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

Takaki Koide, Akiko Aso, Tetuya Yorihuzi, Kazuhiro Nagata

Research output: Contribution to journalArticle

54 Citations (Scopus)

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°C but not at 30°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.

Original languageEnglish
Pages (from-to)27957-27963
Number of pages7
JournalJournal of Biological Chemistry
Volume275
Issue number36
DOIs
Publication statusPublished - 2000 Sep 8
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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