiments we show that E2 conjugated ubiquitin contacts the RING homodimer interface of the IAP (inhibitor of apoptosis) proteins, XIAP (X-linked IAP) and cIAP (cellular IAP) 2. Structural and biochemical analysis of the XIAP RING dimer shows that an aromatic residue at the dimer interface is required for E2~Ub binding and Ub transfer. Mutation of the aromatic residue abolishes Ub transfer, but not interaction with Ub. This indicates that nuleophilic attack on the thioester bond depends on precise contacts between Ub and the RING domain. RING dimerization is a critical activating step for the cIAP proteins; however, our analysis shows that the RING domain of XIAP forms a stable dimer and its E3 ligase activity does not require an activation step.
authors.affiliation: Biochemistry Department, Otago School of Medical Sciences, University of Otago, Dunedin 9054, New Zealand.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Nakatani
authors.forename: Torsten
authors.initials: T
authors.lastname: Kleffmann
authors.forename: Katrin
authors.initials: K
authors.lastname: Linke
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Condon
authors.forename: Mark G
authors.initials: MG
authors.lastname: Hinds
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Day
mesh_terms: D000595:Amino Acid Sequence; D006801:Humans; D008954:Models, Biological; D008958:Models, Molecular; D010649:Phenylalanine / Q000235:genetics / Q000502:physiology; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D054730:Protein Interaction Domains and Motifs / Q000235:genetics / Q000502:physiology; D055503:Protein Multimerization / Q000235:genetics / Q000502:physiology; D013379:Substrate Specificity; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D054875:Ubiquitination / Q000235:genetics / Q000502:physiology; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
chemical_list: D025801:Ubiquitin; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D010649:Phenylalanine; C097309:UBE2D2 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases