Fig. 5. Amino acid sequence numbering of the collagen molecule (as for RCSB codes 1Y0F and 1YGV). The amino acids found at the N and C termini, the MMP1 interaction region, and the cleavage site are enumerated here for each of the three polypeptide chains. It is common practice to number the residue sequence from the estimated beginning of the triple-helical region, excluding the N-terminal sequence. In this study, the N-terminal sequence is included in determining the sequence numbering, as previously in ref. 7.

Fig. 6. Proposed mechanism for collagenase initiated digestion of the collagen fibril. (A) Topological map of five ~67 nm D-repeats of the collagen microfibril, the requisite prior cleavage of the C-terminal telopeptide of the "red layer" (D5) is implied in B. (B) Once the C-telopeptide is cleaved (by a telopeptidase such as collagenase), cleavage could occur at the B (α2) chain (D5). The molecule then degrades, and becomes dissociated from, the microfibril (assisted by gelatinase), leaving the same degree of inaccessibility in three of the remaining D-repeats of the original five first considered (A), within the same microfibril. However, it leaves greater accessibility in the D-period vacated by the C-terminal end of the cleaved collagen molecule. The vacated N-terminal end of the cleaved molecule allows enzyme accessibility within the previously protected neighboring microfibrils (see E and Fig. 3). C) Rapid proteolysis may occur in the C-terminal direction of the microfibril/fibril due to exposure of the α2 peptide in the neighboring D period, C-terminal to the previous monomer's cleavage. (D) Less rapid proteolysis may occur in the N-terminal direction of the fibril (see E). (E) Lateral and exterior initiation of collagenase-mediated fibril digestion, towards the fibril interior and around the ring of a microfibril layer (F). Lateral digestion (anti-clockwise as shown in F, viewed from the C to N terminal prospective) could occur relatively slowly via the C (α1) chain of the microfibril adjacent (in the same D-period) to the first digest (represented by colorless circles). This direction is the same as the microfibril and triple-helix twists (right-handed). As collogenolysis progresses, the loosened matrix may allow removal of the N-terminus of the monomer cleaved in D4 (A and B) of the first microfibril, exposing the C (α1) peptide in an adjacent, second microfibril in D1 (A and B), but towards the fibril interior. Rapid C-terminal-directed cleavage along the length of the second microfibril would follow (see C), whilst removal of the monomer cleaved in D1 would allow the slow initial cleavage in D1 (equivalent to D3) of a third microfibril, followed by rapid microfibril digestion in the C-terminal direction (C), and so on. (F) Fibril schematic showing the path of "slow" lateral digestion described in D and E. This would primarily proceed around the ring of the same microfibril layer because of the exposure of the C (α1) chain (Fig. 3G). The newly exposed microfibril layer remains protected by the C-terminal telopeptides, and further fibril digestion requires the removal of the folded C-telopeptide as before, or via the less vulnerable (and presumably therefore slower reaction route) C (α1). (G) The rapid digestion described in C, enabled by the successive removal of C-telopeptides at D+1, allows a rapid removal of individual microfibrils. This may give the progress of the reaction the appearance of a molecular motor, as collagenase molecules appear to progress along a groove (as a single line of microfibril/s is removed in the N to C terminal direction). Summary: The digestion could proceed rapidly in the C-terminal axial direction for individual microfibrils, due to exposure of the B (α2) chain, and in the same direction as the microfibril and triple-helix twist, and more slowly N-terminal around the fibril circumference, due to the exposure of the less vulnerable C (α1) chain. Collagenolysis would only be able to proceed into the fibril interior after the newly exposed fibril surface layer is penetrated by removal of one or more C-terminal telopeptides and/or in a slower progressing C to N terminal direction via the C (α1) chain, as the matrix is loosened and accessibility is improved. Once this point is reached, collagenolysis could occur more rapidly due to the diminished restrictions from fibril architecture.

Fig. 7. Simplified reaction schematic for fibrillar collagenolysis.

Fig. 8. Consideration of the fibril lattice arrangement with the structure of the microfibril shows which crystal lattice spacing is most likely to be found at the fibril surface. This consideration is in agreement with Hulmes et al. (1). Marked in green, yellow, red, and gray are the: 1.37, 1.33, 1.26, and 3.8 nm lateral packing values respectively. (A) Cross-section of the molecular packing lattice viewed in the overlap region at the level of the cleavage-site. Of the four principal packing values, only the 1.37 and 3.8 nm (green and gray) spacings can be orientated perpendicular to the fibril surface when the structure of the microfibril is considered. Although the 1.37 nm spacing can be orientated at the fibril surface it would introduce systematic absences of collagen molecules (spaces). (B) However, the 3.8-nm spacing leaves only the fifth segment (C-terminal containing) at the surface. (C) The prospective of the tilt of collagen molecules at the fibril surface is indicated with the black arrow. Although the tilt of the molecules is not insignificant when view from this prospective (1.37-nm spacing perpendicular to fibril surface), it is less significant then that seen for the 3.8 nm (D). (D) The tilt of the molecules relative to the fibril long-axis is most pronounced from this prospective (arrow), with the 3.8nm spacing perpendicular to the fibril surface. This seems to represent the observations of the fibril surface offered in Fig. 1 and that of Hulmes et al. (1) (3.8 nm reflection perpendicular to fibril surface).

1. Hulmes DJ, Jesior JC, Miller A, Berthet-Colominas C, Wolff C (1981) Electron microscopy shows periodic structure in collagen fibril cross sections. Proc Natl Acad Sci USA 78:3567-71.