Characterization of a heterodimeric Smac-based peptide that features sequences specific to both the BIR2 and BIR3 domains of the X-linked inhibitor of apoptosis protein.
XIAP, an important regulator of apoptosis, has emerged as a target for the development of cancer therapeutics. The homodimeric Smac protein simultaneously binds to both the BIR2 and BIR3 domains of XIAP. Peptide-based dimeric compounds that mimic the binding mode of Smac show promise as XIAP antagonists. Herein we characterize the first example of a Smac mimetic that incorporates a peptide sequence specific for BIR2. We show that the tetrapeptide motif Ala-Glu-Ala-Val has a higher affinity for BIR2 than the BIR3-specific sequence Ala-Val-Pro-Phe, and we compare the binding characteristics of a heterodimeric peptide containing both tetrapeptide motifs to those of a homodimeric peptide featuring only AVPF. Despite the enhanced affinity of AEAV (relative to AVPF) for BIR2, the heterodimeric peptide displays only a slightly higher affinity for XIAP relative to its homodimeric counterpart. Enhanced affinity of both dimers relative to the tetrapeptide AVPF is largely maintained even when the BIR2 binding groove is modified, implying that hydrophobic contacts afforded by the second peptide motif need not necessarily be made at the BIR2 binding groove to contribute substantial binding energy. Finally, we use mutagenesis to show that the difference in sequence specificity observed between the two domains is primarily owing to steric bulk introduced at the BIR2 site by lysine 206. Replacement of K206 at BIR2 with glycine, the corresponding residue in BIR3, restores the majority of the affinity for the AVPF motif exhibited by BIR3. The implications of these finding in the development of XIAP antagonists are discussed. © 2011 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 98: 122-130, 2012.
Department of Chemistry, Macalester College, St. Paul, MN 55105, USA.
Kelsey F
KF
Speer
Charles L
CL
Cosimini
Kathryn E
KE
Splan
D020816:Amino Acid Motifs; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D019281:Dimerization; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry*; D024101:Mitochondrial Proteins / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D009369:Neoplasms / Q000628:therapy; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000737:chemistry*
D051017:Apoptosis Regulatory Proteins; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D010455:Peptides; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human