Identification of HSP47 Binding Site on Native Collagen and Its Implications for the Development of HSP47 Inhibitors

Abstract

HSP47 (heat shock protein 47) is a collagen-specific molecular chaperone that is essential for procollagen folding and function. Previous studies have shown that HSP47 binding requires a critical Arg residue at the Y position of the (Gly-Xaa-Yaa) repeats of collagen; however, the exact binding sites of HSP47 on native collagens are not fully defined. To address this, we mapped the HSP47 binding sites on collagens through an ELISA binding assay using collagen toolkits, synthetic collagen peptides covering the entire amino acid sequences of collagen types II and III assembled in triple-helical conformation. Our results showed that HSP47 binds to only a few of the GXR motifs in collagen, with most of the HSP47 binding sites identified located near the N-terminal part of the triple-helical region. Molecular modelling and binding energy calculation indicated that residues flanking the key Arg in the collagen sequence also play an important role in defining the high-affinity HSP47 binding site of collagen. Based on this binding mode of HSP47 to collagen, virtual screening targeting both the Arg binding site and its neighboring area on the HSP47 surface, and a subsequent bioassay, we identified two novel compounds with blocking activity towards HSP47 binding of collagen. Overall, our study revealed the native HSP47 binding sites on collagen and provided novel information for the design of small-molecule inhibitors of HSP47.

Structural insights into collagen binding by platelet receptor glycoprotein VI

“…In contrast to structures available to date of proteins in complex with collagen peptides, which all involve a collagen residue with a long side chain, i.e. phenylalanine (VWF-A3 20 , SPARC 17 , and OSCAR 21 ), glutamate (α 2 β 1 40 ), or arginine (Hsp47 41,42 ), our structures reveal a novel collagen-binding mode employing the interaction of a rather flat surface across the D1 β-sheet of GPVI with several modestly-sized hydroxyprolines in collagen.…”

N‐glycoproteomics reveals distinct glycosylation alterations in NGLY1‐deficient patient‐derived dermal fibroblasts

“…SERPINH1, normally resides in the endoplasmic and plays a role in collagen biosynthesis and its folding as a molecular chaperone. 61,62 The protein level of SERPINH1 was not altered. THY1, also known as CD90, is a heavily glycosylated protein with a GPI anchor, which is present on the cell membrane of several cell types including fibroblasts.…”

“…Several other proteins also showed the downregulation at their glycosylation levels such as SERPINH, THY1, and HAPLN1. SERPINH1, normally resides in the endoplasmic and plays a role in collagen biosynthesis and its folding as a molecular chaperone 61,62 . The protein level of SERPINH1 was not altered.…”

Code for Collagen Folding Deciphered

“…Hsp47 binding sites are predominantly located towards the N-terminal half of collagens type I, II and III. ( 12, 20, 70 ) Given the C- to N-terminal propagation, the implication is that Hsp47 binds these collagens after half the triple-helix has already folded. Hsp47 also does not recognize or does so only weakly collagens type XI, XIV, XXII.…”

“…By comparison, only a few binding sites for Hsp47 have formally been identified in human type II and III collagens. ( 20 ) Importantly, incorrect alignment of the chains in the respective triple helices dramatically decreases the number of possible salt bridges in all collagen subtypes. We also find that non-collagenous interruptions are frequently flanked by a higher density of salt bridges.…”