C OMMUNICATION Asymmetric Recruitment of cIAPs by TRAF2 Peter D. Mace 1 , Callum Smits 1 , David L. Vaux 2 , John Silke 2 and Catherine L. Day 1 ⁎ 1 Department of Biochemistry, University of Otago, Dunedin 9054, New Zealand 2 Department of Biochemistry, La Trobe University, Victoria 3086, Australia Received 8 March 2010; received in revised form 23 April 2010; accepted 27 April 2010 Available online 4 May 2010 Cellular inhibitor of apoptosis protein (cIAP) 1 and cIAP2 set the balance between transcription factor and apoptosis signaling downstream of tumor necrosis factor (TNF) receptor superfamily members by acting as ubiquitin E3 ligases for substrates that are part of the TNF receptor complex. To fulfill this role, cIAPs must be recruited to the receptor complex by TNF-receptor- associated factor (TRAF) 2. In this study, we reconstituted the complex between baculoviral IAP repeat (BIR) 1 of cIAP1 and the coiled-coil region of TRAF2, solved the structure of BIR1 from cIAP1, and mapped key binding residues on each molecule using mutagenesis. Biophysical analysis indicates that a single BIR1 domain binds the trimeric TRAF2 coiled-coil domain. This suggests that only one IAP molecule binds to each TRAF trimer and makes it likely that the dimeric cIAPs crosslink two TRAF trimers. © 2010 Elsevier Ltd. All rights reserved. Edited by R. Huber Keywords: coiled coil; TRAF2; IAP; apoptosis; protein interactions Inhibitor of apoptosis proteins (IAPs) are key negative regulators of programmed cell death that function by either directly inhibiting caspases or blocking pathways that activate them. By defini- tion, all IAPs possess at least one baculoviral IAP repeat (BIR) domain, and those that regulate apoptosis also contain a RING domain that confers ubiquitin E3 ligase activity. 1 Among mammalian IAPs, X-linked inhibitor of apoptosis protein (XIAP) is thought to primarily modulate apoptotic signals transmitted via the mitochon- dria, whereas cellular inhibitor of apoptosis protein (cIAP) 1 and cIAP2 are associated with death receptor complexes and modulate signaling by tumor necrosis factor (TNF) superfamily members, as well as that from Toll and innate immune signaling complexes. 2,3 cIAP1 and cIAP2 were first identified as components of the TNF receptor signaling com- plex, to which they are recruited by TNF-receptor- associated factor (TRAF) 2. 4,5 Both cIAP1 and cIAP2 have three BIR domains, followed by a ubiquitin binding domain, a caspase recruitment domain, and a C-terminal RING domain. 6 Al- though the BIR fold is highly conserved, variation at key surface residues and flanking linkers confer specific functions. Notably, XIAP, cIAP1, and cIAP2 all have three BIRs, but only the BIR1 domains from cIAP1 and cIAP2 interact with TRAF2 and are res- ponsible for cIAP recruitment to the TNF receptor complex. 7,8 In contrast, BIR1 of XIAP interacts with TAB1, an adaptor protein that links XIAP to TAK1 (a kinase that activates other kinases and NF-κB transcription factors). 9,10 The six human TRAF proteins serve as adaptor molecules that recruit other proteins to TNF recep- tors, and they have been implicated in the regulation of a variety of processes, including apoptosis and immune function. 11 All contain a domain of approx- imately 250 amino acids, termed the TRAF domain. The TRAF domain from TRAF2 forms a trimer and consists of an N-terminal coiled coil, followed by a C- terminal β-sandwich domain responsible for bind- ing to the TNF receptor 12,13 and to the adaptor protein TRADD. 14 There are variations in the length of the N-terminal coiled-coil region between doi:10.1016/j.jmb.2010.04.055 J. Mol. Biol. (2010) 400,8–15 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved. *Corresponding author. E-mail address: catherine.day@otago.ac.nz. Abbreviations used: cIAP, cellular inhibitor of apoptosis protein; TNF, tumor necrosis factor; TRAF, TNF-receptor- associated factor; BIR, baculoviral IAP repeat; IAP, inhibitor of apoptosis protein; XIAP, X-linked inhibitor of apoptosis protein; TRAFN, N-terminal region of the TRAF domain; GST, glutathione S-transferase; MALLS, multiple-angle laser light scattering; SEC, size-exclusion chromatography; ITC, isothermal titration calorimetry; PDB, Protein Data Bank.