Amino acid determinants of β-hairpin conformation in erythropoeitin receptor agonist peptides derived from a phage display library

Abstract 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).