posted on 2006-12-12, 00:00authored byMichael C. Sweeney, Xianxi Wang, Junguk Park, Yusen Liu, Dehua Pei
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.