# Revealing Accessibility of Cryptic Protein Binding Sites within the Functional Collagen Fibril
## 3 Collagen Interactions and the Fibril Surface  
### Identification of Binding Sequences  
The complex assembly of monomers into microfibrils and fibrils results in one face being surface exposed and available for binding while other segments are buried within the fibril core [54; 53]. Fibrillar collagens have numerous binding partners: interacting with ECM components, binding to cellular receptors, and interacting with enzymes for chemical modification and matrix metalloproteinases (MMPs) during degradation [55; 56; 57; 58; 59; 4]. Despite its presentation as a long rod with a repeating GXX\({}^{\prime}\) sequence, collagen's binding partners recognize specific sequences and motifs across its more than 1000 residue span, and some specifically bind to particular structural conformations. Much progress has been made in determining collagen recognition sequences of cell receptors and interaction partners through the elegant use of triple helical peptides, recombinant and bacterial collagen, and sequence-dependent adhesion assays [5; 60; 61; 62; 63; 64; 65; 66; 67; 68; 69; 70; 71]. The collagen peptide toolkits developed in the Fardale laboratory are libraries of overlapping peptides that span the entire collagenous sequences of homotrimic collagens II and III. Each peptide contains 27 residues from natural collagen sequences, including nine unique residues, flanked on each termini by five GPP repeats ((GPP)) for stabilization of the triple helix [62]. Although, collagen I is a heterotrimer of two \(\alpha\)1(I) chains and one \(\alpha\)2(I) chain, the collagenous domain of the collagen I \(\alpha\)1(I) chain contains 74.5% and 63.0% sequence identity to the collagenous domains of collagens II and III, respectively. Thus, these peptides provide excellent tools for identifying specific sequence motifs recognized by collagen-binding partners and comparing their relative affinities in the triple helical context.  
By evaluating the binding of receptors to the collagen toolkit peptides, specific binding sequences have been determined for collagen binding integrins \(\alpha\)1\(\beta\)1 and \(\alpha\)2\(\beta\)1 [72; 73; 74; 75; 76], discoidin domain receptors (DDR) 1 and 2 [64; 65; 77], von Willebrand factor (VWF) [78; 79], glycoprotein VI (GPVI) [80; 81], leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) [82; 83], osteoclast-associated receptor (OSCAR) [84; 85], secreted protein acidic and rich cysteine (SPARC) [86], _Yersinia_ adhesion A (YadA) [87], procollagen C-proteinases enhancer (PCPE) [43], MMP1 [88], MMP13 [89], and fibromyalgia [90]. Known sequences of collagen binding partners have been previously reviewed and mapped onto fibrillar collagen I and III sequences [5; 56; 57; 91]. Due to challenges of acquiring intact monomeric collagen I, only a few studies exist on protein binding to full collagen I monomers, using microscopy techniques to detect ligand binding. The binding of heparin to procollagen type I was localized by rotary shadowing and electron microscopy (EM) [92]. Binding sites of SPARC to procollagens of types I, II, and III have been visualized by rotary shadowing EM [86] and atomic force microscopy (AFM) [93], and to the collagen I monomer lacking propeptides by AFM [93]. These experiments probe primarily protein interactions with collagen when it is in a long triple-helical rod, in which X and X\({}^{\prime}\) residues are largely exposed and accessible for binding. The relative affinities of the recognition sequences to the collagen triple helix may not be translatable to the full fibril architecture. In the three-dimensional packing of the fibril, some of the interaction domains become \"cryptic\" or buried from the accessible surface [4; 54; 58; 94; 95], yet cellular functions are still carried out, and it remains difficult to understand how molecular partners recognize and interact with their specific binding motifs when packed in the three-dimensional collagen fibril.