title: Conformational requirements of collagenous peptides for recognition by the chaperone protein HSP47.
abstract: The collagen binding chaperone HSP47 interacts with procollagen in the endoplasmic reticulum and plays a crucial role in the biosynthesis of collagen. We recently demonstrated that typical collagen model peptides, (Pro-Pro-Gly)(n), possess sufficient structural information for interaction with HSP47 (Koide, T., Asada, S., and Nagata, K. (1999) J. Biol. Chem. 274, 34523-34526). Here we show that binding of (Gly-Pro-Pro)(n) peptides to HSP47 can be detected using the two-hybrid system in yeast if a trimerizing domain is fused to the C termini of the peptides. Some peptides interacted with HSP47 at a lowered assay temperature at 24 degrees C but not at 30 degrees C, indicating the importance of conformational change of the substrate peptides. To analyze the spectrum of HSP47 substrate sequences, we performed two-hybrid screening of collagen-like peptides in designed random peptide libraries using HSP47 as a bait. In selected peptides, the enrichment ratio calculated for each amino acid residue correlated strongly with the contribution of the residue to triple-helix stability independently determined using synthetic collagen model peptides. Taken together, our results suggest that HSP47 preferentially recognizes collagenous Gly-X-Y repeats in triple-helical conformation. We also demonstrated that screening of combinatorial peptide libraries is a powerful strategy to determine conformational requirements as well as the elucidation of binding motifs in primary structure.
authors.affiliation: Core Research for Evolutional Science and Technology (CREST), Japan.
authors.forename: T
authors.initials: T
authors.lastname: Koide
authors.forename: A
authors.initials: A
authors.lastname: Aso
authors.forename: T
authors.initials: T
authors.lastname: Yorihuzi
authors.forename: K
authors.initials: K
authors.lastname: Nagata
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003094:Collagen / Q000737:chemistry* / Q000378:metabolism*; D016384:Consensus Sequence; D004268:DNA-Binding Proteins; D004926:Escherichia coli; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D006360:Heat-Shock Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D019151:Peptide Library; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D006360:Heat-Shock Proteins; D010446:Peptide Fragments; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D003094:Collagen