## 3 Results and Discussion
### Binding Mechanism Analysis between HSP47 and Native Collagen Peptides  
To elucidate the structural basis underlying the specific binding of these collagen peptides with HSP47, we analyzed their binding characteristics through structural modelling and binding energy calculations. A model of human HSP47 was built based on the reported crystal structure of canine HSP47 complexed with the triple-helical CMP (PPGP\({}^{-7}\)P\({}^{-6}\)GP\({}^{-4}\)T\({}^{-3}\)GP\({}^{-1}\)R\({}^{0}\)GPPPPG, the key arginine residue is numbered as position 0). According to the crystal structure of the HSP47 and CMP complex, there are two copies of HSP47 molecules binding to a triple-helical collagen peptide in the asymmetric unit cell (PDB ID: 3 ZHA). Each HSP47 molecule interacts mainly with one strand of the collagen and these two HSP47 molecules have similar binding interactions with the corresponding collagen strands [24]; therefore, the binding interactions between HSP47 and one strand of collagen were selected for subsequent analysis. The binding sites of CMP on human and canine HSP47 are almost identical, as human and canine HSP47 are highly similar, with only a few different residues. The CMP is 18 residues long, only 8 of which (P\({}^{-7}\)P\({}^{-6}\)GP\({}^{-4}\)T\({}^{-3}\)GP\({}^{-1}\)R\({}^{0}\)) are involved in direct interactions with the \(\beta\)-sheet C region (yellow-colored) of HSP47 (Figure 2). The Arg\({}^{0}\) in the collagen formed a salt bridge to the conserved residue Asp385 in HSP47 (Figure 2b), which was essential for collagen binding, as confirmed by mutagenic study [24]. Based on this model, we then analyzed the structure-activity relationship of the binding between collagen peptides and HSP47 using FoldX software, which is a commonly-used protein stability prediction algorithm that can estimate changes in protein folding free energy caused by residue variations [27]. We applied FoldX to analyze single residue variations at different positions in CMP to estimate changes of the folding free energy compared to the WT complex. A lower folding free energy change (\(\Delta\Delta\)G) indicates favorable binding of HSP47 and CMP. Here, we first validated this method by calculating the binding energies of a set of collagen model peptides with variations at position -3 presented in a  
Figure 2: A model of human HSP47 complexed with triple-helical collagen peptide. (**a**) The model was derived from the crystal structure of canine HSP47 complexed with collagen model peptide (CMP; PDB: 3 ZHA [24]). HSP47 is shown in cartoon with three beta-sheets (A, B, C) colored in pale blue, green, and yellow, respectively, while its reactive center loop is in pink. The surface of HSP47 is shown in pale grey. The triple-helical CMP is shown in blue. (**b**) Close-up of the interaction between human HSP47 and the collagen peptide (PPGP\({}^{-7}\)P\({}^{-6}\)GP\({}^{-4}\)T\({}^{-3}\)GP\({}^{-1}\)R\({}^{0}\)GPPPPG, cyan cartoon and lines). Residues of both HSP47 and CMP that are involved in interactions are shown as sticks. previous report, where the bioactivities of these peptides were measured [31]. This showed that the calculated energy scores of these peptides are largely consistent with the HSP47 binding activities (Figure S3), where the most optimal residue at position -3 is Pro or Thr. Even though the hydroxyl group and the main chain of Thr\({}^{-3}\) formed polar interactions with residue Arg222 and Ser305 seen in the crystal structure of HSP47 (Figure 2b), Pro at this position could bind into the same pocket and has similar binding activities [31]. Other residues such as Hyp and Met at position -3 seemed compatible with HSP47 binding as well (Figure S3), while bulky or charged residue such as Phe, His, Arg, Trp, and Tyr were not. This is consistent with the HSP47 binding peptides identified here from the toolkits with Pro, Hyp, Met (III-14), or Thr (II-10) at position -3 (Table 1). Subsequently, FoldX was applied to analyze the impact of substitutions at position -4 on the HSP47-collagen binding energy.