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. 2008 Jul 25;283(30):21187-97.
doi: 10.1074/jbc.M709319200. Epub 2008 May 15.

Candidate cell and matrix interaction domains on the collagen fibril, the predominant protein of vertebrates

Shawn M Sweeney  1 , Joseph P Orgel, Andrzej Fertala, Jon D McAuliffe, Kevin R Turner, Gloria A Di Lullo, Steven Chen, Olga Antipova, Shiamalee Perumal, Leena Ala-Kokko, Antonella Forlino, Wayne A Cabral, Aileen M Barnes, Joan C Marini, James D San Antonio
Affiliations

Candidate cell and matrix interaction domains on the collagen fibril, the predominant protein of vertebrates

Shawn M Sweeney et al. J Biol Chem. .

Abstract

Type I collagen, the predominant protein of vertebrates, polymerizes with type III and V collagens and non-collagenous molecules into large cable-like fibrils, yet how the fibril interacts with cells and other binding partners remains poorly understood. To help reveal insights into the collagen structure-function relationship, a data base was assembled including hundreds of type I collagen ligand binding sites and mutations on a two-dimensional model of the fibril. Visual examination of the distribution of functional sites, and statistical analysis of mutation distributions on the fibril suggest it is organized into two domains. The "cell interaction domain" is proposed to regulate dynamic aspects of collagen biology, including integrin-mediated cell interactions and fibril remodeling. The "matrix interaction domain" may assume a structural role, mediating collagen cross-linking, proteoglycan interactions, and tissue mineralization. Molecular modeling was used to superimpose the positions of functional sites and mutations from the two-dimensional fibril map onto a three-dimensional x-ray diffraction structure of the collagen microfibril in situ, indicating the existence of domains in the native fibril. Sequence searches revealed that major fibril domain elements are conserved in type I collagens through evolution and in the type II/XI collagen fibril predominant in cartilage. Moreover, the fibril domain model provides potential insights into the genotype-phenotype relationship for several classes of human connective tissue diseases, mechanisms of integrin clustering by fibrils, the polarity of fibril assembly, heterotypic fibril function, and connective tissue pathology in diabetes and aging.

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Figures

FIGURE 1.
FIGURE 1.
Assembly and structure of the type I collagen fibril. A fragment of a single type I collagen triple helix (monomer) is depicted (A). Type I collagen is secreted as a procollagen monomer ≅ 300 nm long, extracellularly, the propeptides (N and C) are cleaved by proteinases (dashed vertical lines, B) and the tropocollagen monomers assemble in a staggered fashion into collagen fibrils (C) where one D-period repeat (expanded two-dimensional view of 67 nm segment of microfibril, box) contains the complete collagen sequence from elements of the five monomers and includes an overlap and gap zone. The subunit structure of the fibril can be considered to be the D-periodic 5-mer referred to as the microfibril (D). Collagen fibrils appear as periodic banded structures by electron microscopy; arrow, left border of overlap zone; particles are heparin-albumin gold conjugates used to map the heparin-binding site (E). The collagen map in Fig. 2 represents an expanded view of one D-period. Modified from a figure reproduced from J. Cell Biol. by copyright permission of The Rockefeller University Press (53).
FIGURE 2.
FIGURE 2.
Ligand binding site and mutation map of the human type I collagen fibril. Protein sequences of the triple helix are shown (GenBankTM, α1(I) accession #NP000079.2 and α2(I) NP_000080.2; proline and hydroxyproline are designated as P, the latter being the third position in glycine-X-Y sequences. Note that Ala-459, α2(I) results from a single nucleotide polymorphism observed in some patient samples in the non-redundant protein data base, whereas α2(I) Pro-459 appears in the NP_000080.2 sequence. Yet, the association of Ala to Pro-459 substitution mutations with intracranial aneurisms (26) justifies its inclusion here. Ligand binding regions are indicated by rectangular boxes adjacent to relevant sequences; ligand binding to type I procollagen or tropocollagen (gray), or to either α1(I) or α2(I) chains (unshaded). Ligand binding hot spots 1, 2, and 3 are enclosed by dashed-lined black boxes. Highlighted sequences include: the α1β1/α2β1 integrin binding site GFPGER502-507 (maroon rectangle); MMP-1 cleavage site (green arrow); N- and C-terminal intermolecular cross-links (purple rectangles). Studies responsible for mapping many of the functional domains on collagen are cited in the first map publication (9) and in the supplemental materials. New sites on the revised map include endo180 (57), HSP47 (58), micro-unfolding and N-anchor domains (59, 60), MMP-interaction domain (11), prolyl-3-hydroxylase substrate P986 (61), SPARC (62), phosphorphoryn (63, 64), von Willebrand Factor (65), discoidin domain receptor 2 (66), and activities of THPs on angiogenesis (13), endothelial cell activation (67), and osteoblast differentiation (68). Mutations were from sources cited under the “Experimental Procedures.” This figure was modified from a previously published illustration (9).
FIGURE 2.
FIGURE 2.
Ligand binding site and mutation map of the human type I collagen fibril. Protein sequences of the triple helix are shown (GenBankTM, α1(I) accession #NP000079.2 and α2(I) NP_000080.2; proline and hydroxyproline are designated as P, the latter being the third position in glycine-X-Y sequences. Note that Ala-459, α2(I) results from a single nucleotide polymorphism observed in some patient samples in the non-redundant protein data base, whereas α2(I) Pro-459 appears in the NP_000080.2 sequence. Yet, the association of Ala to Pro-459 substitution mutations with intracranial aneurisms (26) justifies its inclusion here. Ligand binding regions are indicated by rectangular boxes adjacent to relevant sequences; ligand binding to type I procollagen or tropocollagen (gray), or to either α1(I) or α2(I) chains (unshaded). Ligand binding hot spots 1, 2, and 3 are enclosed by dashed-lined black boxes. Highlighted sequences include: the α1β1/α2β1 integrin binding site GFPGER502-507 (maroon rectangle); MMP-1 cleavage site (green arrow); N- and C-terminal intermolecular cross-links (purple rectangles). Studies responsible for mapping many of the functional domains on collagen are cited in the first map publication (9) and in the supplemental materials. New sites on the revised map include endo180 (57), HSP47 (58), micro-unfolding and N-anchor domains (59, 60), MMP-interaction domain (11), prolyl-3-hydroxylase substrate P986 (61), SPARC (62), phosphorphoryn (63, 64), von Willebrand Factor (65), discoidin domain receptor 2 (66), and activities of THPs on angiogenesis (13), endothelial cell activation (67), and osteoblast differentiation (68). Mutations were from sources cited under the “Experimental Procedures.” This figure was modified from a previously published illustration (9).
FIGURE 3.
FIGURE 3.
Integrin binding site of type I collagen: function and fibrillar environment. A, the α1β1/α2β1 integrin binding site. GFPGER502-507 functions in angiogenesis, endothelial cell activation, and osteoblast differentiation, occupies a PG-clear zone, and coincides with sequences silent for mutations on the α1 and α2 chains of M3 and on other monomers in the fibril (blue boxes, Figs. 2 and 3B). GFPGER502-507 neighbors the major region of non-enzymatic glycation of the fibril (blue stripe) and the glycation substrate, lysine 479 (Fig. 3C). B, GFPGER502-507 and mutation silent zones localize to a narrow region of the fibril surface. GFPGER502-507 (maroon); mutation silent zones numbered sequentially from N to C termini (blue). C, glycation may influence integrin-collagen interactions. Model of α2 integrin subunit I-domain (purple) binding to GFPGER502-507 adjacent to α2(I) fructosyl-lysine 479 (sugar). Note that glycation may potentially affect collagen ligation by the integrin heterodimer (gray arrow), but likely not the I-domain. On collagen: α1 GFPGER502-507 (yellow); mutation silent zone (blue); glycine residues associated with lethal OI mutations (red). Gray arrow, 10 nm is the maximal estimated reach of the integrin heterodimer.
FIGURE 4.
FIGURE 4.
Domain model of the collagen fibril. A, schematic of domains on the collagen fibril D-period. The putative cell interaction domain, including the α1β1/α2β1 integrin binding site GFPGER502-507 (maroon horseshoe); MMP cleavage site (green arrow); and elements of MLBR2 (geometric figures); occupy the fibril overlap zone. The putative matrix interaction domain, including sites for intermolecular cross-linking (X); PG-binding (yellow and pink); and mineralization occupy the remainder of the fibril. B, physical mapping of the cell and matrix interaction domains on the collagen microfibril. Selected elements of the putative cell and matrix interaction domains were mapped, including GFPGER502-507 (maroon); MMP cleavage site (green arrowhead), MMP interaction domain (cyan); lateral edges of the fibronectin binding site (blue); keratan sulfate PG binding sites (yellow); dermatan sulfate PG binding sites (pink); N- and C-terminal intermolecular cross-links (black and blue arrows), respectively. The SPARC binding site (black) was rendered transparent where it overlaps the fibronectin and MMP binding/cleavage sites. The three-dimensional image was derived from an x-ray diffraction structure of the collagen microfibril in situ. The microfibrillar aspect that faces toward the outside of the fibril is at the top of the picture (the C terminus points outside of the fibril, while the N terminus faces inside the fibril). Distances between GFPGER502-507 and domains for MMP interaction, or the N-terminal end of the fibronectin-binding domain were 11.2 and 5.0 nm, respectively. A view of microfibril function in the context of the collagen fibril appears in Fig. 5.
FIGURE 5.
FIGURE 5.
Domain regulation of collagen fibril function. The collagen fibril is proposed to be composed of cell interaction domains (unshaded) and matrix interaction domains (yellow and pink shading). In the cell interaction domain integrin binding sites promote cell adhesion and integrin receptor ligation, clustering, and activation. The confluence of crucial cell and ligand-binding sites on the fibril surface implies their coordinate regulation. Thus, fibril-bound integrins may modulate other ligand-collagen interactions, and MMP cleavage of one monomer may enhance or disrupt ligand-fibril interactions elsewhere. In the matrix interaction domain, PGs and other matrix molecules bind to impart key structural properties to the fibril. Note: the fibril shown in this schematic includes ten microfibrils and spans two and a half, 67 nm wide collagen D-periods; however, the average collagen fibril in vivo contains many more microfibrils than depicted here and may reach hundreds of microns in length (52).
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