Collagens as multidomain proteins

The number of proteins known to contain collagen-like triple helical domains is rapidly increasing. The functions of these domains are to provide molecular rods that separate spatially non-triple helical domains with varied properties and structures and to permit lateral interactions between molecules. Two-thirds of the amino acids of the triple helical domains have their side-chains at the surface of the protein. The triple helix is also a structure that is easily predictable from the primary structure. The structure of several recently discovered collagens are discussed in terms of domains and functions. The triple helical domains have sizes varying from 33 to over 1,000 amino acid residues. The longest uninterrupted triple helices are involved in the formation of the classical quarter-staggered fibrils. Other triple helical domains permit varied molecular aggregates. A very broad spectrum of non-triple helical or globular domains are interspersed by triple helices. Only those located at the extremities of the molecules are large in size, sometimes several hundred kDa, while the domains separating 2 triple helices are small (less than 50 amino acids) and provide the molecules with hinges, proteolytic cleavage sites or other specialized functions like a glycosaminoglycan attachment site. If the assembly of the 3 chains required for the triple helix formation can be controlled in vitro, collagen-like molecules offer an as yet unexploited potential for protein engineering.     

Collagen family of proteins

Collagen molecules are structural macro-molecules of the extracellular matrix that include in their structure one or several domains that have a characteristic triple helical conformation. They have been classified by types that define distinct sets of polypeptide chains that can form homo- and heterotrimeric assemblies. All the collagen molecules participate in supramolecular aggregates that are stabilized in part by interactions between triple helical domains. Fourteen collagen types have been defined so far. They form a wide range of structures. Most notable are 1) fibrils that are found in most connective tissues and are made by alloys of fibrillar collagens (types I, II, III, V, and XI) and 2) sheets constituting basement membranes (type IV collagen), Descemet's membrane (type VIII collagen), worm cuticle, and organic exoskeleton of sponges. Other collagens, present in smaller quantities in tissues, play the role of connecting elements between these major structures and other tissue components. The fibril-associated collagens with interrupted triple helices (FACITs) (types IX, XII, and XIV) appear to connect fibrils to other matrix elements. Type VII collagen assemble into anchoring fibrils that bind epithelial basement membranes and entrap collagen fibrils from the underlying stroma to glue the two structures together. Type VI collagen forms thin-beaded filaments that may interact with fibrils and cells.     

Folding defects in fibrillar collagens

Fibrillar collagens have a long triple helix in which glycine is in every third position for more than 1000 amino acids. The three chains of these molecules are assembled with specificity into several different molecules that have tissue-specific distribution. Mutations that alter folding of either the carboxy-terminal globular peptides that direct chain association, or of the regions of the triple helix that are important for nucleation, or of the bulk of the triple helix, all result in identifiable genetic disorders in which the phenotype reflects the region of expression of the genes and their tissue-specific distribution. Mutations that result in changed amino-acid sequences in any of these regions have different effects on folding and may have different phenotypic outcomes. Substitution for glycine residues in the triple helical domains are among the most common effects of mutations, and the nature of the substituting residue and its location in the chain contribute to the effect on folding and also on the phenotype. More complex mutations, such as deletions or insertions of triple helix, also affect folding, probably because of alterations in helical pitch along the triple helix. These mutations all interfere with the ability of these molecules to form the characteristic fibrillar array in the extracellular matrix and many result in intracellular retention of abnormal molecules.     

Sequence analysis of alpha 1(VI) and alpha 2(VI) chains of human type VI collagen reveals internal triplication of globular domains similar to the A domains of von Willebrand factor and two alpha 2(VI) chain variants that differ in the carboxy terminus.

Amino acid sequences of human collagen alpha 1(VI) and alpha 2(VI) chains were completed by cDNA sequencing and Edman degradation demonstrating that the mature polypeptides contain 1009 and 998 amino acid residues respectively. In addition, they contain small signal peptide sequences. Both chains show 31% identity in the N-terminal (approximately 235 residues) and C-terminal (approximately 430 residues) globular domains which are connected by a triple helical segment (335-336 residues). Internal alignment of the globular sequences indicates a repetitive 200-residue structure (15-23% identity) occurring three times (N1, C1, C2) in each chain. These repeating subdomains are connected to each other and to the triple helix by short (15-30 residues) cysteine-rich segments. The globular domains possess several N-glycosylation sites but no cell-binding RGD sequences, which are exclusively found in the triple helical segment. Sequencing of alpha 2(VI) cDNA clones revealed two variant chains with a distinct C2 subdomain and 3' non-coding region. The repetitive segments C1, C2 and, to a lesser extent, N1 show significant identity (15-18%) to the collagen-binding A domains of von Willebrand factor (vWF) and they are also similar to some integrin receptors, complement components and a cartilage matrix protein. Since the globular domains of collagen VI come into close contact with triple helical segments during the formation of tissue microfibrils it suggests that the globular domains bind to collagenous structures in a manner similar to the binding of vWF to collagen I.     

A peptide study of the relationship between the collagen triple-helix and amyloid

Type XXV collagen, or collagen‐like amyloidogenic component, is a component of amyloid plaques, and recent studies suggest this collagen affects amyloid fibril elongation and has a genetic association with Alzheimer's disease. The relationship between the collagen triple helix and amyloid fibrils was investigated by studying peptide models, including a very stable triple helical peptide (Pro‐Hyp‐Gly)₁₀, an amyloidogenic peptide GNNQQNY, and a hybrid peptide where the GNNQQNY sequence was incorporated between (GPO)_n domains. Circular dichroism and nuclear magnetic resonance (NMR) spectroscopy showed the GNNQQNY peptide formed a random coil structure, whereas the hybrid peptide contained a central disordered GNNQQNY region transitioning to triple‐helical ends. Light scattering confirmed the GNNQQNY peptide had a high propensity to form amyloid fibrils, whereas amyloidogenesis was delayed in the hybrid peptide. NMR data suggested the triple‐helix constraints on the GNNQQNY sequence within the hybrid peptide may disfavor the conformational change necessary for aggregation. Independent addition of a triple‐helical peptide to the GNNQQNY peptide under aggregating conditions delayed nucleation and amyloid fibril growth. The inhibition of amyloid nucleation depended on the Gly‐Xaa‐Yaa sequence and required the triple‐helix conformation. The inhibitory effect of the collagen triple‐helix on an amyloidogenic sequence, when in the same molecule or when added separately, suggests Type XXV collagen, and possibly other collagens, may play a role in regulating amyloid fibril formation. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 795–806, 2012.     

The collagen family members as cell adhesion proteins

The collagen family of extracellular matrix proteins has played a fundamental role in the evolution of multicellular animals. At the present, 28 triple helical proteins have been named as collagens and they can be divided into several subgroups based on their structural and functional properties. In tissues, the cells are anchored to collagenous structures. Often the interaction is indirect and mediated by matrix glycoproteins, but cells also express receptors, which have the ability to directly bind to the triple helical domains in collagens. Some receptors bind to sites that are abundant in all collagens. However, increasing evidence indicates that the coevolution of collagens and cell adhesion mechanisms has given rise to receptors that bind to specific motifs in collagens. These receptors may also recognize the different members of the large collagen family in a selective manner. This review summarizes the present knowledge about the properties of collagen subtypes as cell adhesion proteins.     

Self-association of collagen triple helic peptides into higher order structures

Interest in self-association of peptides and proteins is motivated by an interest in the mechanism of physiologically higher order assembly of proteins such as collagen as well as the mechanism of pathological aggregation such as beta-amyloid formation. The triple helical form of (Pro-Hyp-Gly)(10), a peptide that has proved a useful model for molecular features of collagen, was found to self-associate, and its association properties are reported here. Turbidity experiments indicate that the triple helical peptide self-assembles at neutral pH via a nucleation-growth mechanism, with a critical concentration near 1 mM. The associated form is more stable than individual molecules by about 25 degrees C, and the association is reversible. The rate of self-association increases with temperature, supporting an entropically favored process. After self-association, (Pro-Hyp-Gly)(10) forms branched filamentous structures, in contrast with the highly ordered axially periodic structure of collagen fibrils. Yet a number of characteristics of triple helix assembly for the peptide resemble those of collagen fibril formation. These include promotion of fibril formation by neutral pH and increasing temperature; inhibition by sugars; and a requirement for hydroxyproline. It is suggested that these similar features for peptide and collagen self-association are based on common lateral underlying interactions between triple helical molecules mediated by hydrogen-bonded hydration networks involving hydroxyproline.     

Reconstitution of cytokeratin filaments in vitro: further evidence for the role of nonhelical peptides in filament assembly

The in vitro renaturation and assembly of cytokeratin molecules to form intermediate filaments (IF) illustrates that these molecules contain all of the structural information necessary for IF information. These molecules contain nine structural domains: the amino- and carboxyterminal extra helical regions, and three conserved extra helical segments that separate four helical rod-like domains. Chymotrypsin treatment of these molecules removes the end-peptide domains and inhibits the self-assembly process. We have examined the renaturation and assembly of cytokeratin molecules using solution conditions that favor the presence of intermediate forms of IF organization. Dialysis against low salt buffers revealed the presence of bead-like chains of filaments in which the 6-8-nm beads are separated by a distance of 21 nm. These data suggest that a lateral stagger of protofilaments was among the primary events in IF assembly. Chymotrypsin-modified cytokeratin enriched for alpha-helix barely initiated a turbidity increase at conditions favoring self-assembly. Addition of small amounts of intact cytokeratin accelerated the rate and extent of this reaction. These results indicate that the nonhelical peptides on intact cytokeratin potentiate the assembly of IF by orientating the stagger of laterally associated protofilaments.     

Mosaic structure of globular domains in the human type VI collagen alpha 3 chain: similarity to von Willebrand factor, fibronectin, actin, salivary proteins and aprotinin type protease inhibitors.

Human collagen alpha 3(VI) chain mRNA (approximately 10 kb) was cloned and shown by sequence analysis to encode a 25 residue signal peptide, a large N-terminal globule (1804 residues), a central triple helical segment (336 residues) and a C-terminal globule (803 residues). Some of the sequence was confirmed by Edman degradation of peptides. The N-terminal globular segment consists of nine consecutive 200 residue repeats (N1 to N9) showing internal homology and also significant identity (17-25%) to the A domains of von Willebrand Factor and similar domains present in some other proteins. Deletions were found in the N3 and N9 domains of several cDNA clones suggesting variation of these structures by alternative splicing. The C-terminal globule starts immediately after the triple helical segment with two domains C1 (184 residues) and C2 (248 residues) being similar to the N domains. They are followed by a proline rich, repetitive segment C3 of 122 residues, with similarity to some salivary proteins, and domain C4 (89 residues), which is similar to the type III repeats present in fibronectin and tenascin. The most C-terminal domain C5 (70 residues) shows 40-50% identity to a variety of serine protease inhibitors of the Kunitz type. The whole sequence contains 29 cysteines which are mainly clustered in short segments connecting domains N1, C1, C2 and the triple helix, and in the inhibitor domain. Five putative Arg-Gly-Asp cell-binding sequences are exclusively localized in the triple helical segment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Peptide internalization enabled by folding: triple helical cell‐penetrating peptides

Cell‐penetrating peptides (CPPs) are known as efficient transporters of molecular cargo across cellular membranes. Their properties make them ideal candidates for in vivo applications. However, challenges in the development of effective CPPs still exist: CPPs are often fast degraded by proteases and large concentration of CPPs required for cargo transporting can cause cytotoxicity. It was previously shown that restricting peptide flexibility can improve peptide stability against enzymatic degradation and limiting length of CPP peptide can lower cytotoxic effects. Here, we present peptides (30‐mers) that efficiently penetrate cellular membranes by combining very short CPP sequences and collagen‐like folding domains. The CPP domains are hexa‐arginine (R₆) or arginine/glycine (RRGRRG). Folding is achieved through multiple proline–hydroxyproline–glycine (POG [proline‐hydroxyproline‐glycine])_n repeats that form a collagen‐like triple helical conformation. The folded peptides with CPP domains are efficiently internalized, show stability against enzymatic degradation in human serum and have minimal toxicity. Peptides lacking correct folding (random coil) or CPP domains are unable to cross cellular membranes. These features make triple helical cell‐penetrating peptides promising candidates for efficient transporters of molecular cargo across cellular membranes. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i) to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.     

Characterization of a synthetic peptide from type IV collagen that promotes melanoma cell adhesion, spreading, and motility

The adhesion and motility of tumor cells on basement membranes is a central consideration in tumor cell invasion and metastasis. Basement membrane type IV collagen directly promotes the adhesion and migration of various tumor cell types in vitro. Our previous studies demonstrated that tumor cells adhered and spread on surfaces coated with intact type IV collagen or either of the two major enzymatically purified domains of this protein. Only one of these major domains, the pepsin-generated major triple helical fragment, also supported tumor cell motility in vitro, implicating the involvement of the major triple helical region in type IV collagen-mediated tumor cell invasion in vivo. The present studies extend our previous observations using a synthetic peptide approach. A peptide, designated IV-H1, was derived from a continuous collagenous region of the major triple helical domain of the human alpha 1(IV) chain. This peptide, which has the sequence GVKGDKGNPGWPGAP, directly supported the adhesion, spreading, and motility of the highly metastatic K1735 M4 murine melanoma cell line, as well as the adhesion and spreading of other cell types, in a concentration-dependent manner in vitro. Furthermore, excess soluble peptide IV-H1, or polyclonal antibodies directed against peptide IV-H1, inhibited type IV collagen-mediated melanoma cell adhesion, spreading, and motility, but had no effect on these cellular responses to type I collagen. The full complement of cell adhesion, spreading, and motility promoting activities was dependent upon the preservation of the three prolyl residues in the peptide IV-H1 sequence. These studies indicate that peptide IV-H1 represents a cell-specific adhesion, spreading, and motility promoting domain that is active within the type IV collagen molecule.     

Structural model of the collagen-like region of C1q comprising the kink region and the fibre-like packing of the six triple helices

A detailed three-dimensional model of the collagenous part of C1q was derived by model building and computer-aided energy refinement calculations. The proposed structure is based on the collagen-like (-Gly-Xaa-Yaa-) repeating sequence of 78 to 81 residues in the N-terminal regions of the constituent A, B and C chains, on the mode of disulphide linkage between the 18 chains of C1q, and on its electron microscopically derived gross structure. It is demonstrated that the interruptions of the repeating sequence about half-way along the length of the collagenous regions (Gly36-Ile37-Arg38-Thr39 in the A chain and Ala36-Ile37-Hy138 in the C chain) do not lead to a disruption of the triple helical conformation but rather to a bend of about 60 degrees in an otherwise continuous triple helix. These features are consistent with a flexibility comparable with that of regular triple helices and with the observed low proteolytic susceptibility of the kink region. The azimuthal orientation of the kink is defined approximately by ArgA38 being located in the cap of the knee. Because of this extra residue between two glycine residues, a bad contact that would arise between the methyl group of AlaC36 and the peptide carbonyl of IleA37 in a straight triple helix is relaxed. The model features also a cluster of hydrophobic contacts between large hydrophobic side-chains in the interaction edges between the six collagen triple helices aligned with their about 10 nm long N-terminal regions in the fibril-like endpiece of C1q. The azimuthal orientations of the triple helices were derived by energy calculations of side-chain interactions previously applied to fibre-forming collagens. Independently, the same orientations and interaction edges were derived from the azimuthal orientation of the kink and the electron microscopically observed orientations of the triple helical arms that emerge from the endpiece, and which carry the C-terminal globular binding domains. The structural model has a number of implications for the assembly of the first component of complement from C1q and the zymogen complex C1r2C1s2 and possible mechanisms of its activation.     

Template‐tethered collagen mimetic peptides for studying heterotrimeric triple‐helical interactions

Collagen mimetic peptides (CMPs) have been used to elucidate the structure and stability of the triple helical conformation of collagen molecules. Although CMP homotrimers have been widely studied, very little work has been reported regarding CMP heterotrimers because of synthetic difficulties. Here, we present the synthesis and characterization of homotrimers and ABB type heterotrimers comprising natural and synthetic CMP sequences that are covalently tethered to a template, a tris(2‐aminoethyl) amine (TREN) succinic acid derivative. Various tethered heterotrimers comprising synthetic CMPs [(ProHypGly)₆, (ProProGly)₆] and CMPs representing specific domains of type I collagen were synthesized and characterized in terms of triple helical structure, thermal melting behavior, and refolding kinetics. The results indicated that CMPs derived from natural type I collagen sequence can form stable heterotrimeric helical complexes with artificial CMPs and that the thermal stability and the folding rate increase with the increasing number of helical stabilizing amino acids (e.g. Hyp) in the peptide chains. Covalent tethering enhanced the thermal stability and refolding kinetics of all CMPs; however, their relative values were not affected suggesting that the tethered system can be used for comparative study of heterotrimeric CMP's folding behavior in regards to chain composition and for characterization of thermally unstable CMPs. © 2010 Wiley Periodicals, Inc. Biopolymers 95: 94–104, 2011.     

Molecular dynamics simulation study on the interaction of collagen‐like peptides with gelatinase‐A (MMP‐2)

Although several models have been proposed for the interaction of collagen with gelatinase‐A (matrix metalloproteinases‐2 (MMP‐2)), the extensive role of each domain of gelatinase A in hydrolyzing the collagens with and without interruptions is still elusive. Molecular docking, molecular dynamics (MD) simulation, normal mode analysis (NMA) and framework rigidity optimized dynamics algorithm (FRODAN) based analysis were carried out to understand the function of various domains of MMP‐2 upon interaction with collagen like peptides. The results reveal that the collagen binding domain (CBD) binds to the C‐terminal of collagen like peptide with interruption. CBD helps in unwinding the loosely packed interrupted region of triple helical structure to a greater extent. It can be possible to speculate that the role of hemopexin (HPX) domain is to prevent further unwinding of collagen like peptide by binding to the other end of the collagen like peptide. The catalytic (CAT) domain then reorients itself to interact with the part of the unwound region of collagen like peptide for further hydrolysis. In conclusion the CBD of MMP‐2 recognizes the collagen and aids in unwinding the collagen like peptide with interruptions, and the HPX domain of MMP‐2 binds to the other end of the collagen allowing CAT domain to access the cleavage site. This study provides a comprehensive understanding of the structural basis of collagenolysis by MMP‐2. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 779–794, 2014.     

A novel mutation causes a perinatal lethal form of osteogenesis imperfecta. An insertion in one alpha 1(I) collagen allele (COL1A1)

We have identified an infant with the perinatal lethal form of osteogenesis imperfecta (type II) whose cells synthesize in equal amounts two different pro alpha 1(I) chains of type I procollagen: one chain is normal in length, the other contains an insertion of approximately 50-70 amino acid residues within the triple helical domain defined by amino acids 123-220. The structure of the insertion is consistent with duplication of an approximately 600-base pair segment in one allele of the alpha 1(I) gene (COL1A1). These cells synthesize normal type I procollagen molecules as well as molecules that contain one or two mutant chains. Unlike type I procollagen molecules synthesized by cells from most other infants with osteogenesis imperfecta type II which contain increased lysyl hydroxylation and hydroxylysyl glycosylation along the triple helical domain, the abnormal molecules synthesized by these cells are not overmodified. The lethal effect of this mutation may result from secretion of about one-quarter the normal amount of normal type I procollagen and secretion of a large amount of a molecule which has a lowered melting temperature, is extended asymmetrically, and which has altered structure in domains important for cross-link formation and bone mineralization.     

Visions for novel biophysical elucidations of extracellular matrix networks

The extracellular matrix consists of multifunctional molecules, which are composed of a large numbers of different domains. Clearly these domains and even the entire molecules do not function independently as isolated species, but interact with each other in large networks. In many cases specific regions of the networks may be considered as molecular machines in which the different molecules are arranged in highly defined spatial structures and act in a dynamic, concerted fashion. At present most structural information is limited to single molecules, and dynamics have been measured mainly for pairs of interacting partners in solution. Work needs to be extended to large integrated systems and the functions of molecular machines need to be explored. Electron tomography, fluorescence resonance energy transfer, and other biophysical techniques are very promising.     

Computational analysis of bovine alpha-1 collagen sequences

Bovine collagen alpha-1 is a naturally occurring extracellular matrix protein found in tendons and other connective tissues. It plays a vital role in cell growth, differentiation, attachment, and migration. Recent findings have established that collagen alpha-1 is involved in osteogenesis imperfecta phenotype in cattle but deep information about other members of this large family is not available so far. So with a view to finding a new edge and attempt to figure out a correlation among the well attributed Bovine alpha-1 collagen sequences are executed and analyzed. To do so, comparative analysis among the 28 members of collagen family has been carried out using Computational tools. Consequently, based on the physico-chemical, secondary structural, functional and phylogenetic classifications, we have selected collagen 12, 14 and 20 as targets for pathological conditions. These proteins belong to the FACIT family and significantly showed low glycine and proline content, high instability and aliphatic index. Moreover, FACIT family collagens contain multiple triple helical domains and being members of the FACIT family, bovine collagen 12, 14, 20 do not form fibrils by themselves but they are associated to collagen 1 associated fibrils. These collagen molecules might be crucial candidates to detect and understand the process of matrix remodeling in diseases especially in the arena of cellular compartments.     

Thermodynamic Characterization of Conformational States of Biological Macromolecules using Differential Scanning Calorimetry

Abstract

The Ras superfamily small G proteins are master regulators of a diverse range of cellular processes and act via downstream effector molecules. The first structure of a small G protein–effector complex, that of Rap1A with c-Raf1, was published 20 years ago. Since then, the structures of more than 60 small G proteins in complex with their effectors have been published. These effectors utilize a diverse array of structural motifs to interact with the G protein fold, which we have divided into four structural classes: intermolecular β-sheets, helical pairs, other interactions, and pleckstrin homology (PH) domains. These classes and their representative structures are discussed and a contact analysis of the interactions is presented, which highlights the common effector-binding regions between and within the small G protein families.     

Energetics of intrachain salt-linkage formation in collagen

The energy of formation of salt linkages between Arg or Lys with Asp or Glu in a polypeptide chain having the collagen fold have been estimated using the fully empirical energy minimization scheme AMBER. The polypeptide was considered both in an isolated and a hydrated triple helical state. The collagen fold associated with a one-bonded triple helical conformation allows intrachain salt linkages having stabilization energies of 60-100 kcal when the reacting residues are separated by no more than two intervening residues. The amino end of one side chain always approaches the carboxyl end of the other side chain, and simultaneously approaches the carbonyl oxygen of the intervening backbone residue. The salt linkage conformation and the backbone conformation of the isolated collagen fold in vacuo are maintained when the molecules are in a hydrated triple helix. These results are compatible with a fold-forming role for salt linkages, especially in proline poor regions, during collagen polypeptide synthesis, and with the persistence of intrachain salt linkages throughout molecular and fibril assembly.