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The cell-extrinsic apoptotic pathway triggers programmed cell death in response to certain ligands that bind to cell-surface death receptors. Apoptosis is essential for normal development and homeostasis in metazoans, and furthermore, selective activation of the cell-extrinsic pathway in tumor cells holds considerable promise for cancer therapy. We used phage display to identify peptides and synthetic antibodies that specifically bind to the human proapoptotic death receptor DR5. Despite great differences in overall size and structure, the DR5-binding peptides and antibodies shared a tripeptide motif, which was conserved within a disulfide-constrained loop of the peptides and the third complementarity determining region of the antibody heavy chains. The X-ray crystal structure of an antibody in complex with DR5 revealed that the tripeptide motif is buried at the core of the interface, confirming its central role in antigen recognition. We found that certain peptides and antibodies exhibited potent proapoptotic activity against DR5-expressing SK-MES-1 lung carcinoma cells. These phage-derived ligands may be useful for elucidating DR5 activation at the molecular level and for creating synthetic agonists of proapoptotic death receptors.

Display of peptide libraries on filamentous phage has led to the identification of peptides of the form X 2-5 CX 2 GPXTWXCX 2-5 (where X is a variable residue) that bind to the extra-cellular portion of the erythropoietin receptor (EPO-R). These peptides adopt β-hairpin conformations when co-crystallized with EPO-R. Solution NMR studies reveal that the peptide is conformationally heterogeneous in the absence of receptor due to cis - trans isomerization about the Gly-Pro peptide bond. Replacement of the conserved threonine residue with glycine at the turn i + 3 position produces a stable β-hairpin conformation in solution, although this peptide no longer has activity in an EPO-R-dependent cell proliferation assay. A truncated form of the EPO-R-binding peptide (containing the i + 3 glycine residue) also forms a highly populated, monomeric β-hairpin. In contrast, phage-derived peptide antagonists of insulin-like growth factor binding protein 1 (IGFBP-1) have a high level of sequence identity with the truncated EPO-R peptide (eight of 12 residues) yet adopt a turn-α-helix conformation in solution. Peptides containing all possible pairwise amino acid substitutions between the EPO-R and IGFBP-1 peptides have been analyzed to assess the degree to which the non-conserved residues stabilize the hairpin or helix conformation. All four residues present in the original sequence are required for maximum population of either the β-hairpin or α-helix conformation, although some substitutions have a more dominant effect. The results demonstrate that, within a given sequence, the observed conformation can be dictated by a small subset of the residues (in this case four out of 12).

The small immunoglobulin G (IgG)-binding protein GB1 is a favored model system for the study of individual residue contributions to the stability of β-sheets. Nevertheless, only a few of the many possible combinations of mutations have been characterized, leaving many questions unanswered. In order to allow the simultaneous evaluation of libraries of mutants, we have adapted a phage-display method, called shotgun scanning. This method combines a binding (i.e. stability) selection with high-throughput sequence analysis. Relative folding free energies determined from GB1-phage sequence data agree well with published GB1 thermal stability studies, validating the use of phage display to conduct quantitative stability studies on GB1, and further suggesting that this method is generally applicable to mutational analysis of protein stability. Examination of residue pairing in our large collection of GB1 mutants indicates that specific side-chain–side-chain interactions are much less important to β-sheet stability than individual residue contributions. The discrepancy between this observation and published studies can be traced to anomalous stability of the alanine-substituted GB1 variants typically used as reference states in double mutant-cycle analyses. Finally, the combination of large library sizes and a quantitative stability selection should allow phage-based “computation” to be applied to protein design problems.

Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific mediator of angiogenesis and vasculogenesis. VEGF is involved pathologically in cancer, proliferative retinopathy and rheumatoid arthritis, and as such represents an important therapeutic target. Three classes of disulfide-constrained peptides that antagonize binding of the VEGF dimer to its receptors, KDR and Flt-1, were identified previously using phage display methods. NMR studies of a representative peptide from the most potent class of these peptide antagonists, v107 (GGNECDAIRMWEWECFERL), were undertaken to characterize its interactions with VEGF. v107 has no defined structure free in solution, but binding to VEGF induces folding of the peptide. The solution structure of the VEGF receptor-binding domain-v107 complex was determined using 3940 (1970 per VEGF monomer) internuclear distance and 476 (238 per VEGF monomer) dihedral angle restraints derived from NMR data obtained using samples containing either (13)C/(15)N-labeled protein plus excess unlabeled peptide or (13)C/(15)N-labeled peptide plus excess unlabeled protein. Residual dipolar coupling restraints supplemented the structure determination of the complex and were found to increase significantly both the global precision of VEGF in the complex and the agreement with available crystal structures of VEGF. The calculated ensemble of structures is of high precision and is in excellent agreement with the experimental restraints. v107 has a turn-helix conformation with hydrophobic residues partitioned to one face of the peptide and polar or charged residues at the other face. Contacts between two v107 peptides and the VEGF dimer are mediated by primarily hydrophobic side-chain interactions. The v107-binding site on VEGF overlaps partially with the binding site of KDR and is similar to that for domain 2 of Flt-1. The structure of the VEGF-v107 complex provides new insight into how binding to VEGF can be achieved that may be useful for the design of small molecule antagonists.