Determination of the sequence specificity of XIAP BIR domains by screening a combinatorial peptide library.
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.
Sweeney
Michael C
MC
Ohio State Biochemistry Program, Department of Chemistry, The Ohio State University, Columbus, Ohio 43210, USA.
Wang
Xianxi
X
Park
Junguk
J
Liu
Yusen
Y
Pei
Dehua
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D016384:Consensus Sequence; D017931:DNA Primers; D006801:Humans; D009842:Oligopeptides / Q000737:chemistry; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000737:chemistry* / Q000378:metabolism
D002352:Carrier Proteins; D017931:DNA Primers; D009842:Oligopeptides; D019151:Peptide Library; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D020169:Caspases