Olga Antipova

Collagen fibril architecture, domain organization, and triple-helical conformation govern its proteolysis

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 substrates “interaction domain,” which facilitates the triple-helix unwinding/dissociation function of the enzyme before collagenolysis.

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

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.

Molecular and structural mapping of collagen fibril interactions

The fibrous collagens form the structural basis of all mammalian connective tissues, including the vasculature, dermis, bones, tendons, cartilage, and those tissues that support organs such as the heart, kindneys, liver, and lungs. The helical structure of collagen has been extensively studied but in addition to its helical character, its molecular packing arrangement (in its aggregated or fibrillar form) and the presence of specific amino acid sequences govern collagens in vivo functions. Collagens molecular packing arrangement helps control cellular communication, attachment and movement, and conveys its tissue-specific biomechanical properties. Recent progress in understanding collagens molecular packing, fibrillar structure, domain organization, and extracellular matrix (ECM) interactions in light of X-ray fiber diffraction data provides significant new insights into how the ECM is organized and functions. In this review, the hierarchy of fibrillar collagen structure is discussed in the context of how this organization affects ECM–“ligand” interactions, with specific attention to collagenolysis, integrins, fibronection, glycoprotein VI receptor (GPVI), and proteoglycans (PG). Understanding the complex structure of collagen and its attached ligands should provide new insights into tissue growth, development, regeneration, and disease.

The dentin organic matrix – limitations of restorative dentistry hidden on the nanometer scale

The prevention and treatment of dental caries are major challenges occurring in dentistry. The foundations for modern management of this dental disease, estimated to affect 90% of adults in Western countries, rest upon the dependence of ultrafine interactions between synthetic polymeric biomaterials and nanostructured supramolecular assemblies that compose the tooth organic substrate. Research has shown, however, that this interaction imposes less than desirable long-term prospects for current resin-based dental restorations. Here we review progress in the identification of the nanostructural organization of the organic matrix of dentin, the largest component of the tooth structure, and highlight aspects relevant to understating the interaction of restorative biomaterials with the dentin substrate. We offer novel insights into the influence of the hierarchically assembled supramolecular structure of dentin collagen fibrils and their structural dependence on water molecules. Secondly, we review recent evidence for the participation of proteoglycans in composing the dentin organic network. Finally, we discuss the relation of these complexly assembled nanostructures with the protease degradative processes driving the low durability of current resin-based dental restorations. We argue in favour of the structural limitations that these complexly organized and inherently hydrated organic structures may impose on the clinical prospects of current hydrophobic and hydrolyzable dental polymers that establish ultrafine contact with the tooth substrate.

Decorin Core Protein (Decoron) Shape Complements Collagen Fibril Surface Structure and Mediates Its Binding

Decorin is the archetypal small leucine rich repeat proteoglycan of the vertebrate extracellular matrix (ECM). With its glycosaminoglycuronan chain, it is responsible for stabilizing inter-fibrillar organization. Type I collagen is the predominant member of the fibrillar collagen family, fulfilling both organizational and structural roles in animal ECMs. In this study, interactions between decoron (the decorin core protein) and binding sites in the d and e1 bands of the type I collagen fibril were investigated through molecular modeling of their respective X-ray diffraction structures. Previously, it was proposed that a model-based, highly curved concave decoron interacts with a single collagen molecule, which would form extensive van der Waals contacts and give rise to strong non-specific binding. However, the large well-ordered aggregate that is the collagen fibril places significant restraints on modes of ligand binding and necessitates multi-collagen molecular contacts. We present here a relatively high-resolution model of the decoron-fibril collagen complex. We find that the respective crystal structures complement each other well, although it is the monomeric form of decoron that shows the most appropriate shape complementarity with the fibril surface and favorable calculated energies of interaction. One molecule of decoron interacts with four to six collagen molecules, and the binding specificity relies on a large number of hydrogen bonds and electrostatic interactions, primarily with the collagen motifs KXGDRGE and AKGDRGE (d and e1 bands). This work helps us to understand collagen-decorin interactions and the molecular architecture of the fibrillar ECM in health and disease.

In situ D-periodic molecular structure of type II collagen

Collagens are essential components of extracellular matrices in multicellular animals. Fibrillar type II collagen is the most prominent component of articular cartilage and other cartilage-like tissues such as notochord. Its in situ macromolecular and packing structures have not been fully characterized, but an understanding of these attributes may help reveal mechanisms of tissue assembly and degradation (as in osteo- and rheumatoid arthritis). In some tissues such as lamprey notochord, the collagen fibrillar organization is naturally crystalline and may be studied by x-ray diffraction. We used diffraction data from native and derivative notochord tissue samples to solve the axial, D-periodic structure of type II collagen via multiple isomorphous replacement. The electron density maps and heavy atom data revealed the conformation of the nonhelical telopeptides and the overall D-periodic structure of collagen type II in native tissues, data that were further supported by structure prediction and transmission electron microscopy. These results help to explain the observed differences in collagen type I and type II fibrillar architecture and indicate the collagen type II cross-link organization, which is crucial for fibrillogenesis. Transmission electron microscopy data show the close relationship between lamprey and mammalian collagen fibrils, even though the respective larger scale tissue architecture differs.

Collagen fibril surface displays a constellation of sites capable of promoting fibril assembly, stability, and hemostasis

Fibrillar collagens form the structural basis of organs and tissues including the vasculature, bone, and tendon. They are also dynamic, organizational scaffolds that present binding and recognition sites for ligands, cells, and platelets. We interpret recently published X-ray diffraction findings and use atomic force microscopy data to illustrate the significance of new insights into the functional organization of the collagen fibril. These data indicate that collagens most crucial functional domains localize primarily to the overlap region, comprising a constellation of sites we call the “master control region.” Moreover, the collagens most exposed aspect contains its most stable part—the C-terminal region that controls collagen assembly, cross-linking, and blood clotting. Hidden beneath the fibril surface exists a constellation of “cryptic” sequences poised to promote hemostasis and cell–collagen interactions in tissue injury and regeneration. These findings begin to address several important, and previously unresolved, questions: How functional domains are organized in the fibril, which domains are accessible, and which require proteolysis or structural trauma to become exposed? Here we speculate as to how collagen fibrillar organization impacts molecular processes relating to tissue growth, development, and repair.

Variation in the Helical Structure of Native Collagen

The structure of collagen has been a matter of curiosity, investigation, and debate for the better part of a century. There has been a particularly productive period recently, during which much progress has been made in better describing all aspects of collagen structure. However, there remain some questions regarding its helical symmetry and its persistence within the triple-helix. Previous considerations of this symmetry have sometimes confused the picture by not fully recognizing that collagen structure is a highly complex and large hierarchical entity, and this affects and is effected by the super-coiled molecules that make it. Nevertheless, the symmetry question is not trite, but of some significance as it relates to extracellular matrix organization and cellular integration. The correlation between helical structure in the context of the molecular packing arrangement determines which parts of the amino acid sequence of the collagen fibril are buried or accessible to the extracellular matrix or the cell. In this study, we concentrate primarily on the triple-helical structure of fibrillar collagens I and II, the two most predominant types. By comparing X-ray diffraction data collected from type I and type II containing tissues, we point to evidence for a range of triple-helical symmetries being extant in the molecules native environment. The possible significance of helical instability, local helix dissociation and molecular packing of the triple-helices is discussed in the context of collagens supramolecular organization, all of which must affect the symmetry of the collagen triple-helix.

Measurement of Elastic Modulus of Collagen Type I Single Fiber

Collagen fibers are the main components of the extra cellular matrix and the primary contributors to the mechanical properties of tissues. Here we report a novel approach to measure the longitudinal component of the elastic moduli of biological fibers under conditions close to those found in vivo and apply it to type I collagen from rat tail tendon. This approach combines optical tweezers, atomic force microscopy, and exploits Euler-Bernoulli elasticity theory for data analysis. This approach also avoids drying for measurements or visualization, since samples are freshly extracted. Importantly, strains are kept below 0.5%, which appear consistent with the linear elastic regime. We find, surprisingly, that the longitudinal elastic modulus of type I collagen cannot be represented by a single quantity but rather is a distribution that is broader than the uncertainty of our experimental technique. The longitudinal component of the single-fiber elastic modulus is between 100 MPa and 360 MPa for samples extracted from different rats and/or different parts of a single tail. Variations are also observed in the fibril-bundle / fibril diameter with an average of 325±40 nm. Since bending forces depend on the diameter to the fourth power, this variation in diameter is important for estimating the range of elastic moduli. The remaining variations in the modulus may be due to differences in composition of the fibril-bundles, or the extent of the proteoglycans constituting fibril-bundles, or that some single fibrils may be of fibril-bundle size.

Non-Enzymatic Decomposition of Collagen Fibers by a Biglycan Antibody and a Plausible Mechanism for Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory and destructive joint disorder that affects tens of millions of people worldwide. Normal healthy joints maintain a balance between the synthesis of extracellular matrix (ECM) molecules and the proteolytic degradation of damaged ones. In the case of RA, this balance is shifted toward matrix destruction due to increased production of cleavage enzymes and the presence of (autoimmune) immunoglobulins resulting from an inflammation induced immune response. Herein we demonstrate that a polyclonal antibody against the proteoglycan biglycan (BG) causes tissue destruction that may be analogous to that of RA affected tissues. The effect of the antibody is more potent than harsh chemical and/or enzymatic treatments designed to mimic arthritis-like fibril de-polymerization. In RA cases, the immune response to inflammation causes synovial fibroblasts, monocytes and macrophages to produce cytokines and secrete matrix remodeling enzymes, whereas B cells are stimulated to produce immunoglobulins. The specific antigen that causes the RA immune response has not yet been identified, although possible candidates have been proposed, including collagen types I and II, and proteoglycans (PGs) such as biglycan. We speculate that the initiation of RA associated tissue destruction in vivo may involve a similar non-enzymatic decomposition of collagen fibrils via the immunoglobulins themselves that we observe here ex vivo.