Conformational requirements of collagenous peptides for recognition by the chaperone protein HSP47

J Biol Chem. 2000 Sep 8;275(36):27957-63. doi: 10.1074/jbc.M003026200.


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.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Cloning, Molecular
  • Collagen / chemistry*
  • Collagen / metabolism*
  • Consensus Sequence
  • DNA-Binding Proteins
  • Escherichia coli
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Gene Library
  • Heat-Shock Proteins / metabolism*
  • Molecular Sequence Data
  • Peptide Fragments / chemistry*
  • Peptide Fragments / metabolism
  • Peptide Library*
  • Protein Binding
  • Protein Conformation*
  • Protein Structure, Secondary
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Repetitive Sequences, Amino Acid
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae Proteins*
  • Substrate Specificity
  • Transcription Factors / genetics
  • Transcription Factors / metabolism


  • DNA-Binding Proteins
  • Fungal Proteins
  • GAL4 protein, S cerevisiae
  • Heat-Shock Proteins
  • Peptide Fragments
  • Peptide Library
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors
  • Collagen

Associated data

  • GENBANK/AB044560