Structures of the cIAP2 RING Domain Reveal Conformational Changes Associated with Ubiquitin-conjugating Enzyme (E2) Recruitment

Abstract

Inhibitor of apoptosis (IAP) proteins are key negative regulators of cell death that are highly expressed in many cancers. Cell death caused by antagonists that bind to IAP proteins is associated with their ubiquitylation and degradation. The RING domain at the C terminus of IAP proteins is pivotal. Here we report the crystal structures of the cIAP2 RING domain homodimer alone, and bound to the ubiquitin-conjugating (E2) enzyme UbcH5b. These structures show that small changes in the RING domain accompany E2 binding. By mutating residues at the E2-binding surface, we show that autoubiquitylation is required for regulation of IAP abundance. Dimer formation is also critical, and mutation of a single C-terminal residue abrogated dimer formation and E3 ligase activity was diminished. We further demonstrate that disruption of E2 binding, or dimerization, stabilizes IAP proteins against IAP antagonists in vivo. Inhibitor of apoptosis (IAP) proteins are key negative regulators of cell death that are highly expressed in many cancers. Cell death caused by antagonists that bind to IAP proteins is associated with their ubiquitylation and degradation. The RING domain at the C terminus of IAP proteins is pivotal. Here we report the crystal structures of the cIAP2 RING domain homodimer alone, and bound to the ubiquitin-conjugating (E2) enzyme UbcH5b. These structures show that small changes in the RING domain accompany E2 binding. By mutating residues at the E2-binding surface, we show that autoubiquitylation is required for regulation of IAP abundance. Dimer formation is also critical, and mutation of a single C-terminal residue abrogated dimer formation and E3 ligase activity was diminished. We further demonstrate that disruption of E2 binding, or dimerization, stabilizes IAP proteins against IAP antagonists in vivo. Apoptosis is a tightly controlled process that is required for normal development and homeostasis of cell number that when dysregulated contributes to disease (1Danial N.N. Korsmeyer S.J. Cell. 2004; 116: 205-219Abstract Full Text Full Text PDF PubMed Scopus (4060) Google Scholar). Mammalian “inhibitor of apoptosis” (IAP) 3The abbreviations used are: IAP, inhibitor of apoptosis; XIAP, X-linked IAP; RING, really interesting new gene; BIR, baculoviral IAP repeat; TNF, tumor necrosis factor; CARD, caspase recruitment domain; MALS, multiple angle light-scattering; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin ligase; Ubc, ubiquitin-conjugating; GST, glutathione S-transferase; WT, wild type; 4HT, 4-hydroxy-tamoxifen; MDM, murine double minute; CHIP, C-terminal of Hsp70-interacting protein. proteins, such as XIAP, cIAP1, and cIAP2, are key negative regulators of apoptosis that can prevent apoptosis by directly inhibiting caspases or by blocking pathways that activate them (2Salvesen G.S. Duckett C.S. Nat. Rev. Mol. Cell Biol. 2002; 3: 401-410Crossref PubMed Scopus (1581) Google Scholar). Amplification of the locus bearing cIAP1 and cIAP2 has been observed in liver, lung, and cervical tumors in humans, raising the possibility that inhibition of cell death by IAP proteins contributes to oncogenesis and provoking the design of drugs to antagonize IAP proteins for the treatment of cancer (3Hunter A.M. Lacasse E.C. Korneluk R.G. Apoptosis. 2007; 12: 1543-1568Crossref PubMed Scopus (489) Google Scholar). IAP proteins are characterized by the presence of one or more zinc-binding baculoviral IAP repeat (BIR) domains that function as protein-protein interaction domains. The N-terminal BIR domains of cIAP1 and cIAP2 interact with the TRAF-N domain of tumor necrosis factor-associated factors (4Samuel T. Welsh K. Lober T. Togo S.H. Zapata J.M. Reed J.C. J. Biol. Chem. 2006; 281: 1080-1090Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 5Varfolomeev E. Wayson S.M. Dixit V.M. Fairbrother W.J. Vucic D. J. Biol. Chem. 2006; 281: 29022-29029Abstract Full Text Full Text PDF PubMed Scopus (76) Goo