research papers 58 doi:10.1107/S0907444908039243 Acta Cryst. (2009). D65, 58–66 Acta Crystallographica Section D Biological Crystallography ISSN 0907-4449 The structure of the BIR3 domain of cIAP1 in complex with the N-terminal peptides of SMAC and caspase-9 Raviraj Kulathila, a Brian Vash, a David Sage, a Susan Cornell- Kennon, a Kirk Wright, a James Koehn, a Travis Stams, a Kirk Clark a and Allen Price b * a Novartis Institutes for Biomedical Research Inc., USA, and b Emmanuel College, Boston, USA Correspondence e-mail: priceal@emmanuel.edu # 2009 International Union of Crystallography Printed in Singapore – all rights reserved The inhibitor of apoptosis protein (IAP) family of molecules inhibit apoptosis through the suppression of caspase activity. It is known that the XIAP protein regulates both caspase-3 and caspase-9 through direct protein–protein interactions. Speci- fically, the BIR3 domain of XIAP binds to caspase-9 via a ‘hotspot’ interaction in which the N-terminal residues of caspase-9 bind in a shallow groove on the surface of XIAP. This interaction is regulated via SMAC, the N-terminus of which binds in the same groove, thus displacing caspase-9. The mechanism of suppression of apoptosis by cIAP1 is less clear. The structure of the BIR3 domain of cIAP1 (cIAP1-BIR3) in complex with N-terminal peptides from both SMAC and caspase-9 has been determined. The binding constants of these peptides to cIAP1-BIR3 have also been determined using the surface plasmon resonance technique. The structures show that the peptides interact with cIAP1 in the same way that they interact with XIAP: both peptides bind in a similar shallow groove in the BIR3 surface, anchored at the N-terminus by a charge-stabilized hydrogen bond. The binding data show that the SMAC and caspase-9 peptides bind with comparable affinities (85 and 48 nM, respectively). Received 27 May 2008 Accepted 21 November 2008 PDB References: cIAP1-BIR3, complex with N-terminal peptide from caspase-9, 3d9t; complex with N-terminal peptide from SMAC, 3d9u. 1. Introduction Because of its extreme consequences, programmed cell death, also known as apoptosis, must be tightly regulated. Both the logic and the structural mechanisms underlying the regulation of this process are areas of current research. An important set of proteins which suppress the apoptotic signal are the IAP (inhibitor of apoptosis protein) family. Although some members of this family are known to repress apoptosis through the inhibition of caspases (Shi, 2004), the functions of all the members are not known. These genes (numbering eight in the human genome) are composed of one to three N-terminal BIR (baculoviral repeat) domains, plus accessory C-terminal domains such as RING, CARD and UBC domains. The most studied of the IAPs is XIAP (X-linked IAP), which contains three BIR domains and a single C-terminal RING domain. The BIR1 and BIR2 domains are responsible for inhibiting the effector caspases caspase-3 and caspase-7 by competitive inhibition: a linker region between the two domains binds to the active site of the caspases (Shiozaki & Shi, 2004). The BIR3 domain is responsible for inhibiting the initiator caspase caspase-9 by forming a protein–protein complex with the caspase and holding it in an inactive con- formation (Shiozaki & Shi, 2004). The BIR3–caspase-9 inter- action is regulated by SMAC (also known as DIABLO), a pro-