title: Amino acid determinants of beta-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 beta-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 beta-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 beta-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-alpha-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 beta-hairpin or alpha-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).
authors.affiliation: Department of Protein Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA. skelly@gene.com
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Skelton
authors.forename: Stephen
authors.initials: S
authors.lastname: Russell
authors.forename: Frederic
authors.initials: F
authors.lastname: de Sauvage
authors.forename: Andrea G
authors.initials: AG
authors.lastname: Cochran
mesh_terms: D000595:Amino Acid Sequence; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002942:Circular Dichroism; D019295:Computational Biology; D030562:Databases, Protein; D006801:Humans; D006860:Hydrogen Bonding; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017467:Receptors, Erythropoietin / Q000819:agonists* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion; D013816:Thermodynamics; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D008024:Ligands; D019151:Peptide Library; D010455:Peptides; D017467:Receptors, Erythropoietin; D014764:Viral Proteins; C451499:erythropoietin receptor agonist peptide 18, filamentous phage