biomolecules Article Identification of HSP47 Binding Site on Native Collagen and Its Implications for the Development of HSP47 Inhibitors Haiyan Cai 1,† , Parvathy Sasikumar 2,† , Gemma Little 2 , Dominique Bihan 3 , Samir W. Hamaia 3 , Aiwu Zhou 1 , Jonathan M. Gibbins 2 and Richard W. Farndale 3,4, *   Citation: Cai, H.; Sasikumar, P.; Little, G.; Bihan, D.; Hamaia, S.W.; Zhou, A.; Gibbins, J.M.; Farndale, R.W. Identification of HSP47 Binding Site on Native Collagen and Its Implications for the Development of HSP47 Inhibitors. Biomolecules 2021, 11, 983. https://doi.org/10.3390/ biom11070983 Academic Editor: Maria Stefania Sinicropi Received: 9 June 2021 Accepted: 30 June 2021 Published: 3 July 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Department of Pathophysiology, Shanghai JiaoTong University School of Medicine, Shanghai 200025, China; hycai@shsmu.edu.cn (H.C.); awz20@shsmu.edu.cn (A.Z.) 2 Institute for Cardiovascular & Metabolic Research, School of Biological Sciences, University of Reading, Health and Life Sciences Building, Whiteknights, Reading RG6 6EX, UK; p.sasi-kumar@imperial.ac.uk (P.S.); gemma.little@reading.ac.uk (G.L.); j.m.gibbins@reading.ac.uk (J.M.G.) 3 Department of Biochemistry, University of Cambridge, Downing Site, Cambridge CB2 1QW, UK; dominique.bihan@ucalgary.ca (D.B.); swh23@cam.ac.uk (S.W.H.) 4 CambCol Laboratories Ltd., Ely CB6 1RS, UK * Correspondence: rwf10@cam.ac.uk † Authors contributed equally to this work. 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. Keywords: HSP47 inhibitor; collagen; fibrosis; molecular docking; structural analysis 1. Introduction Collagen is the most abundant protein in mammals and is critical for forming special- ized extracellular networks that bind cells together. The folding, processing, and assembly of collagen is tightly regulated in eukaryotic cells. Procollagen, the precursor molecule of collagen, undergoes extensive posttranslational processing to assemble into a triple-helical collagen, following which mature collagen is then 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 activ- ity [3]. It normally resides in the endoplasmic reticulum (ER) and functions as a collagen- specific molecular chaperone [4–6]. HSP47 associates transiently with procollagen in the ER, thereby preventing premature interactions between nascent procollagen molecules, and dissociates when procollagen transfers to cis-Golgi [7]. HSP47 knockout mice are embryonic-lethal 11.5 days post-coitus [8], and aberrant formation of triple-helical collagen Biomolecules 2021, 11, 983. https://doi.org/10.3390/biom11070983 https://www.mdpi.com/journal/biomolecules