NMR and mutational identification of the collagen-binding site of the chaperone Hsp47.
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.
Institute for Molecular Science and Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Japan.
Maho
M
Yagi-Utsumi
Sumi
S
Yoshikawa
Yoshiki
Y
Yamaguchi
Yohei
Y
Nishi
Eiji
E
Kurimoto
Yoshihito
Y
Ishida
Takayuki
T
Homma
Jun
J
Hoseki
Yoshimi
Y
Nishikawa
Takaki
T
Koide
Kazuhiro
K
Nagata
Koichi
K
Kato
D000818:Animals; D001665:Binding Sites; D002645:Chickens; D003094:Collagen / Q000737:chemistry*; D018360:Crystallography, X-Ray / Q000379:methods; D004252:DNA Mutational Analysis / Q000379:methods*; D005355:Fibrosis / Q000473:pathology; D050958:HSP47 Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013552:Swine; D014364:Tryptophan / Q000737:chemistry
D050958:HSP47 Heat-Shock Proteins; D010455:Peptides; C494660:SERPINH1 protein, human; D006639:Histidine; D014364:Tryptophan; D003094:Collagen