title: Determination of the sequence specificity of XIAP BIR domains by screening a combinatorial peptide library.
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.
authors.affiliation: Ohio State Biochemistry Program, Department of Chemistry, The Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Sweeney
authors.forename: Xianxi
authors.initials: X
authors.lastname: Wang
authors.forename: Junguk
authors.initials: J
authors.lastname: Park
authors.forename: Yusen
authors.initials: Y
authors.lastname: Liu
authors.forename: Dehua
authors.initials: D
authors.lastname: Pei
mesh_terms: 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
chemical_list: D002352:Carrier Proteins; D017931:DNA Primers; D009842:Oligopeptides; D019151:Peptide Library; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D020169:Caspases