The NC2 Domain of Collagen IX Provides Chain Selection and Heterotrimerization

The mechanism of chain selection and trimerization of fibril-associated collagens with interrupted triple helices (FACITs) differs from that of fibrillar collagens that have special C-propeptides. We recently showed that the second carboxyl-terminal non-collagenous domain (NC2) of homotrimeric collagen XIX forms a stable trimer and substantially stabilizes a collagen triple helix attached to either end. We then hypothesized a general trimerizing role for the NC2 domain in other FACITs. Here we analyzed the NC2 domain of human heterotrimeric collagen IX, the only member of FACITs with all three chains encoded by distinct genes. Upon oxidative folding of equimolar amounts of the α1, α2, and α3 chains of NC2, a stable heterotrimer with a disulfide bridge between α1 and α3 chains is formed. Our experiments show that this heterotrimerization domain can stabilize a short triple helix attached at the carboxyl-terminal end and allows for the proper oxidation of the cystine knot of type III collagen after the short triple helix.     

The noncollagenous domain 1 of type X collagen. A novel motif for trimer and higher order multimer formation without a triple helix.

In this study, we test the hypothesis that the carboxyl noncollagenous (NC1) domain of collagen X is sufficient to direct multimer formation without a triple helix. Two peptides containing the NC1 domain of avian collagen X have been synthesized using a bacterial expression system and their properties characterized. One peptide consists only of the NC1 domain, and the other is a chimeric molecule with a noncollagenous A domain of matrilin-1 fused to the N terminus of NC1. The NC1 peptide alone forms a 45-kDa trimer under native conditions, suggesting that NC1 contains all the information for trimerization without any triple helical residues. This trimeric association is highly thermostable without intermolecular disulfide bonds. This indicates that the NC1 domain contributes to the remarkable structural stability of collagen X. Chemical cross-linking of the NC1 trimer results in a series of varying sized multimers, the smallest of which is a trimer. Therefore the NC1 trimer is sufficient to form higher order multimers. The chimeric A-NC1 peptide forms a homotrimer by itself, and a series of heterotrimers with the NC1 peptide via the NC1 domain. Thus the NC1(X) domain directs multimer formation, even in a noncollagenous molecule.     

Collagen triple helix formation can be nucleated at either end

The directional dependence of folding rates for rod-like macromolecules such as parallel alpha-helical coiled-coils, DNA double-helices, and collagen triple helices is largely unexplored. This is mainly due to technical difficulties in measuring rates in different directions. Folding of collagens is nucleated by trimeric non-collagenous domains. These are usually located at the COOH terminus, suggesting that triple helix folding proceeds from the COOH to the NH(2) terminus. Evidence is presented here that effective nucleation is possible at both ends of the collagen-like peptide (Gly-Pro-Pro)(10), using designed proteins in which this peptide is fused either NH(2)- or COOH-terminal to a nucleation domain, either T4-phage foldon or the disulfide knot of type III collagen. The location of the nucleation domain influences triple-helical stability, which might be explained by differences in the linker sequences and the presence or absence of repulsive charges at the carboxyl-terminal end of the triple helix. Triple helical folding rates are found to be independent of the site of nucleation and consistent with cis-trans isomerization being the rate-limiting step.     

Chemical shift assignments of the fibronectin III like domains 7–8 of type VII collagen

Type VII collagen (Col7) is important for skin stability. This is underlined by the severe skin blistering phenotype in the Col7 related diseases dystrophic epidermolysis bullosa and epidermolysis bullosa acquisita (EBA). Col7 has a large N-terminal non-collagenous domain (NC1) that is followed by the triple helical collagenous domain. The NC1 domain has subdomains with homology to adhesion molecules and mediates important interactions within the extracellular matrix. An 185 amino acid long part of the NC1-subdomain termed fibronectin III like domains 7 and 8 (FNIII7-8) was investigated. Antibodies against this region are pathogenic in a mouse model of EBA and one reported missense mutations of Col7 lies within these domains. The nearly complete NMR resonance assignment of recombinant FNIII7-8 of Col7 is reported.     

Trimerization and triple helix stabilization of the collagen XIX NC2 domain

The mechanisms of chain selection and assembly of fibril-associated collagens with interrupted triple helices (FACITs) must differ from that of fibrillar collagens, since they lack the characteristic C-propeptide. We analyzed two carboxyl-terminal noncollagenous domains, NC2 and NC1, of collagen XIX as potential trimerization units and found that NC2 forms a stable trimer and substantially stabilizes a collagen triple helix attached to either end. In contrast, the NC1 domain requires formation of an adjacent collagen triple helix to form interchain disulfide bridges. The NC2 domain of collagen XIX and probably of other FACITs is responsible for chain selection and trimerization.     

Crystal structure of human type III collagen Gly991-Gly1032 cystine knot-containing peptide shows both 7/2 and 10/3 triple helical symmetries

Type III collagen is a critical collagen that comprises extensible connective tissue such as skin, lung, and the vascular system. Mutations in the type III collagen gene, COL3A1, are associated with the most severe forms of Ehlers-Danlos syndrome. A characteristic feature of type III collagen is the presence of a stabilizing C-terminal cystine knot. Crystal structures of collagen triple helices reported so far contain artificial sequences like (Gly-Pro-Pro)(n) or (Gly-Pro-Hyp)(n). To gain insight into the structural properties exhibited by the natural type III collagen triple helix, we synthesized, crystallized, and determined the structure of a 12-triplet repeating peptide containing the natural type III collagen sequence from residues 991 to 1032 including the C-terminal cystine knot region, to 2.3A resolution. This represents the longest collagen triple helical structure determined to date with a native sequence. Strikingly, the Gly(991)-Gly(1032) structure reveals that the central non-imino acid-containing region adopts 10/3 superhelical properties, whereas the imino acid rich N- and C-terminal regions adhere to a 7/2 superhelical conformation. The structure is consistent with two models for the cystine knot; however, the poor density for the majority of this region suggests that multiple conformations may be adopted. The structure shows that the multiple non-imino acids make several types of direct intrahelical as well as interhelical contacts. The looser superhelical structure of the non-imino acid region of collagen triple helices combined with the extra contacts afforded by ionic and polar residues likely play a role in fibrillar assembly and interactions with other extracellular components.     

Type XIII collagen forms homotrimers with three triple helical collagenous domains and its association into disulfide-bonded trimers is enhanced by prolyl 4-hydroxylase

Type XIII collagen is a type II transmembrane protein predicted to consist of a short cytosolic domain, a single transmembrane domain, and three collagenous domains flanked by noncollagenous sequences. Previous studies on mRNAs indicate that the structures of the collagenous domain closest to the cell membrane, COL1, the adjacent noncollagenous domain, NC2, and the C-terminal domains COL3 and NC4 are subject to alternative splicing. In order to extend studies of type XIII collagen from cDNAs to the protein level we have produced it in insect cells by means of baculoviruses. Type XIII collagen alpha chains were found to associate into disulfide-bonded trimers, and hydroxylation of proline residues dramatically enhanced this association. This protein contains altogether eight cysteine residues, and interchain disulfide bonds could be located in the NC1 domain and possibly at the junction of COL1 and NC2, while the two cysteine residues in NC4 are likely to form intrachain bonds. Pepsin and trypsin/chymotrypsin digestions indicated that the type XIII collagen alpha chains form homotrimers whose three collagenous domains are in triple helical conformation. The thermal stabilities (T(m)) of the COL1, COL2, and COL3 domains were 38, 49 and 40 degrees C, respectively. The T(m) of the central collagenous domain is unusually high, which in the light of this domain being invariant in terms of alternative splicing suggests that the central portion of the molecule may have an important role in the stability of the molecule. All in all, most of the type XIII collagen ectodomain appears to be present in triple helical conformation, which is in clear contrast to the short or highly interrupted triple helical domains of the other known collagenous transmembrane proteins.     

The hinge region of type VII collagen is intrinsically disordered

Type VII collagen (Col7) is important for skin integrity. As a major component of the anchoring fibrils, Col7 is essential for linking different skin layers together. The central collagenous domain of Col7 contains several interruptions of the collagen triple helix. The longest interruption is 39 amino acids long and referred to as the hinge region. The hinge region is highly conserved between species. This region was predicted to adopt a coiled coil structure and to serve as the trimerization domain of Col7.To gain insight into the potential function of the hinge region we investigated a heterologous expressed peptide by CD and NMR spectroscopy. CD spectroscopy implies that the hinge region is intrinsically disordered. Resonance assignment was performed and allowed secondary structure analysis based on the chemical shift values. Seven amino acids in the N-terminal moiety show residual α-helical conformation. Subsequent investigation of temperature dependency of amide chemical shifts indicated participation in hydrogen bonding of amino acid residues in the C-terminal moiety of the hinge region. Therefore, the hinge region does not form a coiled coil structure under the employed experimental conditions. The intrinsic disorder of the hinge region might be desired for flexibility to serve as a “hinge” or the hinge region is an important interaction site as typically observed for intrinsically disordered proteins.     

Folding of collagen IV

Collagen IV dimers of two collagen IV molecules connected by their C-terminal globular NC1 domains were isolated by limited digestion with bacterial collagenase from mouse Engelbreth-Holm-Swarm (EHS) sarcoma tissue. The collagenous domains were only 300 nm long as compared to 400 nm of intact collagen IV but the disulfide bonds in the N-terminal region of the major triple helix were retained. Unfolding of the collagenous domains as monitored by circular dichroism occurred in a temperature range of 30 to 44 degrees C with a midpoint at 37 degrees C. The transition is significantly broader than that of the continuous triple helices in collagens I, II and III, a feature which can be explained by the frequent non-collagenous interruptions in the triple-helical domain of collagen IV. Refolding at 25 degrees C following complete unfolding at 50 degrees C was monitored by circular dichroism, selective proteolytic digestion of non-refolded segments and by a newly developed method in which the recovered triple-helical segments were visualized by electron microscopy. Triple-helix formation was found to proceed in a zipper-like fashion from the C-terminal NC1 domains towards the N-terminus, indicating that this domain is essential for nucleations. For collagen IV dimers with intact NC1 domains the rate of triple-helix growth was of comparable magnitude to that of collagen III, demonstrating that the non-collagenous interruptions do not slow down the refolding process where the rate-limiting step is the cis-trans isomerization of proline peptide bonds. Refolding was near to 100% and the refolding products were similar to the starting material as judged by thermal stability and electron microscopic appearance. Removal of the NC1 domains by pepsin or dissociation of their hexametric structures by acetic acid led to a loss of the refolding ability. Instead products with randomly dispersed short triple-helical segments were formed in a slow reaction. In no case, even when the disulfide bonds in the N-terminal region of the triple-helical domain were intact, was refolding from the N- towards the C-terminus observed. Taken together with results in other collagens, this suggests that C to N directionality might be an intrinsic property of triple-helix folding.     

Type XIV collagen, a new homotrimeric molecule extracted from fetal bovine skin and tendon, with a triple helical disulfide-bonded domain homologous to type IX and type XII collagens

Previously undescribed disulfide-bonded collagenous pepsin-derived fragments have been isolated from fetal calf tendon and skin. One fragment, 10.5 kDa after reduction, was shown to be similar but distinct to the COL1 domain of the recently characterized type XII collagen (64% primary structure identity). The similarity includes important features such as size, location of the cysteine residues, and nature and position of an imperfection of the triple helix. From fetal calf skin, two approximately 34-kDa disulfide-bonded trimeric fragments were isolated in the unreduced form. Amino acid sequencing showed that one fragment contained solely the COL1 domain of type XII collagen while the other one only contained the COL1 domain of the new chain. Like type XII collagen, the new chain is therefore part of a homotrimeric molecule and should thus be considered as a distinct collagen type. We propose to call the molecule from which this fragment is derived, type XIV collagen, with a chain composition (alpha 1 (XIV]3. The presence of a domain similar to the COL1 domain of collagens types IX and XII suggests that type XIV collagen belongs to the group of fibril-associated collagens with interrupted triple helices (FACIT). Two other fragments, 13.5 and 17 kDa after reduction, were also purified. They were shown to contain the same triple helical domain with different pepsin cleavage sites at the amino terminus. Several tryptic peptides were sequenced, and the derived sequences could be aligned with the COL2 domain of type XII collagen or with flanking sequences in the NC2 and NC3 domains (61% sequence identity). These fragments are very likely to be also derived from type XIV collagen.     

Transformation of the mechanism of triple-helix peptide folding in the absence of a C-terminal nucleation domain and its implications for mutations in collagen disorders

Folding abnormalities of the triple helix have been demonstrated in collagen diseases such as osteogenesis imperfecta in which the mutation leads to the substitution of a single Gly in the (Gly-X-Y)n sequence pattern by a larger residue. Model peptides can be used to clarify the details of normal collagen folding and the consequences of the interruption of that folding by a Gly substitution. NMR and CD studies show that placement of a (GPO)4 nucleation domain at the N terminus rather than the C terminus of a native collagen sequence allows the formation of a stable triple helix but alters the folding mechanism. Although C- to N-terminal directional folding occurs when the nucleation domain is at the C terminus, there is no preferential folding direction when the nucleation domain is at the N terminus. The lack of zipper-like directional folding does not interfere with triple-helix formation, and when a Gly residue is replaced by Ser to model an osteogenesis imperfecta mutation, the peptide with the N-terminal (GPO)4 domain can still form a good triple helix N-terminal to the mutation site. These peptide studies raise the possibility that mutant collagen could fold in a C to N direction in a zipper-like manner up to the mutation site and that completion of the triple helix N-terminal to the mutation would involve an alternative mechanism.     

Trimerization of collagen IX alpha-chains does not require the presence of the COL1 and NC1 domains

Collagen IX is a heterotrimer of three alpha-chains, which consists of three COL domains (collagenous domains) (COL1-COL3) and four NC domains (non-collagenous domains) (NC1-NC4), numbered from the C-terminus. Although collagen IX chains have been shown to associate via their C-terminal NC1 domains and form a triple helix starting from the COL1 domain, it is not known whether chain association can occur at other sites and whether other collagenous and non-collagenous regions are involved. To address this question, we prepared five constructs, two long variants (beginning at the NC4 domain) and three short variants (beginning at the COL2 domain), all ending at the NC2 domain (or NC2 replaced by NC1), to study association and selection of collagen IX alpha-chains. Both long variants were able to associate with NC1 or NC2 at the C-terminus and form various disulfide-bonded trimers, but the specificity of chain selection was diminished compared with full-length chains. Trimers of the long variant ending at NC2 were shown to be triple helical by CD. Short variants were not able to assemble into disulfide-bonded trimers even in the presence of both conserved cysteine residues from the COL1-NC1 junction. Our results demonstrate that collagen IX alpha-chains can associate in the absence of COL1 and NC1 domains to form a triple helix, but the COL2-NC2 region alone is not sufficient for trimerization. The results suggest that folding of collagen IX is a co-operative process involving multiple COL and NC domains and that the COL1-NC1 region is important for chain specificity.     

Synthesis of heterotrimeric collagen models containing Arg residues in Y-positions and analysis of their conformational stability

An Arg residue incorporated into the Y-position of collagenous host-guest peptide Ac-(Gly-Pro-Hyp)(3)-Gly-Pro-Y-(Gly-Pro-Hyp)(4)-Gly-Gly-NH(2) is reported to stabilize the triple helical structure as well as a 4(R)-hydroxyproline (Hyp) residue. Here, we synthesized heterotrimeric collagen models containing Arg in Y-positions utilizing the cystine knot strategy. Analysis of their thermal transition temperatures using circular dichroism spectrometry demonstrated unexpected decrease in the triple helical stability as the number of Arg increased. The obtained results indicated that an Arg residue in a Y-position is not always an equivalent of a Hyp residue, and that it possesses a potential helix destabilizing effect.     

The Self-assembly of a Mini-fibril with Axial Periodicity from a Designed Collagen-mimetic Triple Helix

In this work we describe the self-assembly of a collagen-like periodic mini-fibril from a recombinant triple helix. The triple helix, designated Col108, is expressed in Escherichia coli using an artificial gene and consists of a 378-residue triple helix domain organized into three pseudo-repeating sequence units. The peptide forms a stable triple helix with a melting temperature of 41 °C. Upon increases of pH and temperature, Col108 self-assembles in solution into smooth mini-fibrils with the cross-striated banding pattern typical of fibrillar collagens. The banding pattern is characterized by an axially repeating feature of ∼35 nm as observed by transmission electron microscopy and atomic force microscopy. Both the negatively stained and the positively stained transmission electron microscopy patterns of the Col108 mini-fibrils are consistent with a staggered arrangement of triple helices having a staggering value of 123 residues, a value closely connected to the size of one repeat sequence unit. A mechanism is proposed for the mini-fibril formation of Col108 in which the axial periodicity is instigated by the built-in sequence periodicity and stabilized by the optimized interactions between the triple helices in a 1-unit staggered arrangement. Lacking hydroxyproline residues and telopeptides, two factors implicated in the fibrillogenesis of native collagen, the Col108 mini-fibrils demonstrate that sequence features of the triple helical domain alone are sufficient to “code” for axially repeating periodicity of fibrils. To our knowledge, Col108 is the first designed triple helix to self-assemble into periodic fibrils and offers a unique opportunity to unravel the specific molecular interactions of collagen fibrillogenesis.     

Type XIV collagen is a variant of undulin.

We have isolated undulin, an extracellular matrix protein associated with the surface of collagen fibrils, from chicken embryos. The protein showed a molecular mass of about 600 kDa and is composed of three 210-kDa subunits linked by reducible as well as non-reducible bonds. In contrast to human undulin which reportedly is devoid of collagenous sequences, the chicken protein contained a short triple-helical segment that was sensitive to digestion by bacterial collagenase. Screening of an expression library with affinity-purified antibodies yielded two cDNA clones specific for chicken undulin. Analysis of the amino acid sequence deduced from the nucleotide sequence of these clones showed that the human and the chicken protein shared 71% sequence identity. At the amino-terminus both polypeptides contained several similar repeats related to the type III modules found in fibronectin. Towards the carboxyl terminus, however, the two sequences diverged substantially from each other. While the human sequence terminated in a proline-rich segment, the chicken sequence continued with a domain related to von Willebrand factor, with a domain similar to the noncollagenous domain NC4 of type IX collagen and with a typical collagenous triple helix. A short segment of this sequence was found to be identical with the published sequence of a bovine peptide derived from type XIV collagen. Our protein must therefore represent chicken type XIV collagen. One way to explain these results is the possibility that undulin exists in at least two alternatively spliced variants, one lacking the collagenous domain, as described initially for human undulin, and one containing the triple-helical domain, as found in type XIV collagen.     

Procollagen VII self-assembly depends on site-specific interactions and is promoted by cleavage of the NC2 domain with procollagen C-proteinase

Procollagen VII is a homotrimer of 350-kDa proalpha1(VII) chains. Each chain has a central collagenous domain flanked by a noncollagenous amino-terminal NC1 domain and a carboxy-terminal NC2 domain. After secretion from cells, procollagen VII molecules form antiparallel dimers with a 60 nm overlap. These dimers are stabilized by disulfide bonds formed between cysteines present in the NC2 domain and cysteines present in the triple-helical domain. Electron microscopy has provided direct evidence for the existence of collagen VII dimers, but the dynamic process of dimer formation is not well understood. In the present study, we tested the hypothesis that, during dimer formation, the NC2 domain of one procollagen VII molecule specifically recognizes and binds to the triple-helical region adjacent to Cys-2625 of another procollagen VII molecule. We also investigated the role of processing of the NC2 domain by the procollagen C-proteinase/BMP-1 in dimer assembly. We engineered mini mouse procollagen VII variants consisting of intact NC1 and NC2 domains and a shortened triple helix in which the C-terminal region encompassing Cys-2625 was either preserved or substituted with the region encompassing Cys-1448 derived from the N-terminal part of the triple-helical domain. The results indicate that procollagen VII self-assembly depends on site-specific interactions between the NC2 domain and the triple-helical region adjacent to Cys-2625 and that this process is promoted by the cleavage of the NC2 by procollagen C-proteinase/BMP1.     

The collagen-like peptide (GER)15GPCCG forms pH-dependent covalently linked triple helical trimers

A collagen-like peptide with the sequence (GER)(15) GPCCG was synthesized to study the formation of a triple helix in the absence of proline residues. This peptide can form a triple helix at acidic and basic pH, but is insoluble around neutral pH. The formation of a triple helix can be used to covalently oxidize the cysteine residues into a disulfide knot. Three disulfide bonds are formed between the three chains as has been found at the carboxyl-terminal end of the type III collagen triple helix. This is a new method to covalently link collagen-like peptides with a stereochemistry that occurs in nature. The peptide undergoes a reversible, cooperative triple helix coil transition with a transition midpoint (T(m)) of 17 to 20 degrees C at acidic pH and 32 to 37 degrees C at basic pH. At acidic pH there was little influence of the T(m) on the salt concentration of the buffer. At basic pH increasing the salt concentration reduced the T(m) to values comparable to the stability at acidic pH. These experiments show that the tripeptide unit GER which occurs frequently in collagen sequences can form a triple helical structure in the absence of more typical collagen-like tripeptide units and that charge-charge interactions play a role in the stabilization of the triple helix of this peptide.     

Structure formation in the C terminus of type III collagen guides disulfide cross-linking

In type III collagen the main triple-helical domain is followed by a disulfide knot and the C-terminal propeptide, which are both essential for nucleation, stabilization and registration of the triple helix. We demonstrate that oxidative inter-chain disulfide bridging does not occur between the knot sequences GlyProCysCysGly of dissociated randomly coiled chains. N-terminal fusion of the obligatory trimeric domain of mini-fibritin is able to direct this process efficiently, demonstrating a folded precursor mechanism in which the thiol groups have to be properly placed for the formation of native disulfide bonds. The natural C-propeptide domain may act in a similar way as the mini-fibritin domain. After disulfide linkage and triple-helix formation the catalyzing mini-fibritin domain was removed by thrombin cleavage. In this way a short but stable triple-helical collagen fragment was expressed in Escherichia coli for structural and functional studies.     

Trimerization Domain of the Collagen Tail of Acetylcholinesterase

In the collagen-tailed forms of cholinesterases, each subunit of a specific triple helical collagen, ColQ, may be attached through a proline-rich domain (PRAD) situated in its N-terminal noncollagenous region, to tetramers of acetylcholinesterase (AChE) or butyrylcholinesterase (BChE). This heteromeric assembly ensures the functional anchoring of AChE in extracellulare matrices, for example, at the neuromuscular junction. In this study, we analyzed the influence of deletions in the noncollagenous C-terminal region of ColQ on its capacity to form a triple helix. We show that an 80-residue segment located downstream of the collagenous regions contains the trimerization domain, that it can form trimers without the collagenous regions, and that a pair of cysteines located at the N-boundary of this domain facilitates oligomerization, although it is not absolutely required. We further show that AChE subunits can associate with nonhelical collagen ColQ monomers, forming ColQ-associated tetramers (G₄-Q), which are secreted or are anchored at the cell surface when the C-terminal domain of ColQ is replaced by a GPI-addition signal.     

Vascular Ehlers-Danlos Syndrome Mutations in Type III Collagen Differently Stall the Triple Helical Folding

Vascular Ehlers-Danlos syndrome (EDS) type IV is the most severe form of EDS. In many cases the disease is caused by a point mutation of Gly in type III collagen. A slower folding of the collagen helix is a potential cause for over-modifications. However, little is known about the rate of folding of type III collagen in patients with EDS. To understand the molecular mechanism of the effect of mutations, a system was developed for bacterial production of homotrimeric model polypeptides. The C-terminal quarter, 252 residues, of the natural human type III collagen was attached to (GPP)₇ with the type XIX collagen trimerization domain (NC2). The natural collagen domain forms a triple helical structure without 4-hydroxylation of proline at a low temperature. At 33 °C, the natural collagenous part is denatured, but the C-terminal (GPP)₇-NC2 remains intact. Switching to a low temperature triggers the folding of the type III collagen domain in a zipper-like fashion that resembles the natural process. We used this system for the two known EDS mutations (Gly-to-Val) in the middle at Gly-910 and at the C terminus at Gly-1018. In addition, wild-type and Gly-to-Ala mutants were made. The mutations significantly slow down the overall rate of triple helix formation. The effect of the Gly-to-Val mutation is much more severe compared with Gly-to-Ala. This is the first report on the folding of collagen with EDS mutations, which demonstrates local delays in the triple helix propagation around the mutated residue.