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

Haiyan Cai,Richard W Farndale,Parvathy Sasikumar,Samir W Hamaia,Dominique Bihan,Aiwu Zhou,Gemma Little,Jonathan M Gibbins

doi:10.3390/biom11070983

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.

Highlights

Collagen is the most abundant protein in mammals and is critical for forming specialized extracellular networks that bind cells together
The results showed that Col003 and its analogs and the compounds reported by others preferred binding to the large subpocket1 near Asp385 on heat shock protein 47 (HSP47); for compound
18 amino acids along the triple-helical sequence of human collagen II or III with each successive peptide, and a 9-amino acid overlap is included between adjacent peptides

Summary

Introduction

Collagen is the most abundant protein in mammals and is critical for forming specialized extracellular networks that bind cells together. Procollagen, the precursor molecule of collagen, undergoes extensive posttranslational processing to assemble into a triple-helical collagen, following which mature collagen is secreted [1,2]. During these processes, several molecular chaperones and enzymes such as prolyl hydroxylase and heat shock protein 47 (HSP47) are involved. HSP47 is a member of the serpin family, but it lacks serine protease inhibitory activity [3] It normally resides in the endoplasmic reticulum (ER) and functions as a collagenspecific molecular chaperone [4,5,6]. HSP47 knockout mice are embryonic-lethal 11.5 days post-coitus [8], and aberrant formation of triple-helical collagen

Methods

Results

Conclusion