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, 105 (8), 2824-9

Collagen Fibril Architecture, Domain Organization, and Triple-Helical Conformation Govern Its Proteolysis

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Collagen Fibril Architecture, Domain Organization, and Triple-Helical Conformation Govern Its Proteolysis

Shiamalee Perumal et al. Proc Natl Acad Sci U S A.

Abstract

We describe the molecular structure of the collagen fibril and how it affects collagen proteolysis or "collagenolysis." The fibril-forming collagens are major components of all mammalian connective tissues, providing the structural and organizational framework for skin, blood vessels, bone, tendon, and other tissues. The triple helix of the collagen molecule is resistant to most proteinases, and the matrix metalloproteinases that do proteolyze collagen are affected by the architecture of collagen fibrils, which are notably more resistant to collagenolysis than lone collagen monomers. Until now, there has been no molecular explanation for this. Full or limited proteolysis of the collagen fibril is known to be a key process in normal growth, development, repair, and cell differentiation, and in cancerous tumor progression and heart disease. Peptide fragments generated by collagenolysis, and the conformation of exposed sites on the fibril as a result of limited proteolysis, regulate these processes and that of cellular attachment, but it is not known how or why. Using computational and molecular visualization methods, we found that the arrangement of collagen monomers in the fibril (its architecture) protects areas vulnerable to collagenolysis and strictly governs the process. This in turn affects the accessibility of a cell interaction site located near the cleavage region. Our observations suggest that the C-terminal telopeptide must be proteolyzed before collagenase can gain access to the cleavage site. Collagenase then binds to the substrate's "interaction domain," which facilitates the triple-helix unwinding/dissociation function of the enzyme before collagenolysis.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Molecular substructure of the D-periodic collagen fibril. (A) A field emission gun–scanning electron microscope image of nonstained, lyophilized fibrils, adapted from ref. . The blue arrows mark three successive “X3” ridges, which correspond to the C terminus of the collagen molecule (19). The tilted surface of the fibril surface and the location of the bulging X3 ridge are similar to that of the microfibril's tilt and bulging C terminus (B). To scale, Insets B and C correspond to elements shown in B and C (below). (B) Single D-period of a microfibril. The C terminus (folded structure marked with a “C”) points toward the outside of the fibril. The N terminus is marked with an “N.” (C) Expansion of box in A. Surface accessibility of two fibril–ligand interaction sites. The surface view shows a section of the fibril surface where one C-telopeptide, and its connecting triple helix, has been removed, allowing access for MMP1 to the B (α2) peptide chain (red arrow). The cleavage site is represented as a yellow band, whereas the rest of monomer 4 is dark gray. Although the cleavage site is partly solvent-accessible, MMP1 (red) is unable to approach it while the C-telopeptide remains intact because of steric hindrance. A central integrin binding site (S. Sweeney and J. San Antonio, personal communication, and ref. 29) is illustrated in green. The B (α2) chain is accessible within the 2- to 3-nm-deep solvent pit on the solvent surface (see C), but neither of the α1 chains are, unless further proteolysis were to fully expose monomer 3 (see D). This may explain the poor binding of integrin to intact fibrils (34). (D) Fibril model based on the parameters of refs. and . See key for identification of each segment by color. (E) Schematic of a section of the fibril surface shown in C and outlined in the small box at the bottom of D. The red arrow indicates the location of the cleavage site shown in C. The green arrow points to the same monomer 3 as C. Monomer 3 carries the ligand attachment sites needed for cell surface interactions [an integrin binding site (S. Sweeney and J. San Antonio, personal communication, and ref. 29)]. Strong cell surface interaction may require a prior, limited proteolysis of the fibril (25, 26), because the site is not otherwise fully accessible without the removal of monomers 5 (clear) and 4 (red number).
Fig. 2.
Fig. 2.
Disassociation of peptide chains: difference between the relaxed and stringent models of the collagen triple helix. Sequence numbering includes the N-telopeptide. A and C show the difference of the “from helix center” distances, a measure of triple-helix dissociation of the peptides. The magnitude of dissociation of the three peptide chains, A (α1), B (α2), and C (α1), are shown, along with the average, standard deviation (σ), two times standard deviation (2*σ), or zero, as indicated. (A) The cleavage site region (flanked by 10-aa residues N- and C-terminal). (B) End-on view of central section of cleavage site region. The black dot represents the triple-helix center, the cyan line is the radius of stringent model at 791A Cα, and the red line is the radius of the relaxed model 791A Cα. The difference between the two radiuses is used as the measure of native collagen's triple-helix disassociation (shown as graphs in A and C). (C) As A, except the entire triple helix.
Fig. 3.
Fig. 3.
Molecular accessibility of the collagenase cleavage site at the fibril surface. (A) End-on view of the triple helix surrounding the cleavage site on monomer 4 (both stringent and relaxed models are shown). Monomer 4 is shown in the same orientation in all figure elements. (B) Lateral packing of collagen molecules in the region of the collagenase cleavage site (see Fig. 1E). The 3 × 4 microfibrils have been related by their crystal symmetry to give a representation of the fibril surface. It is apparent that there is no possibility of MMP1 interaction with monomer 4 from the N-terminal side of the fibril (monomer 1) (bottom left corner) because it is buried within the fibril interior (see also Fig. 1D). Four areas of possible collagenase–collagen fibril interaction have been marked with boxes, which are shown in greater detail in elements C–G. (C–G) Solvent-accessible surface views of the boxed regions of B and MMP1 attempting to dock with each area of the fibril. Monomers 1–3 and 5 are blue, and monomer 4 is as in A. MMP1 is red. MMP1 has been located as close to the monomer 4 cleavage site as possible, while avoiding steric clashes. For the areas represented in C, F, and G, MMP1 can come no closer than 15 Å from the cleavage site. (E) Prior removal of the C-terminal telopeptide allows access to the proteolytically vulnerable B (α2) chain of the cleavage site. The change in relative accessibility, by removal of the C terminus, is shown in the difference between E and F (marked by a yellow arrow). (F) Access to the less proteolytically vulnerable C (α1) chain would also become possible after the removal of one microfibril or at least the C-terminal fragment from one monomer (see also Fig. 1).
Fig. 4.
Fig. 4.
MMP1 at the collagenase-interaction domain (CID). The collagen B (α2) peptide chain is facing, and inside, the catalytic cleft while the two α1 chains interact with residues around, and in, the positively charged shallow surface pocket between modules 3 and 4 of the hemopexin domain (20). The relatively disassociated state of the triple helix represented here is the naturally occurring condition at approximately room temperature in situ (7). (Left) Domain organization of MMP1 while in contact with the fibrillar substrate. (Right) Expanded view rotated ≈30° from A, showing the solvent-accessible surface of MMP1, colored according to relative charge (spectrum toward red is acidic and toward blue is basic). The blue dashed arrows indicate the direction in which attraction/repulsion forces may operate to “pull” the two α1 chains in opposite directions, causing further dissociation on the triple helix at the cleavage site.

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