NMR and mutational identification of the collagen-binding site of the chaperone Hsp47

PLoS One. 2012;7(9):e45930. doi: 10.1371/journal.pone.0045930. Epub 2012 Sep 25.

Abstract

Heat shock protein 47 (Hsp47) acts as a client-specific chaperone for collagen and plays a vital role in collagen maturation and the consequent embryonic development. In addition, this protein can be a potential target for the treatment of fibrosis. Despite its physiological and pathological importance, little is currently known about the collagen-binding mode of Hsp47 from a structural aspect. Here, we describe an NMR study that was conducted to identify the collagen-binding site of Hsp47. We used chicken Hsp47, which has higher solubility than its human counterpart, and applied a selective (15)N-labeling method targeting its tryptophan and histidine residues. Spectral assignments were made based on site-directed mutagenesis of the individual residues. By inspecting the spectral changes that were observed upon interaction with a trimeric collagen peptide and the mutational data, we successfully mapped the collagen-binding site in the B/C β-barrel domain and a nearby loop in a 3D-homology model based upon a serpin fold. This conclusion was confirmed by mutational analysis. Our findings provide a molecular basis for the design of compounds that target the interaction between Hsp47 and procollagen as therapeutics for fibrotic diseases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • Chickens
  • Collagen / chemistry*
  • Crystallography, X-Ray / methods
  • DNA Mutational Analysis / methods*
  • Fibrosis / pathology
  • HSP47 Heat-Shock Proteins / chemistry*
  • HSP47 Heat-Shock Proteins / metabolism
  • Histidine / chemistry
  • Magnetic Resonance Spectroscopy / methods*
  • Models, Molecular
  • Molecular Conformation
  • Mutagenesis, Site-Directed
  • Peptides / chemistry
  • Protein Conformation
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Swine
  • Tryptophan / chemistry

Substances

  • HSP47 Heat-Shock Proteins
  • Peptides
  • SERPINH1 protein, human
  • Histidine
  • Tryptophan
  • Collagen

Grant support

This work was supported in part by Grants-in-Aid for Scientific Research (20059030, 21370050) (http://www.jsps.go.jp/english/e-grants/index.html) and the Nanotechnology Network Project of the Ministry of Education, Culture, Sports, Science and Technology of Japan (https://nanonet.nims.go.jp/english/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.