Determination of the Sequence Specificity of XIAP BIR Domains by Screening a Combinatorial Peptide Library † Michael C. Sweeney, ‡ Xianxi Wang, § Junguk Park, | Yusen Liu, § and Dehua Pei* ,‡,| Ohio State Biochemistry Program, Department of Chemistry, and Children’s Research Institute, Department of Pediatrics, College of Medicine and Public Health, The Ohio State UniVersity, Columbus, Ohio 43210 ReceiVed August 29, 2006; ReVised Manuscript ReceiVed October 3, 2006 ABSTRACT: Inhibitor of apoptosis (IAP) proteins regulate programmed cell death by inhibiting members of the caspase family of proteases. The X-chromosome-linked IAP (XIAP) contains three baculovirus IAP repeat (BIR) domains, which bind directly to the N-termini of target proteins including those of caspases-3, -7, and -9. In the present study, we defined the consensus sequences of the motifs that interact with the three BIR domains in an unbiased manner. A combinatorial peptide library containing four random residues at the N-terminus was constructed and screened using BIR domains as probes. We found that the BIR3 domain binds a highly specific motif containing an alanine or valine at the N-terminus (P1 position), an arginine or proline at the P3 position, and a hydrophobic residue (Phe, Ile, and Tyr) at the P4 position. The BIR2-binding motif is less stringent. Although it still requires an N-terminal alanine, it tolerates a wide variety of amino acids at P2-P4 positions. The BIR1 failed to bind to any peptides in the library. SPR analysis of individually synthesized peptides confirmed the library screening results. Database searches with the BIR2- and BIR3-binding consensus sequences revealed a large number of potential target proteins. The combinatorial library method should be readily applicable to other BIR domains or other types of protein modular domains. Apoptosis, or programmed cell death, fulfills an essential requirement in normal physiology by eliminating undesirable cells during embryogenesis, immune cell development, viral infection, and after environmental insult. As might be expected from its irreversible nature and severe consequence, apoptosis must be tightly regulated. Both insufficient and excessive apoptotic activity can lead to human diseases such as developmental abnormalities and cancers (1). The caspas- es, which are a family of cysteine proteases responsible for the initiation and execution phases of cellular dismember- ment, must be held in check until the arrival of appropriate signals. Their regulation is achieved at several levels beginning with their synthesis as inactive zymogens. Under the control of accessory proteins, proteolytic processing yields active caspases consisting of an R22 subunit stoi- chiometry (2). The activated proteases are further regulated by a family of inhibitor of apoptosis (IAP) 1 proteins (3, 4). A common feature of IAPs is that they contain one to three baculovirus IAP repeat (BIR) domains (5). The BIR domain, which is composed of ∼70 amino acids, mediates protein- protein interactions by interacting with specific N-terminal sequences of its partner proteins (6, 7). The best characterized member of the IAP family is the human X-linked IAP (XIAP), which contains three N-terminal BIR domains (BIR1, BIR2, and BIR3) and a canonical C-terminal RING finger domain (5). The BIR3 domain of XIAP binds directly to the small subunit of processed caspase-9, the initiator caspase of the mitochondrial pathway of apoptosis (8, 9). Structural studies show that the BIR domain interacts with the N-terminal tetrapeptide of caspase-9, ATPF, and seques- ters caspase-9 in a catalytically inactive monomeric state (8, 10). The BIR2 domain of XIAP, together with the preceding linker sequence, binds to and inhibits the executioner caspases-3 and -7 (11-13). Caspase inhibition by XIAP is in turn regulated by a small protein, second mitochondrial activator of caspases (SMAC), which binds to the BIR2 and BIR3 domains of XIAP, thereby sequestering the inhibitor and releasing the caspase activity (6, 7, 14, 15). Interestingly, the BIR1 domain of XIAP, despite sharing 41% sequence identity with BIR2, has never been observed in any protein- protein interactions. The physiological functions of other IAPs are much less understood. In addition to their putative function in inhibition of apoptosis, IAPs have been suggested to play additional roles. For example, survivin, which consists largely of a single BIR domain, has been shown to be associated with cytokinesis (16, 17). It is therefore of great interest to determine what other proteins may be recognized by the BIR † This work was supported by grants from the National Institutes of Health (GM062820 to D.P. and AI057798 to Y.L.). * To whom correspondence should be addressed at the Department of Chemistry, The Ohio State University. Telephone: (614) 688-4068. Fax: (614) 292-1532. E-mail: pei.3@osu.edu. ‡ Ohio State Biochemistry Program. § Children’s Research Institute. | Department of Chemistry. 1 Abbreviations: BCIP, 5-bromo-4-chloro-3-indolyl phosphate; BIR domain, baculovirus IAP repeat domain; IAP, inhibitor of apoptosis; MBP, maltose-binding protein; Nic-OSU, N-hydroxysuccinimidyl nico- tinate; PED/MS, partial Edman degradation/mass spectrometry; PITC, phenyl isothiocyanate; SA-AP, streptavidin-alkaline phosphatase; SMAC, second mitochondrial activator of caspases; SPR, surface plasmon resonance; TFA, trifluoroacetic acid; XIAP, X-linked inhibitor of apoptosis. 14740 Biochemistry 2006, 45, 14740-14748 10.1021/bi061782x CCC: $33.50 © 2006 American Chemical Society Published on Web 11/09/2006