Specific recognition of the collagen triple helix by chaperone HSP47: minimal structural requirement and spatial molecular orientation.
The unique folding of procollagens in the endoplasmic reticulum is achieved with the assistance of procollagen-specific molecular chaperones. Heat-shock protein 47 (HSP47) is an endoplasmic reticulum-resident chaperone that plays an essential role in normal procollagen folding, although its molecular function has not yet been clarified. Recent advances in studies on the binding specificity of HSP47 have revealed that Arg residues at Yaa positions in collagenous Gly-Xaa-Yaa repeats are critical for its interactions (Koide, T., Takahara, Y., Asada, S., and Nagata, K. (2002) J. Biol. Chem. 277, 6178-6182; Tasab, M., Jenkinson, L., and Bulleid, N. J. (2002) J. Biol. Chem. 277, 35007-35012). In the present study, we further examined the client recognition mechanism of HSP47 by taking advantage of systems employing engineered collagen model peptides. First, in vitro binding studies using conformationally constrained collagen-like peptides revealed that HSP47 only recognized correctly folded triple helices and that the interaction with the corresponding single-chain polypeptides was negligible. Second, a binding study using heterotrimeric model clients for HSP47 demonstrated a minimal requirement for the number of Arg residues in the triple helix. Finally, a cross-linking study using photoreactive collagenous peptides provided information about the spatial orientation of an HSP47 molecule in the chaperone-collagen complex. The obtained results led to the development of a new model of HSP47-collagen complexes that differs completely from the previously proposed "flying capstan model" (Dafforn, T. R., Della, M., and Miller, A. D. (2001) J. Biol. Chem. 276, 49310-49319).
Koide
Takaki
T
Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata 950-2081, Japan. koi@niigata-pharm.ac.hp
Asada
Shinichi
S
Takahara
Yoshifumi
Y
Nishikawa
Yoshimi
Y
Nagata
Kazuhiro
K
Kitagawa
Kouki
K
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001667:Binding, Competitive; D015153:Blotting, Western; D002845:Chromatography; D002942:Circular Dichroism; D003094:Collagen / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D050958:HSP47 Heat-Shock Proteins / Q000737:chemistry*; D006801:Humans; D051379:Mice; D008956:Models, Chemical; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D020349:Surface Plasmon Resonance; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D050958:HSP47 Heat-Shock Proteins; D018832:Molecular Chaperones; D010455:Peptides; D011994:Recombinant Proteins; D003094:Collagen; D001120:Arginine