A structure-activity relationship study elucidating the mechanism of sequence-specific collagen recognition by the chaperone HSP47.

Heat-shock protein 47 (HSP47) is a chaperone that facilitates the proper folding of procollagen. Our previous studies showed that the high-affinity HSP47-binding motif in the collagen triple helix is Xaa-(Thr/Pro)-Gly-Xaa-Arg-Gly. In this study, we further investigated structural requirements for the HSP47-binding motif, using synthetic triple-helical collagen-model peptides with systematic amino acid substitutions at either the Thr/Pro (=Yaa(-3)) or the Arg (=Yaa(0)) position. Results obtained from in vitro binding assays indicated that HSP47 detects the side-chain structure of Arg at the Yaa(0)-position, while the Yaa(-3) amino acid serves as the secondary recognition site that affects affinity to HSP47.

Specific recognition of the collagen triple helix by chaperone HSP47. II. The HSP47-binding structural motif in collagens and related proteins.

The endoplasmic reticulum-resident chaperone heat-shock protein 47 (HSP47) plays an essential role in procollagen biosynthesis. The function of HSP47 relies on its specific interaction with correctly folded triple-helical regions comprised of Gly-Xaa-Yaa repeats, and Arg residues at Yaa positions have been shown to be important for this interaction. The amino acid at the Yaa position (Yaa(-3)) in the N-terminal-adjoining triplet containing the critical Arg (defined as Arg(0)) was also suggested to be directly recognized by HSP47 (Koide, T., Asada, S., Takahara, Y., Nishikawa, Y., Nagata, K., and Kitagawa, K. (2006) J. Biol. Chem. 281, 3432-3438). Based on this finding, we examined the relationship between the structure of Yaa(-3) and HSP47 binding using synthetic collagenous peptides. The results obtained indicated that the structure of Yaa(-3) determined the binding affinity for HSP47. Maximal binding was observed when Yaa(-3) was Thr. Moreover, the required relative spatial arrangement of these key residues in the triple helix was analyzed by taking advantage of heterotrimeric collagen-model peptides, each of which contains one Thr(-3) and one Arg(0). The results revealed that HSP47 recognizes the Yaa(-3) and Arg(0) residues only when they are on the same peptide strand. Taken together, the data obtained led us to define the HSP47-binding structural epitope in the collagen triple helix and also define the HSP47-binding motif in the primary structure. A motif search against human protein database predicted candidate clients for this molecular chaperone. The search result indicated that not all collagen family proteins require the chaperoning by HSP47.

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).

Synthesis of heterotrimeric collagen models containing Arg residues in Y-positions and analysis of their conformational stability.

An Arg residue incorporated into the Y-position of collagenous host-guest peptide Ac-(Gly-Pro-Hyp)(3)-Gly-Pro-Y-(Gly-Pro-Hyp)(4)-Gly-Gly-NH(2) is reported to stabilize the triple helical structure as well as a 4(R)-hydroxyproline (Hyp) residue. Here, we synthesized heterotrimeric collagen models containing Arg in Y-positions utilizing the cystine knot strategy. Analysis of their thermal transition temperatures using circular dichroism spectrometry demonstrated unexpected decrease in the triple helical stability as the number of Arg increased. The obtained results indicated that an Arg residue in a Y-position is not always an equivalent of a Hyp residue, and that it possesses a potential helix destabilizing effect.

Xaa-Arg-Gly triplets in the collagen triple helix are dominant binding sites for the molecular chaperone HSP47.

HSP47 is an essential procollagen-specific molecular chaperone that resides in the endoplasmic reticulum of procollagen-producing cells. Recent advances have revealed that HSP47 recognizes the (Pro-Pro-Gly)(n) sequence but not (Pro-Hyp-Gly)(n) and that HSP47 recognizes the triple-helical conformation. In this study, to better understand the substrate recognition by HSP47, we synthesized various collagen model peptides and examined their interaction with HSP47 in vitro. We found that the Pro-Arg-Gly triplet forms an HSP47-binding site. The HSP47 binding was observed only when Arg residues were incorporated in the Yaa positions of the Xaa-Yaa-Gly triplets. Amino acids in the Xaa position did not largely affect the interaction. The recognition of the Arg residue by HSP47 was specific to its side-chain structure because replacement of the Arg residue by other basic amino acids decreased the affinity to HSP47. The significance of Arg residues in HSP47 binding was further confirmed by using residue-specific chemical modification of types I and III collagen. Our results demonstrate that Xaa-Arg-Gly sequences in the triple-helical procollagen molecule are dominant binding sites for HSP47 and enable us to predict HSP47-binding sites in homotrimeric procollagen molecules.