Joseph P. R. O. Orgel

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.

The In Situ Supermolecular Structure of Type I Collagen

BACKGROUND The proteins belonging to the collagen family are ubiquitous throughout the animal kingdom. The most abundant collagen, type I, readily forms fibrils that convey the principal mechanical support and structural organization in the extracellular matrix of connective tissues such as bone, skin, tendon, and vasculature. An understanding of the molecular arrangement of collagen in fibrils is essential since it relates molecular interactions to the mechanical strength of fibrous tissues and may reveal the underlying molecular pathology of numerous connective tissue diseases. RESULTS Using synchrotron radiation, we have conducted a study of the native fibril structure at anisotropic resolution (5.4 A axial and 10 A lateral). The intensities of the tendon X-ray diffraction pattern that arise from the lateral packing (three-dimensional arrangement) of collagen molecules were measured by using a method analogous to Rietveld methods in powder crystallography and to the separation of closely spaced peaks in Laue diffraction patterns. These were then used to determine the packing structure of collagen by MIR. CONCLUSIONS Our electron density map is the first obtained from a natural fiber using these techniques (more commonly applied to single crystal crystallography). It reveals the three-dimensional molecular packing arrangement of type I collagen and conclusively proves that the molecules are arranged on a quasihexagonal lattice. The molecular segments that contain the telopeptides (central to the function of collagen fibrils in health and disease) have been identified, revealing that they form a corrugated arrangement of crosslinked molecules that strengthen and stabilize the native fibril.

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.

Evidence for Novel β-Sheet Structures in Iowa Mutant β-Amyloid Fibrils

Asp23-to-Asn mutation within the coding sequence of beta-amyloid, called the Iowa mutation, is associated with early onset, familial Alzheimers disease and cerebral amyloid angiopathy, in which patients develop neuritic plaques and massive vascular deposition predominantly of the mutant peptide. We examined the mutant peptide, D23N-Abeta40, by electron microscopy, X-ray diffraction, and solid-state NMR spectroscopy. D23N-Abeta40 forms fibrils considerably faster than the wild-type peptide (k = 3.77 x 10(-3) min(-1) and 1.07 x 10(-4) min(-1) for D23N-Abeta40 and the wild-type peptide WT-Abeta40, respectively) and without a lag phase. Electron microscopy shows that D23N-Abeta40 forms fibrils with multiple morphologies. X-ray fiber diffraction shows a cross-beta pattern, with a sharp reflection at 4.7 A and a broad reflection at 9.4 A, which is notably smaller than the value for WT-Abeta40 fibrils (10.4 A). Solid-state NMR measurements indicate molecular level polymorphism of the fibrils, with only a minority of D23N-Abeta40 fibrils containing the in-register, parallel beta-sheet structure commonly found in WT-Abeta40 fibrils and most other amyloid fibrils. Antiparallel beta-sheet structures in the majority of fibrils are indicated by measurements of intermolecular distances through (13)C-(13)C and (15)N-(13)C dipole-dipole couplings. An intriguing possibility exists that there is a relationship between the aberrant structure of D23N-Abeta40 fibrils and the unusual vasculotropic clinical picture in these patients.

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 in situ conformation and axial location of the intermolecular cross-linked non-helical telopeptides of type I collagen.

BACKGROUND Type I collagen contains specific lysine and hydroxylysine residues that are critical in the formation of intermolecular cross-links crucial for the normal configuration and stability of the 67 nm axial repeat of collagen fibrils in the extracellular matrix. The major cross-linkage sites are believed to occur between the non-helical terminal regions (telopeptides) and helical segments of adjacent collagen molecules. In this X-ray fibre diffraction study the tissue has been maintained in the hydrated fibrillar state, whilst detailed structural information was obtained using highly collimated synchrotron radiation. RESULTS The axial component of the X-ray diffraction patterns extends more than twice as far in reciprocal space than that of any already published. The structure-factor phases were calculated using the multiple isomorphous addition method, avoiding model-based approaches, and produced an electron-density profile of the molecular arrangement projected on to the fibre axis to 0.54 nm resolution. This corresponds to the phasing of 124 orders of the meridional diffraction pattern. CONCLUSIONS The axially projected electron-density profile and the electron-density difference maps showed that both the N- and C-terminal telopeptides are contracted structures. This profile puts narrow constraints on the possible conformations of the C-terminal telopeptide; the best fit to the electron-density profile is when the alpha1 chains adopt a folded conformation with a sharp hairpin turn around residues 13 and 14 of the 25-residue telopeptide. Our results reveal for the first time the location, parallel to the fibril axis, of the intermolecular cross-links in normal hydrated tissue. These cross-links are essential for the biological function of the tissue.

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.