# 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
Moreover, the SASA is not uniform within each D-segment along the length of the D-period due to the inherent twisting of monomers within the fibril (see schematic at the top of Figure 3A,B). For example, from surface A, D2 has moderate accessibility in the overlap region and in the N-terminal half of the gap region, but no accessibility in the C-terminal end of the gap region (Figure 3A). The twisting out of the D3 at this end of the gap makes it lowly accessible, and it is non-accessible in the remainder of the D-period. Conversely, surface B gives moderate accessibility to the overlap region and the N-terminal half of the gap region of D3, and moderate accessibility of D2 in the overlap region (Figure 3B). In this orientation, the twisting out of D2 at the C-terminal end of the gap region gives it higher accessibility than even D1, which is at the surface and has the highest exposure for most of the D-period. The variability of SASA both between D-segments and along the D-period makes the exposure of binding sites complex. The accessibility from the fibril surface is highly determinant of interactions with molecular partners. We therefore analyzed the accessibility of thirteen collagen-binding ligands from both surfaces A and B (Figure 3). The SASA highlights the highly accessible binding sites near the surface in either orientation. Quantification of the residue-specific SASA of the 3 \(\times\) 3 collagen fibril SRU model in both possible orientations provides a measure of accessibility of interaction domains when either face is exposed. Presentation of the C-terminus to the surface provides direct access to essential fibril interaction sites, including lysyl oxidase (LOX) [57], GPVI [107; 80], and decoron, the core protein of decorin [106]. LOX interaction sites are on both D1 and D5 for formation of intermolecular cross-links that regulate the collagen fibril assembly. LOX deaminates lysines and hydroxylysines in preparation of cross-link formation between triple helices to stabilize collagen fibrils [108]. GPVI is a glycoprotein on platelet membranes that interacts with collagen to activate the platelets and induce platelet aggregation [107]. GPVI recognizes and binds to short (GPO)\({}_{\text{n}}\) tandem repeats on collagen. The only GPO repeat motif longer than (GPO)\({}_{2}\) in the collagen I sequence is in D5 at the very C-terminus of the collagenous domain and contains (GPO)\({}_{5}\) in the \(\alpha\)1(I) chain and (GPO)\({}_{4}\) in the \(\alpha\)2(I) chain. Decorin is a proteoglycan that interacts with collagen I fibrils to stabilize the suprafibrillar assembly [106; 109]. The core protein of decorin, decoron, recognizes two binding motifs in the gap region of D4, KXGDRGE, and AKGDRGE [106; 109]. An X-ray crystal structure of decoron has been solved and docked onto the collagen I fibril when D4 is exposed [106]. Access of these sites in the mature fibril of collagen would allow these proteins to carry out their cellular functions. Furthermore, in this orientation there is a potential means of collagen fibril cleavage by MMPs. MMPs are the only enzymes that are capable of cleaving collagen molecules. Degradation of collagen fibrils is essential for fibril turnover, tissue repair, and development. MMP1, MMP8,  
Figure 3: Residue-specific modified 5 Å SASA calculations with respect to surfaces (**A**) A [98] and (**B**) B [58]. The D-segments, D1–D5, are indicated and colored as in Figures 1 and 2. The locations of collagen ligands are shown by gray boxes with heparin [104], PEDF [105] and LOX [57] in D1 and D5; SPARC [86], DDRs [64; 65] and VWF [78] in D2; OSCAR [84; 85] and YadA [87] in D3; MMPs [68], fibronectin [70], and the decorin core protein, decoron [106], in D4; and GPVI [80] in D5. Integrin high and low affinity motifs are represented by black and hashed boxes, respectively, and labeled by the first three residues of the indicated six-residue binding motif. The width of the boxes corresponds to the length of the identified recognition sequence.