Biochemical basis for enhanced binding of peptide dimers to X-linked inhibitor of apoptosis protein.
XIAP (X-linked inhibitor of apoptosis protein) is involved in the mediation of programmed cell death and, therefore, is a target for the development of cancer therapeutics. Peptide mimetics based upon Smac, the natural binding partner of XIAP, and specifically, dimeric peptides, have shown great promise in drug development. In the present work, the basis for enhanced dimer efficacy has been explored. Comparisons are made between the peptide binding site on the BIR3 domain of XIAP alone (residues 238-358) and a less truncated construct that includes both BIR2 and BIR3 domains (residues 151-350). This contingency differentially enhances the binding of dimeric tetrapeptides, potentially by providing additional hydrophobic binding surface. The effect of BIR2 on the BIR3 binding site is sustained, even if the BIR2 binding site is disrupted by mutagenesis, as shown by both a fluorescent competition assay and a polarity sensitive dye, badan. FRET measurements reveal an observed separation of >or=45 A between the BIR2 and BIR3 peptide binding pockets, thereby precluding a direct simultaneous interaction of the dimer molecules with both binding domains. Furthermore, variations in the linker length between dimeric tetrapeptides did not show a predictable trend in binding affinities, suggesting that local concentration effects were also an unlikely explanation for the enhanced dimeric affinities. Taken together, the results suggest that enhanced binding of dimeric peptides likely reflects the increased hydrophobic surface area on or near the BIR3 site and have significant ramifications for the design of therapeutics that target this class of proteins.
Splan
Kathryn E
KE
Department of Chemistry, Macalester College, 1600 Grand Avenue, St. Paul, Minnesota 55105, USA. splank@macalester.edu
Allen
John E
JE
McLendon
George L
GL
D015081:2-Naphthylamine / Q000031:analogs & derivatives; D000595:Amino Acid Sequence; D017209:Apoptosis; D001665:Binding Sites; D032701:Biomimetics; D015815:Cell Adhesion Molecules; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D005982:Glutathione Transferase / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014170:Transformation, Genetic; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C122155:6-bromoacetyl-2-dimethylaminonaphthalene; D015815:Cell Adhesion Molecules; D005456:Fluorescent Dyes; D010455:Peptides; D011993:Recombinant Fusion Proteins; D051636:X-Linked Inhibitor of Apoptosis Protein; D006639:Histidine; D015081:2-Naphthylamine; D005982:Glutathione Transferase