# Revealing Accessibility of Cryptic Protein Binding Sites within the Functional Collagen Fibril
## 4 Computational Studies of the Collagen Fibril
### SASA of Collagen I Fibril Reveals Exposure of Partner Binding Sites from the Fibril Surface
Abbreviations: PEDF, pigment epithelium-derived factor; LOX, lysyl oxidase; SPARC, secreted protein acidic and rich in cysteine; DDRs, discoidin domain receptors; VWF, von Willebrand factor; OSCAR, osteoclast-associated immunoglobulin-like receptor; Yad A, _Yersinia_ adhesin A; MMPs, matrix metalloproteinases; GPVI, glycoprotein VI. MMP13, and membrane type-MMP1 (MT-MMP1) cleave collagen \(\alpha\)-chains at a single, specific Gly-Ile/Leu cleavage site, dissecting the triple helix into three-quarter and one-quarter length fragments. MMPs have been proposed to cleave one \(\alpha\)-chain at a time, starting specifically with the \(\alpha\)2 chain in collagen I [110; 111]. The MMP cleavage site is located in the overlap region on segment D4, and therefore is accessible upon displacement of the C-telopeptide. In an _in silico_ study, [PERSON] et al. proposed how collagenolysis can occur in the collagen fibril. From the C-terminal fibril surface, the \(\alpha\)2 chain is oriented toward the outside of the fibril and has a greater dissociation from the center of the triple helix, making it more vulnerable to MMP cleavage [98]. Moreover, the protection of the site of collagenolysis by the C-telopeptide increases MMP specificity, as it will only be able to cleave when the C-telopeptide is displaced [98; 112]. Degradation of the collagen fibril is known to occur, and thus access of MMPs to their cleavage site is essential. On the other hand, surface B favors access to high affinity integrin binding motifs and platelet receptor recognition sites. In this orientation, known platelet interaction sites, including those for integrin \(\alpha\)2\(\beta\)1 and VWF, are more accessible to their binding partners. Both integrin \(\alpha\)2\(\beta\)1 and VWF have been shown to be critical receptors for the platelet adhesion to collagen [55; 113]. Many specific binding sequences for platelet and other cell receptors have been determined from adhesion assays to fully exposed triple helical collagen II and III peptides. Moreover, [PERSON] et al. showed that through solid-phase cell adhesion assays and immunoelectron microscopy, that integrin \(\alpha\)2\(\beta\)1 does in fact adhere to mature collagen I fibrils from bovine skin and facilitates cellular responses, such as cell spreading and formation of cellular projections [114]. This suggests that the integrin \(\alpha\)2I domain must have access to at least one of its recognition domains on the collagen I fibril. In which sequence the integrin \(\alpha\)2I actually binds in collagen I fibrils has yet to be determined. In Figure 3, black and hashed boxes indicate integrin high and moderate affinity binding motifs, respectively. High affinity integrin \(\alpha\)2\(\beta\)1 binding sequences, GROGER and GLOGER [58; 62; 74; 76; 115], are on D1 and would be directly on the surface of the collagen I fibril available for \(\alpha\)2I domain binding. High affinity integrin binding site GROGER and moderate affinity binding site GMOGER, on the N-terminal half of D3 also become more accessible with surface B exposed, allowing accessibility to all high affinity integrin \(\alpha\)2I binding sites. Furthermore, only in this orientation is it possible to gain accessibility to the single VWF binding motif, GVMGFO, on the C-terminal end of D2, which is overlapped with the binding sequences for SPARC and DDRs [64; 65; 78; 86]. Although surface B provides more access to high affinity integrin binding motifs and the GVMGFO motif shared by VWF, DDRs, and SPARC binding partners, this surface does not allow access to the MMP cleavage and binding sites or GPVI and decoron interaction motifs. Yet, integrin binding from surface A is not ruled out, as moderate affinity binding motifs, GQRGER and GASGER, are accessible from this surface, especially if the C-telopeptide is dynamic. The dynamics of the C-telopeptide and proteolysis by MMPs might provide access to functional domains otherwise buried within the fibril, and would therefore support the surface exposure of the C-terminus, as proposed by [PERSON] et al.