# Revealing Accessibility of Cryptic Protein Binding Sites within the Functional Collagen Fibril
## 2 The Complex Collagen Architecture  
At the foundation of all collagens, there is a collagenous domain composed of repeating Gly-Xaa-Xaa\({}^{\prime}\) sequences, where Xaa and Xaa\({}^{\prime}\) are often proline (Pro/P) and post-translationally modified hydroxyproline (Hyp/O), respectively. Fibrillar collagens (I-III, V, XI, XXIV, and XXVII) have an uninterrupted GXX\({}^{\prime}\) sequence. The linear collagenous sequences are over 1000 amino acids long, termed '\(\alpha\)-chains', and fold into left-handed polyproline type II (PPII) helices. Three \(\alpha\)-chains supercoil around a common axis, staggered by one residue, to form the characteristic collagen triple helix. Within the triple helix, the Gly residues are buried on the interior, while the X and \(X^{\prime}\) residues are largely exposed to the solvent and are responsible for the recognition of collagen binding partners [37]. Throughout this review, the triple helix will be considered the monomeric unit. Collagen I is a heterotrimic triple helix composed of two \(\alpha\)I(I) and one \(\alpha\)2(I) chains. The triple helix presents with a long, rod-like profile, with dimensions of approximately 300 nm long and 1.5 nm in diameter [37; 38; 39; 40] (Figure 1A). Collagen is produced as procollagen, containing propeptides in the N and C-termini. Upon secretion into the ECM, the propeptides are cleaved, exposing the telopeptides that are adjacent to the collagenous domain and initiating collagen assembly into large highly organized supermolecular fibril structures [41; 42; 43]. These fibrils are not amyloid in conformation, but have differently highly organized hierarchical structures. The buildup of the collagen architecture is presented in Figure 1.  
Figure 1: Collagen assembly from triple helical monomers to the collagen fibril architecture. **(A)** Monomer triple helix: Collagen I is a heterotrimer composed of two \(\alpha\)1 and one \(\alpha\)2 chains that supercoil into a long rod-like triple helix with approximate dimensions of 300 \(\times\) 1.5 nm. The monomer consists of 4.46 D-periods, numbered D1–D5; D5 is 0.46D; **(B)** Microfibril: Five monomers bundle into pentameric microfibrils, in which one D-period contains the entire collagen sequence from D1 to D5. The short D5 divides the D-period into two distinct regions; the “overlap” region contains segments D1–D5, while the “gap” region is devoid of D5. This creates the characteristic D-banding pattern that can be observed by microscopy. The schematic D-band is rendered in Pymol; **(C)** Fibril surface: A 90\({}^{\circ}\) rotation about the long triple-helical axis reveals the fibril surface. As an example, “surface A” is shown here. For clarity, three D-periods are shown along the \(y\)-axis and one along the \(x\)-axis. Toward fibril assembly, five monomers bundle into microfibrils. Within the microfibrils, the monomers are staggered axially by 67 nm when hydrated [44]. This consistent 67 nm dimension is called the 'D-period' and sets the periodicity of the fibril assembly. The \(\approx\)300 nm triple helix spans the length of 4.46 D-periods and is divided into D-segments, D1-5 (distinguished by color in Figure 1), with D5 being a short 0.46D [45]. Since the monomers of the microfibril are offset by one D-period, the microfibril can be cut into units one D-period long, which contain one copy of each D-segment, D1-5, each from a different monomer (Figure 1B). This unit is the SRU, and the smallest building block of the collagen fibril. Inter-microfibrillar crosslinking between D1 and D5 stabilizes the fibril assembly [46]. Due to D5 being less than half of the length of the other D-segments, the SRU is divided into two distinct sections that are defined by their molecular densities: the 'overlap' region, which contains D5, and the 'gap' region, devoid of D5 (Figure 1B).