The crucial role of trimerization domains in collagen folding

The International Journal of Biochemistry & Cell Biology 44 (2012) 21–32

Contents lists available at SciVerse ScienceDirect

The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel

Review

The crucial role of trimerization domains in collagen folding Sergei P. Boudko a,b , Jürgen Engel c,∗ , Hans Peter Bächinger a,b,∗∗ a

Research Department of Shriners Hospital for Children, 3101 SW Sam Jackson Park Road, Portland, OR 97239, USA Department of Biochemistry and Molecular Biology, Oregon Health & Science University, 3191 SW Sam Jackson Park Road, Portland, OR 97239, USA c Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland b

a r t i c l e

i n f o

Article history: Received 30 July 2011 Received in revised form 27 September 2011 Accepted 27 September 2011 Available online 5 October 2011 Keywords: Protein folding Collagen triple helix Trimerization domain Protein structure Chain selection

a b s t r a c t Collagens contain large numbers of Gly-Xaa-Yaa peptide repeats that form the characteristic triple helix, where the individual chains fold into a polyproline II helix and three of these helices form a right-handed triple helix. For the proper folding of the triple helix collagens contain trimerization domains. These domains ensure a single starting point for triple helix formation and are also responsible for the chain selection in heterotrimeric collagens. Trimerization domains are non-collagenous domains of very different structures. The size of trimerization domains varies from 35 residues in type IX collagen to around 250 residues for the fibrillar collagens. These domains are not only crucial for biological functions, but they are also attractive tools for generating recombinant collagen fragments of interest as well as for general use in protein engineering and biomaterial design. Here we review the current knowledge of the structure and function of these trimerization domains. © 2011 Elsevier Ltd. All rights reserved.

Contents 1. 2.

3. 4. 5. 6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Trimerization domains of collagens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Fibril-forming collagens I and III . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Basement membrane collagen IV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Network-forming collagens VIII and X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Multiplexins (collagens XV and XVIII) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. FACIT-collagens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Beaded filament and anchoring collagens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Minicollagens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8. Trimerization domains with homology to those found in collagens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The foldon trimer from phage fibritin as an experimental tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Use of trimerizing domains for biomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mutations in trimerization domains cause diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Trimerization domains are non-collagenous peptide segments located next to triple helix forming Gly-Xaa-Yaa repeats in colla-

∗ Corresponding author. ∗∗ Corresponding author at: Research Department of Shriners Hospital for Children, 3101 SW Sam Jackson Park Road, Portland, OR 97239, USA. Fax: +1 503 221 3451. E-mail addresses: [email protected] (J. Engel), [email protected] (H.P. Bächinger). 1357-2725/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2011.09.009

21 22 22 23 23 25 26 27 28 28 28 29 29 30 30

gens. They have a high trimerization potential and are essential for the formation of the initial encounter of the three peptide chains making up collagen. In this way they prevent the formation of misaligned collagen triple helices. In the presence of trimerization domains triple helix formation starts at defined sites whereas in their absence helix nucleation may occur at different places at which the collagen chains meet in a statistical way. Indeed staggered chains with mismatched ends are frequently observed misfolding products of collagens whose trimerization domains were removed (Fig. 1). In contrast to globular proteins misfolding of the collagen triple helix is a likely event because of the repeating

22

S.P. Boudko et al. / The International Journal of Biochemistry & Cell Biology 44 (2012) 21–32

which have been employed are deletion studies using efficiency of collagen folding as assays. In many cases candidate domains were isolated or recombinantly expressed and their trimerization potential was studied. Frequently the three-dimensional structure of a trimerization domain was solved by X-ray crystallography and the molecular interactions driving trimerization were explored. Trimerization domains from proteins not belonging to the collagen family were used in chimeric constructs with collagen or collagen model peptides. They exhibited a similar function as real collagen trimerization domains proving that the trimerization is the essential feature and more specific effects are less important. Interesting synthetic products were designed using different fibrous sequence motifs and trimerization domains. In the following we shall describe the function of trimerization domains in different collagens and other proteins.

2. Trimerization domains of collagens 2.1. Fibril-forming collagens I and III

Fig. 1. Three scenarios for collagen folding. (A) Processed gelatin without trimerization domains forms multi molecular aggregates with short patches of misaligned triple helix. (B) Nascent collagen single chains with trimerization domains form trimeric molecules with correct chain register and aligned triple helix. (C) Chain selection and trimerization by heterotypic trimerization domains leads to the formation of heterotrimeric collagen molecules with 2 identical chains.

structure whose stability is relatively insensitive to lateral shifts by one or more Gly-Xaa-Yaa repeats. Many collagens are highly defined heterotrimers of two or three different polypeptide chains. The required high specificity of chain selection is difficult to achieve by the restricted specificity of recognition between the triple helical parts of the collagens. The stabilization of the triple helix is mainly based on backbone interactions and is only partly influenced by interactions between side chains. Specificity of heterotrimer formation of trimerization domains is by far more effective and offers a powerful tool for chain selection. Trimerization domains have been found in many different proteins containing collagen triple helices. These include members of the large family of mammalian collagens, several annelid collagens and the more recently studied bacterial collagens. Related domains were also detected in proteins not commonly named collagens such as complement factor C1q, mannose binding protein, macrophage scavenger receptor and acetylcholine esterase. Trimerization domains are of different folding types. Several are variants of ␣-helical coiled-coil domains, others have globular folds of different types. It is therefore not trivial to find out which of the non-collagenous domains has a trimerization function. Strategies,

Up to about 1980 in the history of research on collagen folding only the processed form of skin collagen I was known (Fig. 2). This product was isolated by extraction from skin tissue and did not contain the amino- and carboxyl-terminal propeptides. At late steps of biosynthesis these are removed by highly specific enzymes after folding of the protein. Since the intact collagen I was not known at this time it was believed that the processed collagen should refold to its native structure after heat denaturation and cooling to native conditions. Results did not show the expected refolding. Instead it was found that products of refolding were very different at different temperatures and only a very small fraction of true native molecules with high stability were recovered in trimeric form. At low temperatures the largest fraction showed a wide distribution of molecular weights larger than that of native collagen but much lower stabilities than the native starting material (Engel and Prockop, 1991). The puzzle was only solved when intact form of collagen I became known. The carboxyl-terminal non-collagenous domain was identified as the domain which aligns and registers the three identical ␣-chains. For technical reasons most refolding studies were performed with the rather similar collagen III, which contains a disulfide linkage at the C-terminus of its triple helix (Bächinger et al., 1978; Bruckner et al., 1978). This so-called disulfide knot is formed after formation of non-covalent interactions between three carboxyl-terminal non-collagenous domains and acts as a permanent linker even after removal of the latter. It was demonstrated that the folding of the triple helix of collagen III started at the C-terminus and proceeded to the N-terminus in a zipper like fashion. This conclusion followed from refolding experiments in which the process was terminated by addition of trypsin after different times of folding. Trypsin is known to digest the polypeptide chains, which are not protected by the triple helical structure (Bruckner and Prockop, 1981). Products were analyzed

Fig. 2. Scheme of collagen I. Collagen I is initially synthesized in its premature form that contains large globular domains at both N- and C-termini (N- and C-propeptides). Later, the propeptides are cleaved by specific enzymes (N- and C-proteinases) and the mature collagen molecules are deposited into fibrils. The mature collagen still contains non-triple helical short peptides called N- and C-telopeptides. It is the C-propetide that was shown to be the trimerization domain in collagen I and other fibrillar collagens.

S.P. Boudko et al. / The International Journal of Biochemistry & Cell Biology 44 (2012) 21–32

by SDS-PAGE and by electron microscopy. It was found that the length of refolded triple helices was increasing linearly with time up to the formation of helices with full length. All refolding products contained the disulfide knot at their carboxyl termini. The effect of mutations within the C-propeptides strongly suggests that they are crucial for the correct interaction of the three polypeptide chains and for their correct folding (Bateman et al., 1989; Chessler et al., 1993; Pace et al., 2001). Many questions remain to be answered concerning the mechanism of their interaction in the hetero-assembly of collagen I and the homo-assembly of collagen III. The three-dimensional structure of the C-propeptide domains is not known. An ␣-helical coiled-coil like region was predicted to exist in the N-terminal part of the C-propeptide and named “oligomerization domain” (McAlinden et al., 2003). The existence of this domain was not confirmed by energy minimization modeling and molecular dynamics but interactions were predicted between most C-terminal globular regions with a ␤structure (Malone et al., 2005). The most interesting question to be addressed in future studies is the mechanism by which the correct ratio of ␣1 and ␣2 chains is determined in collagen I. In cells pro ␣1(I) chains can form homotrimers or heterotrimers with pro ␣2(I) chains but the latter cannot form homotrimers (Myllyharju et al., 1997). This global finding may be explained by the specificity of interactions between the C-propeptides. It is interesting to compare the determinants of chain selection in collagen I with the more advanced work on collagen IX (see paragraph “FACIT collagens”). In collagen IX the NC2 domain was found to be responsible for the selective interaction of three different chains. It has a similar trimerizing function as the NC1 domain of collagen I but differs completely in size and structure. The large variance of different trimerization domains is a general feature. It is even possible to replace oligomerization domains by completely different domains from other proteins. For collagen I the C-propeptide domain was replaced by a transmembrane domain without effecting the trimer formation or triple helix folding (Bulleid et al., 1997). Related observation were made with the trimeric phage protein foldon, which qualified as a very effective trimerizatin domain for collagen model peptides (see paragraph “The foldon trimer from phage fibritin as an experimental tool”).

23

The C-terminal domains of collagen IV have a dual function. They are not only involved in nucleation of triple helix folding but also in the assembly of two collagen molecules. Two trimers associate to a hexamer with a pseudo symmetry plane between the trimers. Thus each trimer links three ␣-chains forming a triple helix and two triple helices are associated in an antiparallel direction. This assembly step together with the linkage of four molecules at their N-termini (Risteli et al., 1980) leads to a two-dimensional network, which was compared with a chicken-wire fence (Timpl et al., 1981). It should be mentioned that the chicken-wire network is only the basic connecting mode and that the real supramolecular structure of the collagen IV network is more complex (Yurchenco and Schittny, 1990). The structure of the NC1-hexamer has been elucidated at high resolution by X-ray crystallography (Sundaramoorthy et al., 2002; Than et al., 2002). The hexamer is composed of two trimeric caps which interact through a large interface (Fig. 3). Each monomer consists of two very similar subdomains, where each contains a finger-like hairpin loop that inserts into a six-stranded ␤-sheet of a neighboring subdomain. It is believed by some authors that the monomers recognize each other through a domain-swapping mechanism in which the ␤-hairpin of one monomer is swapped into the docking site of the other. A high binding strength was found between ␣1- and ␣2-monomers and this was related to the stoichiometry of the assembly to ␣1␣1␣2 heterotrimers (Khoshnoodi et al., 2006). Interactions between the two trimers to a hexamer span larger distances than those within the caps but involve an unusually large interaction surface. In addition a novel type of covalent cross-link spanning this interface was discovered in the crystal structure (Than et al., 2002). The side chains of a Met and a Lys residue are linked connecting the ␣1- and ␣2-chains of opposite trimers. Initially these covalent bonds were not seen in the parallel crystallographic work by Sundaramoorthy et al. (2002) but later the discrepancy was solved and the novel cross-link was identified as a sulfilimine bond (Than et al., 2002; Vanacore et al., 2005, 2009). The three cross-links between the two caps further stabilize the NC1 hexamer. It may also be speculated, that the various enzymes involved in forming the link have a regulatory role in collagen IV assembly. 2.3. Network-forming collagens VIII and X

2.2. Basement membrane collagen IV Collagen IV is a major constituent of basement membranes in which it forms a two-dimensional network. The about 400 nm long triple-helical regions with their characteristic Gly-X-Y repeats are interrupted by approximately 20 short non-collagenous sequences in which Gly does not occur in every third position (reviewed by Kühn, 1995). The interruptions do not disturb the folding of the triple helix but give rise to kinks in the rod-like helix (Hofmann et al., 1984). The interruptions in the three chains form a trimeric structure after folding of the triple helix but it is not known whether some of them have a trimerization potential on their own. Collagen IV contains only a small N-terminal domain but a rather large C-terminal domain of 230 residues. As demonstrated by electron microscopy folding always starts at the C-terminus demonstrating that the C-terminal domain is the domain responsible for helix assembly and nucleation (Dölz et al., 1988; Davis et al., 1989). In addition the C-terminal domain may serve the function of chain selection. A family of six homologous ␣-chains of collagen IV are known but only 3 specific protomers out of 76 possible combinations are formed (Khoshnoodi et al., 2006). Furthermore a high affinity and selectivity of the assembly of isolated NC1-domains of different ␣-chains was demonstrated (Weber et al., 1988; Boutaud et al., 2000).

Collagen VIII is a major component of Descemet’s membrane (Labermeier et al., 1983), a specialized basement membrane separating the corneal endothelium and stroma, and is also present in vascular subendothelial matrices, heart, liver, kidney, and lung, as well as in malignant tumors (Shuttleworth, 1997). Collagen X is expressed specifically by hypertrophic chondrocytes during endochondral ossification in the developing vertebrate embryo (Schmid and Linsenmayer, 1983; Kwan et al., 1991; Kielty et al., 1985; Gibson and Flint, 1985). In the adult animal, collagen X expression is reactivated during fracture repair (Grant et al., 1987) and in osteoarthritis (von der Mark et al., 1992). Type VIII collagen is composed of highly conserved ␣1 and ␣2 chains, while type X collagen has a single ␣1-chain. Both types contain a collagenous domain of ∼150 tripeptide units flanked by a N-terminal NC2 domain of either 117 residues for type VIII or 37 residues for type X and a C-terminal NC1 domain of 161–173 residues (Muragaki et al., 1991; Ninomiya et al., 1986). While early reports suggested the presence of heterotrimers in Descemet’s membrane, a recent detailed study found only co-existing ␣1 and ␣2 homotrimers (Greenhill et al., 2000). As for the type IV collagen, the C-terminal NC1 domains of collagens VIII and X are responsible for trimerization and retained in their suprastructures. The chains assemble into

24

S.P. Boudko et al. / The International Journal of Biochemistry & Cell Biology 44 (2012) 21–32

Fig. 3. Collagen IV trimerization domain. (A) Within each NC1 trimer, the monomers recognize one another through a domain-swapping mechanism in which a ␤-hairpin motif (Hairpin) of one monomer is swapped into a docking site (Docking) of its swapping partner. (B) Cartoon representation of the trimerization domain viewed down the 3-fold rotation axis and rotated by 90◦ about the horizontal axis. The figure is generated using the program PyMol (www.pymol.org) and the coordinates from the PDB data bank (PDB ID: 1LI1).

distinct homotrimers that further assemble into hexagonal lattices (Greenhill et al., 2000; Stephan et al., 2004; Zhang and Chen, 1999). The crystal structures of the NC1 homotrimerization domains of both type VIII and X collagen were determined (Bogin et al., 2002; Kvansakul et al., 2003). They are similar to each other and also share high similarity with the globular domain (gC1q) of the complement protein C1q (Shapiro and Scherer, 1998; Gaboriaud et al., 2003) and C1q-like domains in other proteins. The NC1 trimer has the shape of a squat truncated cone, with the N- and C-termini of the polypeptide chains emerging from the base of the cone (Fig. 4). The apex is formed by the tight association of three loops, one from each monomer. Each subunit consists of a ten-stranded ␤-sandwich with jellyroll topology (Shapiro and Scherer, 1998), the strands of which are labeled A, A , B, B , and C–H. Strands A, H, C and F are mostly buried, whereas strands A , B, B , G, D and E form the solvent-accessible surface of the NC1 trimer. Trimer formation results in the creation of a central solvent-filled channel, which is lined predominantly by strand F. Intersubunit contacts made along the channel are hydrophilic near the apex and become progressively more hydrophobic towards the base of the NC1 trimer. The most striking feature of the NC1 surfaces of type VIII and X collagens is the presence of three strips of partially exposed hydrophobic residues. A possible mechanism for polygonal lattice formation in type VIII and X collagens has been suggested in which

the three hydrophobic strips on the surface of the NC1 trimer initiate the supramolecular assembly (Bogin et al., 2002; Kvansakul et al., 2003). Although the overall structures of the NC1 domains of type VIII and X collagens are very similar, there is a striking and unexpected difference: the collagen X NC1 trimer contains four buried calcium ions, which are absent in the type VIII collagen NC1 structure. The difference is due to a single amino acid replacement in the solvent channel, from T629 in collagen X to K692 in type VIII collagen, which allows the side chains of K692 to replace the calcium ions while maintaining a similarly dense network of interactions near the apex of the NC1 trimer (Kvansakul et al., 2003). It is well known that collagen X is involved in bone calcification and the presence of Ca2+ ions in its trimerization domain may indicate a calcium regulation of its assembly. The NC1 domains of the mouse ␣1(VIII) and ␣2(VIII) chains are 71.5% identical in sequence. Most of the differences are accounted for by solvent-exposed residues on the trimer surface, which are unlikely to have an influence on chain recognition. Four nonconservative substitutions, however, affect residues that are buried in subunit interfaces: E620 of the ␣1 chain is changed to valine in the ␣2 chain, M684–M685 is changed to alanine–threonine, and Y741 is changed to cysteine. Those small changes on its own do not explain why homotrimers are preferred for collagen VIII in vivo (Kvansakul et al., 2003).

S.P. Boudko et al. / The International Journal of Biochemistry & Cell Biology 44 (2012) 21–32

25

Fig. 5. Domain organization of collagen XVIII C-terminal end. Shown is a triple helical part of the COL1 domain and the full-length NC1 domain. The NC1 domain compromised of a trimerization domain, a hinge region and the endostatin domain.

Fig. 4. Collagen X trimerization domain crystal structure (PDB ID: 1GR3). (A) Cartoon representation of the trimerization domain viewed down the 3-fold rotation symmetry axis. The ␤ strands (shown in green) in one subunit are labeled A, A , B, B , and C–H. Ca2+ ions are represented as pink spheres. (B) As in (A), but rotated by 90◦ about the horizontal axis. Adapted from Bogin et al. (2002) with the permission.

2.4. Multiplexins (collagens XV and XVIII) Collagens XV and XVIII are the only two members of the multiplexin family (Pihlajaniemi and Rehn, 1995). Both collagens are homotrimers composed of single ␣-chains that contain a central frequently interrupted collagenous domain flanked by N- and Cterminal non-collagenous domains (Myers et al., 1992; Rehn et al., 1994; Rehn and Pihlajaniemi, 1994). Human type XVIII exists as three variants with differing N-terminal NC domains, but have identical interrupted collagenous and NC1 domains. Type XVIII is a heparan sulfate proteoglycan (Halfter et al., 1998) and type XV can carry chondroitin/dermatan sulfate alone or chondroitin/dermatan sulfate and heparan sulfate chains (Amenta et al., 2005). Type XVIII collagen localizes in epidermal and vascular basement membrane, similar to other heparan sulfate proteoglycans found in basement membranes (Marneros and Olsen, 2005). Type XV is not an integral basement membrane component; rather, it localizes to areas peripheral to basement membranes and associates directly with collagen fibers/fibrils in a manner consistent with other chondroitin sulfate proteoglycans (Amenta et al., 2005; Iozzo, 1999). The highest level of sequence homology between type XV and XVIII lies in their NC1 domain. The NC1 domains of collagens XV and XVIII are organized into a trimerization domain, a hinge region

and an endostatin domain (Sasaki et al., 1998; Sasaki et al., 2000) (Fig. 5). Initially, the recombinantly expressed full-length murine NC1 domain was shown to be a trimer by sieve chromatography, although the isolated endostatin domain was found to be a monomer for both collagen types, XV and XVIII (Sasaki et al., 1998). The NC1 domain of collagen XV was also found to form a trimer according to sieve chromatography (Sasaki et al., 2000). Moreover, the NC1 domain of collagen XVIII was sensitive to endogenous proteolysis, which caused the appearance of electrophoretic bands of 30–32 (apparently the endostatin domain) and 5 kDa in significant amounts. The 5-kDa fragment showed the original N-terminus and was eluted at a position of ∼12 kDa in molecular sieve chromatography, which indicated its trimeric organization (Sasaki et al., 1998). Thus, it was suggested that the trimerization domain in collagens XV and XVIII is located in the beginning of the NC1 domain. Independently, chemical cross-linking experiments on the recombinantly expressed human collagen XVIII NC1 domain also confirmed its trimeric nature (Kuo et al., 2001). Remarkably, the trimeric nature of the NC1 domain in the Caenorhabditis elegans multiplexin homologue was also demonstrated by chemical crosslinking (Ackley et al., 2001). Lastly, a set of the full-length NC1 domains and the endostatin domains of murine collagens XV and XVIII were expressed in 293 cells, purified, and analyzed on an analytical ultracentrifuge to determine their oligomeric states. The isolated endostatin domains of both collagens XV and XVIII were found to be monomers, while the full-length NC1 domains were trimers (Boudko et al., 2009). The proposed trimerization domain of murine collagen XVIII was successfully used to exogenously trimerize a single-chain antibody as part of a fusion molecule (Sanchez-Arevalo Lobo et al., 2006). Bacterially expressed constructs containing the proposed trimerization domain of human type XVIII collagen were subjected to trypsin digestion, which has lead to an identification of a trypsin-resistant non-collagenous trimeric fragment of ∼50 residues (Boudko et al., 2009). The fragment was remade recombinantly in significant amounts that allowed its biochemical analysis as well as the determination of the crystal structure (Boudko et al., 2009). Each subunit of the trimer has four ␤-strands, one ␣-helix, and a short 310 helix (Fig. 6). Four ␤-strands of each chain form a mixed parallel and antiparallel ␤-sheet, which faces the ␤-sheets of two adjacent chains. The side chains of all three ␤-sheets form a hydrophobic interior of the trimer. The ␣-helix and 310 helix are solvent exposed on one side and interact with all four ␤-strands of the same chain on the other site as well as with the C-terminal end of the adjacent chain. Two types of hydrophobic core are found in the structure. The first type forms an interface between the ␤-sheet and helices of each chain, which stabilizes the monomeric subunit, and the second type forms the central interior, which must be the driving force

26

S.P. Boudko et al. / The International Journal of Biochemistry & Cell Biology 44 (2012) 21–32

monomer or trimer hydrophobic core (Boudko et al., 2009). It now seems likely that this fold can accommodate different sequences with the conserved hydrophobic pattern. Both trimerization domains have very high association potentials; they form trimers at very low concentrations (56 and 119 pM half-transition concentrations for XVIII and XV, respectively) (Boudko et al., 2009; Wirz et al., 2011). 2.5. FACIT-collagens

Fig. 6. Structure of the collagen XVIII trimerization domain (PDB ID: 3HSH). (A) Cartoon representation of the trimerization domain viewed down the 3-fold rotation axis. (B) As in (A), but rotated by 90◦ about the horizontal axis. (C) The secondary elements topology diagram of the single chain. ␣-Helix (labeled with 1) and 310 -helix (labeled with 2) are shown as red cylinders, ␤-strands (labeled A–D) are shown as green arrows. Adapted from Boudko et al. (2009) with the permission.

for trimer formation. Two residues contribute to the hydrophobic core of the adjacent monomeric subunit and thus stabilize both the monomeric and trimeric structures. No solvent molecules are observed in either hydrophobic core. Interchain hydrogen bonds are also involved in establishing the trimer interface. The crystal structure of the trimerization domain of type XV collagen and its biochemical analysis is now also available (Wirz et al., 2011). Despite having only 32% sequence identity, the type XV structure is remarkably similar to that of type XVIII collagen. Strikingly, the trimerization domain of multiplexins from sea squirt to humans reveals little sequence homology, with the highest levels of homology involving key residues now known to be in the

The fibril-associated collagens with interrupted triple helices (FACITs) include types IX, XII, XIV, XVI, XIX, XX, XXI, and XXII. Collagen IX (Fig. 7) is a heterotrimer composed of three different ␣-chains, and all others are homotrimers. All ␣-chains are characterized by short collagenous domains interrupted by several non-collagenous domains (Myllyharju and Kivirikko, 2004; Ricard-Blum and Ruggiero, 2005). The FACITs have relatively short NC1 domains of varying length: 37 residues for human collagen XIV, fewer than 30 residues for human collagen IX, and even fewer than 20 residues for human collagen XIX. The FACITs share a remarkable sequence homology at their COL1/NC1 junctions by having two strictly conserved cysteine residues separated by four residues. These cysteines form interchain disulfide bonds, a so-called cystine knot, but only after the triple helix is formed (Mazzorana et al., 1993; Lesage et al., 1996; Mazzorana et al., 2001; Boudko et al., 2008). Earlier attempts to assign the trimerizing function to the NC1 domains were unsuccessful (Labourdette and van der Rest, 1993; Mechling et al., 1996). Initially, reassociation of chains of a pepsin-resistant low molecular weight fragment of bovine collagen IX was tested in vitro (Labourdette and van der Rest, 1993). The low molecular weight fragment includes the sequence of COL1 and the beginning of NC1 with intact disulfides. Upon reduction and reassociation, followed by the formation of disulfide-bonded multimers, only a negligible amount of ␣1␣2␣3 was observed. Another in vitro study was focused on either NC1 sequences or NC1 sequences extended with short fragments of COL1 (Mechling et al., 1996). Although experiments with just NC1 sequences did not produce any significant amount of multimers, the extended sequences were partially successful and yielded ∼10% of disulfide-bonded heterotrimeric ␣1␣2␣3. In other words, the NC1 domain cannot efficiently trimerize itself and requires exogenous alignment of three chains. It was pointed out that the second C-terminal non-collagenous domain (NC2) in different FACITs has a sequence pattern that is characteristic for the coiled coil structure, although with some irregularities (McAlinden et al., 2003) (Fig. 8). Coiled coils are often able to associate chains into different oligomers, including trimers (Moutevelis and Woolfson, 2009). The trimerizing potential of the NC2 domain in FACITs was originally demonstrated for collagen XIX (Boudko et al., 2008). The NC2 domain of homotrimeric collagen XIX is ∼50 residue long and forms trimers as observed by analytical centrifugation. The thermal stability of this domain was surprisingly high: no trimer dissociation was observed upon heating up to 90 ◦ C in phosphate buffer. The reversible heat denaturation was only achievable at the presence

Fig. 7. Domain organization of FACIT collagen IX. Three collagenous (COL1–COL3) and four non-collagenous (NC1–NC4) domains are numbered from the carboxyl terminus. It is the NC2 domain that has chain selection and heterotrimerization activity.

S.P. Boudko et al. / The International Journal of Biochemistry & Cell Biology 44 (2012) 21–32

27

Fig. 8. Sequence and putative coiled coil-like structure of the NC2 domains of collagens IX and XIX. Characteristic coiled coil-heptad repeats are labeled with a–g. Hydrophobic residues in positions a and d are highlighted. Original data can be found in McAlinden et al. (2003).

of significant amounts of guanidine hydrochloride. A collagen like sequence of (GPP)10 attached to one or the other end of the NC2 domain of collagen XIX formed the triple helical structure with significantly increased stability. The concentration independent melting temperature of the (GPP)10 part was either 68 or 55 ◦ C for N- or C-terminal location (compare it with 25 ◦ C of the isolated (GPP)10 peptide at 1 mg/ml concentration). Although the NC2 domain is naturally localized between two triple helical domains (COL1 and COL2) it has remarkably different effects on stabilizing the triple helix either N- or C-terminally. The weaker stabilizing effect on the C-terminal side can possibly be compensated by the cystine knot formation at the COL1/NC1 junction (Boudko et al., 2008). Collagen IX (Fig. 7) is the most intriguing FACIT collagen in terms of its chain selection and heterotrimerization properties. It also has the shortest sequences of the NC2 domain (∼35 residues). Chains ␣1 and ␣3, but not ␣2, have single cysteines (Fig. 8). In vitro mixing of all three individually expressed chains of the NC2 domain under reoxidative conditions results in a very efficient formation of a stable heterotrimer with chains ␣1 and ␣3 cross-linked by a disulfide bond (Boudko et al., 2010). The far UV CD spectrum of the heterotrimeric NC2 domain of collagen IX is very similar to that of the homotrimeric NC2 domain of collagen XIX (Boudko et al., 2008, 2010). Both spectra are characteristic of the high content of ␣-helices. This is again in accordance with the ␣-helical coiled coil predictions within NC2 domains of different FACITs (McAlinden et al., 2003) (Fig. 8). Nevertheless, it remains unclear whether these structures form the classical coiled coil or a new structure that can accommodate (or even facilitate) staggering of the collagen triple helix. Because only the ␣1 chain is disulfide-linked to the ␣3 chain and the ␣2 chain is free in the NC2 domain of collagen IX, the complex is in dissociation/association equilibrium and the dissociation constant can be calculated from the measured standard enthalpy and entropy (Boudko et al., 2010); it is in nM range (1–30 nM for 25–37 ◦ C temperature interval), which is considered to be as strong as good antigen/antibody binding. Of course it does not reflect the initial binding affinity of all three nascent chains, where the disulfide bonds have not yet formed. It also does not provide the information on how specific is the initial association and what role disulfide bond formation can play in establishing/locking the specific complex. Nevertheless, under reoxidative conditions the correct heterotrimeric complex is formed with a yield greater than 80% (Boudko et al., 2010). Small, effective and collagen-specific homo- (NC2 of collagen XIX) and hetero-trimerization (NC2 of collagen IX) domains of FACITs open the prospect of easy production of native collagen fragments even with chain composition control. 2.6. Beaded filament and anchoring collagens Type VI collagen is another network-forming molecule. It has a ubiquitous tissue distribution. In general, the chain composition

of the molecule is ␣1(VI)␣2(VI)␣3(VI). However, recently, three new ␣ chains of type VI were found in mice and two in humans (Fitzgerald et al., 2008; Gara et al., 2008). All the recently discovered chains are very similar to the ␣3(VI) chain. In the type VI collagen ␣ chains, the relatively short collagenous domain (∼330 residues) is flanked on both sides by one or few domains, which are similar to von Willebrand factor A (vWFA) domain. To build the collagen VI suprastructure, two trimeric molecules form a dimer, the dimers form a tetramer, and finally the tetramers associate end to end to yield a beaded string (Engel et al., 1985). Recombinant deletion studies have demonstrated that whereas the fifth C-terminal domain (C5) of the ␣3 chain is required for extracellular microfibril formation (Lamande et al., 2006), the first C-terminal domain (C1), in all chains, is sufficient for chain recognition and trimeric protomer assembly (Ball et al., 2001; Lamande et al., 2002). Specific clusters of vWFA domains were demonstrated by electron tomography (Baldock et al., 2003). Interestingly, putative coiledcoil motifs were found within the C1 vWFA domain (McAlinden et al., 2003). Because all known three-dimensional structures of vWFA domains conform to a Rossman type fold with internal ␤sheets and exposed ␣-helices, it was also hypothesized that these putative coiled-coil sequences would also be surface-located. In this way, three adjacent von Willebrand factor A domains might trimerize via coiled-coil interactions (McAlinden et al., 2003). So far no experimental evidence has been reported for this hypothesis. It is also worth mentioning here that there is one report on the trimeric solution structure of the vWFA domain of Factor B protein (Banham et al., 2006). The authors used the pulsed electron paramagnetic resonance (EPR) method of double electron electron resonance (DEER) to measure the distance between solvent-exposed cysteines 267 labeled with the nitroxide. The measured distance (6.15 nm) is in agreement with that in the crystal structure where trimers were observed (Bhattacharya et al., 2004). Notably, the trimer interface in this crystal packing is formed not through the coiled coil. So far it remains unclear in what way the trimerization occurs in the type VI collagen. Collagen VII is composed of three identical ␣ chains, each consisting of a 145-kDa central collagenous triple-helical domain flanked by a large 145-kDa amino-terminal, noncollagenous domain (NC1), and a small 34-kDa carboxyl-terminal noncollagenous domain (NC2) (Sakai et al., 1986). Within the extracellular space, collagen VII molecules form antiparallel, tailto-tail dimers stabilized by disulfide bonding through a small carboxyl-terminal NC2 overlap between two molecules. A portion of the NC2 domain is then proteolytically removed (BrucknerTuderman et al., 1995). The antiparallel dimers then aggregate laterally to form anchoring fibrils with large globular NC1 domains at both ends of the structure. NC1 domains have been suggested to interact at one end with basement membrane zone components, and at the other ends with type IV collagen in “anchoring plaques”. Gene defects in collagen VII cause dystrophic epidermolysis bullosa, a group of heritable mechano-bullous skin diseases characterized

28

S.P. Boudko et al. / The International Journal of Biochemistry & Cell Biology 44 (2012) 21–32

by skin fragility, separation of the epidermis from the dermis (blister formation), milia and scarring (Uitto and Christiano, 1994). Two regions were identified within collagen VII sequence with a potential to form a coiled coil structure and possibly trimerize molecules (McAlinden et al., 2003). One is located N-terminal to the triple-helical region within von Willebrand factor A domain. The other starts a non-triple-helical interruption (also called a hinge region) in the long collagenous domain and contains 3 heptad repeats. So far no experimental data are available to confirm their trimerization potential. The recombinant minicollagen VII that lacks 678 amino acids within the central collagenous domain (including the 39-residue hinge region) was shown to form a stable triple-helical conformation (Chen et al., 2000). It indicates that the hinge region is not essential for the formation and maintenance of a stable triple helix. In contrast, the minicollagen VII has the enhanced proteolytic stability over the authentic type VII collagen. The non-helical hinge region contains a site highly susceptible to degradation by proteases that can explain this phenomenon. On the other hand it was shown that the carboxyl-terminal NC2 and adjacent 186-amino acid of collagenous domains are sufficient for the triple-helical assembly of type VII collagen (Chen et al., 2001), which indicates the possible trimerizing role of the NC2 domain. Nevertheless, the exact region within the NC2 domain, which is responsible for the trimerization is not yet identified. 2.7. Minicollagens The smallest collagens known so far are the minicollagens with only 12–16 Gly-X-Y repeats in their triple helical segment. They are major constituents of the capsule wall of nematocysts in Cnidaria, an animal family which includes the freshwater polyp Hydra, corals and jellyfish. Nematocysts are explosive organelles used for capture of prey and for defense. A high osmotic pressure develops inside the capsules. Upon triggering of a mechanosensor long tubular structures are expelled with such a high speed, that they are able to penetrate into the tissue of the prey. Consequently the capsule walls have a very high mechanical strength, which is provided by a dense assembly of minicollagens and other capsule components. The family of minicollagens consists of 17 members in Hydra and considerable variance in the design of the proteins is observed (David et al., 2008). The best characterized member, minicollagen1, consists of a 12 nm long triple helix, flanked at both ends by polyproline or polyhydroxyproline stretches and terminal cysteine-rich domains (CRDs). The CRDs of minicollagen-1 are very small and show a conserved cysteine pattern CXXCXXXCXXXCXXXCC. The three-dimensional structure of the N- and C-terminal CRDs of minicollagen-1 were elucidated by NMR spectroscopy. Despite identical cysteine patterns, different disulfide bridges and significantly different structures were found for the C-terminal domain (Pokidysheva et al., 2004) and the N-terminal homolog (Milbradt et al., 2005; Meier et al., 2007). Meier et al. have shown that the N- and C-terminal CRDs are connected in sequence space by two mutations with a transition form having a single mutation sampling both structures in equilibrium. The isolated CRDs showed little potential to associate and they are probably not involved in trimerization of the three chains of minicollagen-1. Disulfide reshuffling is however a likely mechanism of association. No interchain disulfide bridges were found in minicollagen-1 but oxidative reshuffling of a fusion protein of minicollagen-1 led to high molecular weight aggregates (Özbek et al., 2002). It was speculated that a switch from intra- to intermolecular disulfide bonds leads to the transition of the capsule wall to its insoluble state with high tensile strength (Engel et al., 2001). The details of this mechanism have to be explored. It has to be considered that minicollagens may not only self-associate but also

interact with different minicollagens and other components of the nematocyst as non-collagenous capsule proteins share the CRDs with minicollagens (Özbek et al., 2004). 2.8. Trimerization domains with homology to those found in collagens Trimerization domains are not restricted to collagens but are also found in many other proteins. Several of these domains have a common origin as indicated by their homology. A striking example is the gC1q domain. It is the trimerization domain of collagens VIII and X (see section on network forming collagens) but also serves the same function in complement component C1q (Kishore and Reid, 1999) and other gC1q domain containing proteins including adipocyte complement-related protein ACPR30 (Shapiro and Scherer, 1998), EMILIN1 (Verdone et al., 2008, 2009) and multimerin (Hayward et al., 1995; Hayward and Kelton, 1995). The three-dimensional structures of gC1q domains in ACPR30 and EMILIN1 were solved. Their basic structure is very similar to that of the domain in collagen X (Fig. 7) but significant differences point to differences in functions. One of these differences, the Ca-cluster was mentioned in the section on network forming collagens and is unique to collagen X. In addition to its trimerization potential different other functions were assigned to the gC1q homologs in different proteins. In C1q six gC1q domains are attached to the C-termini of six collagen stalks, which are assembled to a six-fold microfibril at their N-terminal end. Thus the C1q molecule looks like a space-ferry. It recognizes cluster of IgG or IgM by interactions of the gC1q domains with the Fc-regions of the antibody molecules. In EMILIN1 the gC1q is not connected to a collagen segment. Its structure was also solved and revealed the unique feature of a surface loop binding to ␣4␤1 integrin. Finally it should be mentioned that the gC1q domains are homologous to members of the tumor necrosis factor family. A complete phylogenetic analysis for the gC1q domain is still missing but genomic data indicate that genes encoding only the globular domain gC1q are more ancient than larger proteins including gC1q and date back even to prokaryotes (Réty et al., 2005; Carland and Gerwick, 2010). A wide distribution in many different and unrelated proteins is also observed for the coiled-coil domains (Lupas and Gruber, 2005). In view of these data it would not be surprising if also other trimerization domains, initially discovered in collagens, would be found in non-collagenous proteins. Apparently collagens adopted trimerization domains from different sources in order to facilitate chain registration and folding. 3. The foldon trimer from phage fibritin as an experimental tool Recombinant expression of collagens and its fragments is often a big challenge due to several problems. First of all collagen biosynthesis requires specific post-translational enzymes, in particular prolyl 4-hydroxylase. 4-Hydroxyproline-deficient variants are markedly unstable already at room temperature. The second major problem is the necessity to align three collagen chains and thus prevent unaligned random annealing of chains, which leads to gelatin formation. The second problem becomes significant for longer collagen fragments with more than 10–20 tripeptide units. A natural way to stabilize collagen-like peptides would be the usage of one of the natural collagen trimerization domains. Until very recently, there were no good collagen-specific trimerization candidates with suitable properties, i.e. light weight, effective trimerizing potential, high expression level, solubility upon overexpression, no need for post-translational modifications, and potential expression

S.P. Boudko et al. / The International Journal of Biochemistry & Cell Biology 44 (2012) 21–32

in bacteria. Today such natural candidates are available, these are the trimerization domains of multiplexins (Boudko et al., 2009; Wirz et al., 2011) or FACITs (Boudko et al., 2008, 2010). A decade ago Frank et al. (2001) reported a method to stabilize collagen-like peptides by fusing them to the N terminus of the bacteriophage T4 fibritin foldon domain. Fibritin is a triplestranded, parallel, segmented ␣-helical coiled-coil protein, where the C-terminal globular domain (foldon) is required for correct trimerization and folding of the protein (Efimov et al., 1994). The foldon domain is rather small (∼30 residues) and has a ␤-propeller structure as determined by X-ray crystallography (Tao et al., 1997). Importantly, the foldon domain is capable of trimerization in the absence of the coiled-coil part of fibritin and actually can be used for exogenous trimerization of alien sequences (Letarov et al., 1999). The isolated foldon domain and the chimeric protein (GPP)10 foldon were overexpressed in a soluble form in Escherichia coli. It was known that single (GPP)10 chains are able to form a collagen triple helix of low stability (melting temperature 24 ◦ C at 20 ␮M chain concentration) with high concentration dependence. The stability of the collagen peptide fused to the N-terminus of foldon dramatically increased to 75 ◦ C and became concentration independent. Such high stability was achieved by the high intrinsic concentration of the C-terminal ends of collagen chains (∼1 M). This stabilization could also be explained in terms of entropy, i.e. the foldon domain substantially restricts the freedom of the unfolded collagen chains and aligns them. Moreover, the triple helix folding rate in (GPP)10 foldon was substantially (by two orders!) increased and demonstrated the concentration independent first order kinetics, as opposed to an apparent 2.5 order for free chains (Boudko et al., 2002). Such a dramatic difference is explained by an exogenous collagen triple helix nucleation by the trimeric foldon domain. Interestingly, effective nucleation was possible at both ends of the collagen-like peptide (GPP)10 , using designed proteins in which this peptide was fused either N- or C-terminal to a nucleation domain, T4-phage fibritin foldon (Frank et al., 2003) (also reviewed below). Very recently, the (GPP)10 foldon peptide was also used to study kinetic hysteresis in collagen folding-unfolding (Mizuno et al., 2010). The “foldon” technology was also successfully applied for a production of full-length collagen types I and III, where the natural 30-kDa C-propeptides bearing the trimerization function were replaced by 29-amino acid sequence of fibritin foldon (Pakkanen et al., 2003). The termini of the three chains in a foldon trimer do not match the staggered register of the collagen triple helix, which leads to a substantial kink between the helix and foldon seen in the crystal structure (Fig. 9) (Stetefeld et al., 2003). It is still unclear how nature accommodates the staggered collagen triple helix with their natural trimerization domains, for which only the 3-fold rotational symmetry was observed. During the last decade the “foldon” technology has expanded beyond the collagen field. The trimerization potential of fibritin foldon has been widely used to trimerize and stabilize a variety of other different fibrous and non-fibrous motifs and domains that are reviewed by Papanikolopoulou et al. (2008).

4. Use of trimerizing domains for biomaterials In polymer chemistry cross-linking agents are of major importance for the design of materials with special properties. This also holds for the design of biomaterials. The preparation of nanorods with the help of foldon (Papanikolopoulou et al., 2008) is an excellent example. A review on the building of biomaterials from ␣-helical and collagen-like peptides was published by Woolfson (2010). An interesting concept was proposed by Raman et al. (2006)

29

Fig. 9. Cartoon representation of the crystal structure of (GPP)10 foldon (PDB ID: 1NAY). The angle between the central axis of the triple-helical part and the 3-fold rotation axis of the foldon domain is 62.5◦ . Adapted from Stetefeld et al., 2003 with the permission.

in which regular polyhedral particles are formed by a combination of coiled-coil domains of different strand number. It is of particular interest that the collagen XVIII trimerization domain was used to develop a new system of multivalent antibodies, termed “trimerbodies” (Sanchez-Arevalo Lobo et al., 2006). The trimerbody is comprised of scFv fragments linked to the amino-terminal ends of the trimerization domain through a flexible linker. It has to be anticipated that many new developments will be seen in the near future.

5. Mutations in trimerization domains cause diseases Mutations in collagens lead to a large variety of inherited diseases of connective tissue. A subset of these mutations is localized in the trimerization domains. Mutations in the genes of type I collagen (COL1A1 and COL1A2) lead to Osteogenesis Imperfecta, a brittle bone disease with mild to lethal phenotypes. Several mutations in the carboxyl-terminal propeptide have been reported (Lamande et al., 1995; Pace et al., 2001; Chessler et al., 1993; Pace et al., 2008). As expected these mutations result in slow assembly of trimeric molecules and secretion of overmodified type I collagen molecules. Similar findings have been reported for type III collagen (COL3A1) where mutations in the carboxyl-terminal propeptide lead to Ehlers-Danlos Syndrome (Schwarze et al., 2001; Pope et al., 2010; Pickup and Pollanen, 2011) and in type II collagen (COL2A1) lead to Stickler syndrome (Ahmad et al., 1995). Mutations in the carboxyl-terminal propeptide of type V collagen (COL5A1 and COL5A2) were shown to lead to Ehlers-Danlos Syndrome (De Paepe et al., 1997; Mitchell et al., 2009) and spontaneous cervical artery dissection (Grond-Ginsbach et al., 2002). Schmid metaphyseal chondrodysplasia is caused by mutations in type X collagen (COL10A1). Almost all mutations are localized in the NC1 domain, either as missense mutations or mutations that introduce premature termination signals (Bateman et al., 2005). The missense mutations have been classified in three categories. Mutations that disturb the folding of the monomer, mutations that prevent trimerization and mutations that prevent the supramolecular assembly of type X collagen (Bateman et al., 2005; Wilson et al., 2005), all resulting in a functional haploinsufficiency of type X collagen.

30

S.P. Boudko et al. / The International Journal of Biochemistry & Cell Biology 44 (2012) 21–32

6. Summary and conclusions The formation of the collagen triple helix requires noncollagenous domains for chain selection, chain arrangement (chain stagger) and efficient folding. A single cell can synthesize several types of collagens and proper chain selection plays an important role in the biosynthesis of collagens. Types I, IV, V, VI, IX and XI collagens are heterotrimers composed of two or three different polypeptide chains. Because the three chains of the triple helix are staggered by one amino acid residue, there are three chain arrangements possible for a triple helix consisting of two different polypeptide chains or six chain arrangements for a triple helix consisting of three different polypeptide chains. Only limited experimental evidence is available for the chain stagger of collagens due to the size and shape of these molecules. For types I and IV collagens chain staggers have been reported (Golbik et al., 2000; Orgel et al., 2006), but for the other heterotrimeric collagens the stagger is unknown. The trimerization domains are likely to be responsible not only for chain selection but also for the selection of a specific stagger. The chain arrangement of homotrimeric collagens needs less specification and trimerization domains with three fold symmetry have been identified in these cases. The trimerization domains also increase the stability of the adjacent triple helix (Frank et al., 2001, 2003; Boudko et al., 2008, 2010). In experiments with trimerization domains linked to collagenous peptides, the stability is significantly increased and the increase depends on the location of the trimerization domain. If a given trimerization domain is attached at the carboxyl-terminal end of the collagenous peptide, a higher increase is observed than for the same domain attached to the amino-terminal end (Frank et al., 2003). The reason for this is not clear, but it could be a result of the prevalence of trimerization domains at the carboxyl-terminal end in natural collagens. However the rate of folding is not affected by the location of the trimerization domain (Frank et al., 2003). Trimerization domains increase the rate of folding of the triple helix by several orders of magnitudes mainly by eliminating concentration dependant association steps of the chains and misalignment of the chains. The size of trimerization domains varies from 35 residues in type IX collagen to 230 residues in type IV collagen and around 250 residues for the fibrillar collagens I, II, III, V, and XI. The presence of the larger trimerization domains in fibrillar collagens might also serve as an inhibitor of premature fibril formation. Effective fibril formation is achieved only after cleavage of the carboxyl-terminal propeptide. However, there is no correlation between the size and affinity of a trimerization domain and its involvement in hetero or homo trimerization. Very high affinities of trimerization domains are present in some collagen types that have low concentrations in the synthesizing cells. Trimerization domains are non-collagenous domains of very different structure. The term non-collagenous domain is often misleadingly interpreted as a uniform protein domain. In addition to their trimerization potential the domains may exhibit important additional and domain specific functions. Some examples of their role in the formation of multimolecular assemblies were mentioned. This field is much broader than described in the present review. Finally it may be speculated how collagens with trimerization domains evolved. The shortest exon encoding a collagen triple helix is 6 Gly-Xaa-Yaa repeats long (Exposito et al., 1993). This is the minimum length for forming a stable triple helix and short triple helical segments may have been formed in early proteins. For such short collagens folding and mismatching was no problem and even to-day minicollagens exist without the need for trimerization domains. As soon as many exons combined to longer collagens the formation of incorrectly folded products was disturbing and

fusion with a trimerization domain turned out to be a selective advantage. References Ackley BD, Crew JR, Elamaa H, Pihlajaniemi T, Kuo CJ, Kramer JM. The NC1/endostatin domain of Caenorhabditis elegans type XVIII collagen affects cell migration and axon guidance. J Cell Biol 2001;152:1219–32. Ahmad NN, Dimascio J, Knowlton RG, Tasman WS. Stickler syndrome. A mutation in the nonhelical 3 end of type II procollagen gene. Arch Ophthalmol 1995;113:1454–7. Amenta PS, Scivoletti NA, Newman MD, Sciancalepore JP, Li D, Myers JC. Proteoglycan-collagen XV in human tissues is seen linking banded collagen fibers subjacent to the basement membrane. J Histochem Cytochem 2005;53:165–76. Bächinger HP, Bruckner P, Timpl R, Engel J. The role of cis-trans isomerization of peptide bonds in the coil ↔ triple helix conversion of collagen. Eur J Biochem 1978;90:605–13. Baldock C, Sherratt MJ, Shuttleworth CA, Kielty CM. The supramolecular organization of collagen VI microfibrils. J Mol Biol 2003;330:297–307. Ball SG, Baldock C, Kielty CM, Shuttleworth CA. The role of the C1 and C2 a-domains in type VI collagen assembly. J Biol Chem 2001;276:7422–30. Banham JE, Timmel CR, Abbott RJ, Lea SM, Jeschke G. The characterization of weak protein–protein interactions: evidence from DEER for the trimerization of a von Willebrand Factor A domain in solution. Angew Chem Int Ed Engl 2006;45:1058–61. Bateman JF, Lamande SR, Dahl HH, Chan D, Mascara T, Cole WG. A frameshift mutation results in a truncated nonfunctional carboxyl-terminal pro alpha 1(I) propeptide of type I collagen in osteogenesis imperfecta. J Biol Chem 1989;264:10960–4. Bateman JF, Wilson R, Freddi S, Lamande SR, Savarirayan R. Mutations of COL10A1 in Schmid metaphyseal chondrodysplasia. Hum Mutat 2005;25:525–34. Bhattacharya AA, Lupher Jr ML, Staunton DE, Liddington RC. Crystal structure of the A domain from complement factor B reveals an integrin-like open conformation. Structure (Camb) 2004;12:371–8. Bogin O, Kvansakul M, Rom E, Singer J, Yayon A, Hohenester E. Insight into Schmid metaphyseal chondrodysplasia from the crystal structure of the collagen X NC1 domain trimer. Structure (Camb) 2002;10:165–73. Boudko S, Frank S, Kammerer RA, Stetefeld J, Schulthess T, Landwehr R, et al. Nucleation and propagation of the collagen triple helix in single-chain and trimerized peptides: transition from third to first order kinetics. J Mol Biol 2002;317:459–70. Boudko SP, Engel J, Bächinger HP. Trimerization and triple helix stabilization of the collagen XIX NC2 domain. J Biol Chem 2008;283:34345–51. Boudko SP, Sasaki T, Engel J, Lerch TF, Nix J, Chapman MS, et al. Crystal structure of human collagen XVIII trimerization domain: a novel collagen trimerization fold. J Mol Biol 2009;392:787–802. Boudko SP, Zientek KD, Vance J, Hacker JL, Engel J, Bächinger HP. The NC2 domain of collagen IX provides chain selection and heterotrimerization. J Biol Chem 2010;285:23721–31. Boutaud A, Borza DB, Bondar O, Gunwar S, Netzer KO, Singh N, et al. Type IV collagen of the glomerular basement membrane. Evidence that the chain specificity of network assembly is encoded by the noncollagenous NC1 domains. J Biol Chem 2000;275:30716–24. Bruckner-Tuderman L, Nilssen O, Zimmermann DR, Dours-Zimmermann MT, Kalinke DU, Gedde-Dahl Jr T, et al. Immunohistochemical and mutation analyses demonstrate that procollagen VII is processed to collagen VII through removal of the NC-2 domain. J Cell Biol 1995;131:551–9. Bruckner P, Bächinger HP, Timpl R, Engel J. Three conformationally distinct domains in the amino-terminal segment of type III procollagen and its rapid triple helix ↔ coil transition. Eur J Biochem 1978;90:595–603. Bruckner P, Prockop DJ. Proteolytic enzymes as probes for the triple-helical conformation of procollagen. Anal Biochem 1981;110:360–8. Bulleid NJ, Dalley JA, Lees JF. The C-propeptide domain of procollagen can be replaced with a transmembrane domain without affecting trimer formation or collagen triple helix folding during biosynthesis. EMBO J 1997;16:6694–701. Carland TM, Gerwick L. The C1q domain containing proteins: where do they come from and what do they do? Dev Comp Immunol 2010;34:785–90. Chen M, Keene DR, Costa FK, Tahk SH, Woodley DT. The carboxyl terminus of type VII collagen mediates antiparallel dimer formation and constitutes a new antigenic epitope for epidermolysis Bullosa acquisita autoantibodies. J Biol Chem 2001;276:21649–55. Chen M, O’Toole EA, Muellenhoff M, Medina E, Kasahara N, Woodley DT. Development and characterization of a recombinant truncated type VII collagen “minigene”. Implication for gene therapy of dystrophic epidermolysis bullosa. J Biol Chem 2000;275:24429–35. Chessler SD, Wallis GA, Byers PH. Mutations in the carboxyl-terminal propeptide of the pro alpha 1(I) chain of type I collagen result in defective chain association and produce lethal osteogenesis imperfecta. J Biol Chem 1993;268:18218–25. David CN, Özbek S, Adamczyk P, Meier S, Pauly B, Chapman J, et al. Evolution of complex structures: minicollagens shape the cnidarian nematocyst. Trends Genet 2008;24:431–8. Davis JM, Boswell BA, Bächinger HP. Thermal stability and folding of type IV procollagen and effect of peptidyl-prolyl cis-trans-isomerase on the folding of the triple helix. J Biol Chem 1989;264:8956–62.

S.P. Boudko et al. / The International Journal of Biochemistry & Cell Biology 44 (2012) 21–32 De Paepe A, Nuytinck L, Hausser I, Anton-Lamprecht I, Naeyaert JM. Mutations in the COL5A1 gene are causal in the Ehlers–Danlos syndromes I and II. Am J Hum Genet 1997;60:547–54. Dölz R, Engel J, Kühn K. Folding of collagen IV. Eur J Biochem 1988;178:357–66. Efimov VP, Nepluev IV, Sobolev BN, Zurabishvili TG, Schulthess T, Lustig A, et al. Fibritin encoded by bacteriophage T4 gene wac has a parallel triple-stranded alpha-helical coiled-coil structure. J Mol Biol 1994;242:470–86. Engel J, Furthmayr H, Odermatt E, von der Mark H, Aumailley M, Fleischmajer R, et al. Structure and macromolecular organization of type VI collagen. Ann N Y Acad Sci 1985;460:25–37. Engel J, Prockop DJ. The zipper-like folding of collagen triple helices and the effects of mutations that disrupt the zipper. Annu Rev Biophys Biophys Chem 1991;20:137–52. Engel U, Pertz O, Fauser C, Engel J, David CN, Holstein TW. A switch in disulfide linkage during minicollagen assembly in Hydra nematocysts. EMBO J 2001;20:3063–73. Exposito JY, van der Rest M, Garrone R. The complete intron/exon structure of Ephydatia mulleri fibrillar collagen gene suggests a mechanism for the evolution of an ancestral gene module. J Mol Evol 1993;37:254–9. Fitzgerald J, Rich C, Zhou FH, Hansen U. Three novel collagen VI chains, alpha4(VI), alpha5(VI), and alpha6(VI). J Biol Chem 2008;283:20170–80. Frank S, Boudko S, Mizuno K, Schulthess T, Engel J, Bächinger HP. Collagen triple helix formation can be nucleated at either end. J Biol Chem 2003;278:7747–50. Frank S, Kammerer RA, Mechling D, Schulthess T, Landwehr R, Bann J, et al. Stabilization of short collagen-like triple helices by protein engineering. J Mol Biol 2001;308:1081–9. Gaboriaud C, Juanhuix J, Gruez A, Lacroix M, Darnault C, Pignol D, et al. The crystal structure of the globular head of complement protein C1q provides a basis for its versatile recognition properties. J Biol Chem 2003;278:46974–82. Gara SK, Grumati P, Urciuolo A, Bonaldo P, Kobbe B, Koch M, et al. Three novel collagen VI chains with high homology to the alpha3 chain. J Biol Chem 2008;283:10658–70. Gibson GJ, Flint MH. Type X collagen synthesis by chick sternal cartilage and its relationship to endochondral development. J Cell Biol 1985;101:277–84. Golbik R, Eble JA, Ries A, Kühn K. The spatial orientation of the essential amino acid residues arginine and aspartate within the alpha1beta1 integrin recognition site of collagen IV has been resolved using fluorescence resonance energy transfer. J Mol Biol 2000;297:501–9. Grant WT, Wang GJ, Balian G. Type X collagen synthesis during endochondral ossification in fracture repair. J Biol Chem 1987;262:9844–9. Greenhill NS, Ruger BM, Hasan Q, Davis PF. The alpha1(VIII) and alpha2(VIII) collagen chains form two distinct homotrimeric proteins in vivo. Matrix Biol 2000;19:19–28. Grond-Ginsbach C, Wigger F, Morcher M, von Pein F, Grau A, Hausser I, et al. Sequence analysis of the COL5A2 gene in patients with spontaneous cervical artery dissections. Neurology 2002;58:1103–5. Halfter W, Dong S, Schurer B, Cole GJ. Collagen XVIII is a basement membrane heparan sulfate proteoglycan. J Biol Chem 1998;273:25404–12. Hayward CP, Hassell JA, Denomme GA, Rachubinski RA, Brown C, Kelton JG. The cDNA sequence of human endothelial cell multimerin. A unique protein with RGDS, coiled-coil, and epidermal growth factor-like domains and a carboxyl terminus similar to the globular domain of complement C1q and collagens type VIII and X. J Biol Chem 1995;270:18246–51. Hayward CP, Kelton JG. Multimerin. Curr Opin Hematol 1995;2:339–44. Hofmann H, Voss T, Kühn K, Engel J. Localization of flexible sites in thread-like molecules from electron micrographs. Comparison of interstitial, basement membrane and intima collagens. J Mol Biol 1984;172:325–43. Iozzo RV. The biology of the small leucine-rich proteoglycans. Functional network of interactive proteins. J Biol Chem 1999;274:18843–6. Khoshnoodi J, Sigmundsson K, Cartailler JP, Bondar O, Sundaramoorthy M, Hudson BG. Mechanism of chain selection in the assembly of collagen IV: a prominent role for the alpha2 chain. J Biol Chem 2006;281:6058–69. Kielty CM, Kwan AP, Holmes DF, Schor SL, Grant ME. Type X collagen, a product of hypertrophic chondrocytes. Biochem J 1985;227:545–54. Kishore U, Reid KB. Modular organization of proteins containing C1q-like globular domain. Immunopharmacology 1999;42:15–21. Kühn K. Basement membrane (type IV) collagen. Matrix Biol 1995;14:439–45. Kuo CJ, LaMontagne Jr KR, Garcia-Cardena G, Ackley BD, Kalman D, Park S, et al. Oligomerization-dependent regulation of motility and morphogenesis by the collagen XVIII NC1/endostatin domain. J Cell Biol 2001;152:1233–46. Kvansakul M, Bogin O, Hohenester E, Yayon A. Crystal structure of the collagen alpha1(VIII) NC1 trimer. Matrix Biol 2003;22:145–52. Kwan AP, Cummings CE, Chapman JA, Grant ME. Macromolecular organization of chicken type X collagen in vitro. J Cell Biol 1991;114:597–604. Labermeier U, Demlow TA, Kenney MC. Identification of collagens isolated from bovine Descemet’s membrane. Exp Eye Res 1983;37:225–37. Labourdette L, van der Rest M. Analysis of the role of the COL1 domain and its adjacent cysteine-containing sequence in the chain assembly of type IX collagen. FEBS Lett 1993;320:211–4. Lamande SR, Chessler SD, Golub SB, Byers PH, Chan D, Cole WG, et al. Endoplasmic reticulum-mediated quality control of type I collagen production by cells from osteogenesis imperfecta patients with mutations in the pro alpha 1 (I) chain carboxyl-terminal propeptide which impair subunit assembly. J Biol Chem 1995;270:8642–9. Lamande SR, Mörgelin M, Adams NE, Selan C, Allen JM. The C5 domain of the collagen VI alpha3(VI) chain is critical for extracellular microfibril

31

formation and is present in the extracellular matrix of cultured cells. J Biol Chem 2006;281:16607–14. Lamande SR, Mörgelin M, Selan C, Jobsis GJ, Baas F, Bateman JF. Kinked collagen VI tetramers and reduced microfibril formation as a result of Bethlem myopathy and introduced triple helical glycine mutations. J Biol Chem 2002;277:1949–56. Lesage A, Penin F, Geourjon C, Marion D, van der Rest M. Trimeric assembly and three-dimensional structure model of the FACIT collagen COL1-NC1 junction from CD and NMR analysis. Biochemistry 1996;35:9647–60. Letarov AV, Londer YY, Boudko SP, Mesyanzhinov VV. The carboxy-terminal domain initiates trimerization of bacteriophage T4 fibritin. Biochemistry (Mosc) 1999;64:817–23. Lupas AN, Gruber M. The structure of alpha-helical coiled coils. Adv Protein Chem 2005;70:37–78. Malone JP, Alvares K, Veis A. Structure and assembly of the heterotrimeric and homotrimeric C-propeptides of type I collagen: significance of the alpha2(I) chain. Biochemistry 2005;44:15269–79. Marneros AG, Olsen BR. Physiological role of collagen XVIII and endostatin. FASEB J 2005;19:716–28. Mazzorana M, Cogne S, Goldschmidt D, Aubert-Foucher E. Collagenous sequence governs the trimeric assembly of collagen XII. J Biol Chem 2001;276: 27989–98. Mazzorana M, Gruffat H, Sergeant A, van der Rest M. Mechanisms of collagen trimer formation. Construction and expression of a recombinant minigene in HeLa cells reveals a direct effect of prolyl hydroxylation on chain assembly of type XII collagen. J Biol Chem 1993;268:3029–32. McAlinden A, Smith TA, Sandell LJ, Ficheux D, Parry DA, Hulmes DJ. Alpha-helical coiled-coil oligomerization domains are almost ubiquitous in the collagen superfamily. J Biol Chem 2003;278:42200–7. Mechling DE, Gambee JE, Morris NP, Sakai LY, Keene DR, Mayne R, et al. Type IX collagen NC1 domain peptides can trimerize in vitro without forming a triple helix. J Biol Chem 1996;271:13781–5. Meier S, Jensen PR, Adamczyk P, Bächinger HP, Holstein TW, Engel J, et al. Sequencestructure and structure-function analysis in cysteine-rich domains forming the ultrastable nematocyst wall. J Mol Biol 2007;368:718–28. Milbradt AG, Boulegue C, Moroder L, Renner C. The two cysteine-rich head domains of minicollagen from Hydra nematocysts differ in their cystine framework and overall fold despite an identical cysteine sequence pattern. J Mol Biol 2005;354:591–600. Mitchell AL, Schwarze U, Jennings JF, Byers PH. Molecular mechanisms of classical Ehlers–Danlos syndrome (EDS). Hum Mutat 2009;30:995–1002. Mizuno K, Boudko SP, Engel J, Bächinger HP. Kinetic hysteresis in collagen folding. Biophys J 2010;98:3004–14. Moutevelis E, Woolfson DN. A periodic table of coiled-coil protein structures. J Mol Biol 2009;385:726–32. Muragaki Y, Jacenko O, Apte S, Mattei MG, Ninomiya Y, Olsen BR. The alpha 2(VIII) collagen gene. A novel member of the short chain collagen family located on the human chromosome 1. J Biol Chem 1991;266:7721–7. Myers JC, Kivirikko S, Gordon MK, Dion AS, Pihlajaniemi T. Identification of a previously unknown human collagen chain, alpha 1(XV), characterized by extensive interruptions in the triple-helical region. Proc Natl Acad Sci U S A 1992;89:10144–8. Myllyharju J, Kivirikko KI. Collagens, modifying enzymes and their mutations in humans, flies and worms. Trends Genet 2004;20:33–43. Myllyharju J, Lamberg A, Notbohm H, Fietzek PP, Pihlajaniemi T, Kivirikko KI. Expression of wild-type and modified proalpha chains of human type I procollagen in insect cells leads to the formation of stable [alpha1(I)]2alpha2(I) collagen heterotrimers and [alpha1(I)]3 homotrimers but not [alpha2(I)]3 homotrimers. J Biol Chem 1997;272:21824–30. Ninomiya Y, Gordon M, van der Rest M, Schmid T, Linsenmayer T, Olsen BR. The developmentally regulated type X collagen gene contains a long open reading frame without introns. J Biol Chem 1986;261:5041–50. Orgel JP, Irving TC, Miller A, Wess TJ. Microfibrillar structure of type I collagen in situ. Proc Natl Acad Sci U S A 2006;103:9001–5. Özbek S, Pertz O, Schwager M, Lustig A, Holstein T, Engel J. Structure/function relationships in the minicollagen of Hydra nematocysts. J Biol Chem 2002;277:49200–4. Özbek S, Pokidysheva E, Schwager M, Schulthess T, Tariq N, Barth D, et al. The glycoprotein NOWA and minicollagens are part of a disulfide-linked polymer that forms the cnidarian nematocyst wall. J Biol Chem 2004;279:52016–23. Pace JM, Kuslich CD, Willing MC, Byers PH. Disruption of one intra-chain disulphide bond in the carboxyl-terminal propeptide of the proalpha1(I) chain of type I procollagen permits slow assembly and secretion of overmodified, but stable procollagen trimers and results in mild osteogenesis imperfecta. J Med Genet 2001;38:443–9. Pace JM, Wiese M, Drenguis AS, Kuznetsova N, Leikin S, Schwarze U, et al. Defective C-propeptides of the proalpha2(I) chain of type I procollagen impede molecular assembly and result in osteogenesis imperfecta. J Biol Chem 2008;283:16061–7. Pakkanen O, Hamalainen ER, Kivirikko KI, Myllyharju J. Assembly of stable human type I and III collagen molecules from hydroxylated recombinant chains in the yeast Pichia pastoris. Effect of an engineered C-terminal oligomerization domain foldon. J Biol Chem 2003;278:32478–83. Papanikolopoulou K, van Raaij MJ, Mitraki A. Creation of hybrid nanorods from sequences of natural trimeric fibrous proteins using the fibritin trimerization motif. Methods Mol Biol 2008;474:15–33. Pickup MJ, Pollanen MS. Traumatic subarachnoid hemorrhage and the COL3A1 gene: emergence of a potential causal link. Forensic Sci Med Pathol 2011;7:192–7.

32

S.P. Boudko et al. / The International Journal of Biochemistry & Cell Biology 44 (2012) 21–32

Pihlajaniemi T, Rehn M. Two new collagen subgroups: membrane-associated collagens and types XV and XVII. Prog Nucleic Acid Res Mol Biol 1995;50:225–62. Pokidysheva E, Milbradt AG, Meier S, Renner C, Haussinger D, Bächinger HP, et al. The structure of the Cys-rich terminal domain of Hydra minicollagen, which is involved in disulfide networks of the nematocyst wall. J Biol Chem 2004;279:30395–401. Pope FM, Holden S, Ferguson DJP, Sawle P, Nesbitt M, Pollitt R, et al.Abstracts of the British Human Genetics Conference 2010. J Med Genet 2010;47(Suppl. 1):20. Raman S, Machaidze G, Lustig A, Aebi U, Burkhard P. Structure-based design of peptides that self-assemble into regular polyhedral nanoparticles. Nanomedicine 2006;2:95–102. Rehn M, Hintikka E, Pihlajaniemi T. Primary structure of the alpha 1 chain of mouse type XVIII collagen, partial structure of the corresponding gene, and comparison of the alpha 1(XVIII) chain with its homologue, the alpha 1(XV) collagen chain. J Biol Chem 1994;269:13929–35. Rehn M, Pihlajaniemi T. Alpha 1(XVIII), a collagen chain with frequent interruptions in the collagenous sequence, a distinct tissue distribution, and homology with type XV collagen. Proc Natl Acad Sci U S A 1994;91:4234–8. Ricard-Blum S, Ruggiero F. The collagen superfamily: from the extracellular matrix to the cell membrane. Pathol Biol (Paris) 2005;53:430–42. Risteli J, Bächinger HP, Engel J, Furthmayr H, Timpl R. 7-S collagen: characterization of an unusual basement membrane structure. Eur J Biochem 1980;108:239–50. Réty S, Salamitou S, Garcia-Verdugo I, Hulmes DJ, Le Hégarat F, Chaby R, et al. The crystal structure of the Bacillus anthracis spore surface protein BclA shows remarkable similarity to mammalian proteins. J Biol Chem 2005;280:43073–8. Sakai LY, Keene DR, Morris NP, Burgeson RE. Type VII collagen is a major structural component of anchoring fibrils. J Cell Biol 1986;103:1577–86. Sanchez-Arevalo Lobo VJ, Cuesta AM, Sanz L, Compte M, Garcia P, Prieto J, et al. Enhanced antiangiogenic therapy with antibody-collagen XVIII NC1 domain fusion proteins engineered to exploit matrix remodeling events. Int J Cancer 2006;119:455–62. Sasaki T, Fukai N, Mann K, Göhring W, Olsen BR, Timpl R. Structure, function and tissue forms of the C-terminal globular domain of collagen XVIII containing the angiogenesis inhibitor endostatin. EMBO J 1998;17:4249–56. Sasaki T, Larsson H, Tisi D, Claesson-Welsh L, Hohenester E, Timpl R. Endostatins derived from collagens XV and XVIII differ in structural and binding properties, tissue distribution and anti-angiogenic activity. J Mol Biol 2000;301:1179–90. Schmid TM, Linsenmayer TF. A short chain (pro)collagen from aged endochondral chondrocytes. Biochemical characterization. J Biol Chem 1983;258:9504–9. Schwarze U, Schievink WI, Petty E, Jaff MR, Babovic-Vuksanovic D, Cherry KJ, et al. Haploinsufficiency for one COL3A1 allele of type III procollagen results in a phenotype similar to the vascular form of Ehlers–Danlos syndrome, Ehlers–Danlos syndrome type IV. Am J Hum Genet 2001;69:989–1001. Shapiro L, Scherer PE. The crystal structure of a complement-1q family protein suggests an evolutionary link to tumor necrosis factor. Curr Biol 1998;8:335–8. Shuttleworth CA. Type VIII collagen. Int J Biochem Cell Biol 1997;29:1145–8. Stephan S, Sherratt MJ, Hodson N, Shuttleworth CA, Kielty CM. Expression and supramolecular assembly of recombinant alpha1(viii) and alpha2(viii) collagen homotrimers. J Biol Chem 2004;279:21469–77. Stetefeld J, Frank S, Jenny M, Schulthess T, Kammerer RA, Boudko S, et al. Collagen stabilization at atomic level. Crystal structure of designed (GlyProPro)(10)foldon. Structure (Camb) 2003;11:339–46.

Sundaramoorthy M, Meiyappan M, Todd P, Hudson BG. Crystal structure of NC1 domains. Structural basis for type IV collagen assembly in basement membranes. J Biol Chem 2002;277:31142–53. Tao Y, Strelkov SV, Mesyanzhinov VV, Rossmann MG. Structure of bacteriophage T4 fibritin: a segmented coiled coil and the role of the C-terminal domain. Structure (Camb) 1997;5:789–98. Than ME, Henrich S, Huber R, Ries A, Mann K, Kühn K, et al. The 1.9-A˚ crystal structure of the noncollagenous (NC1) domain of human placenta collagen IV shows stabilization via a novel type of covalent Met-Lys cross-link. Proc Natl Acad Sci U S A 2002;99:6607–12. Timpl R, Wiedemann H, van Delden V, Furthmayr H, Kühn K. A network model for the organization of type IV collagen molecules in basement membranes. Eur J Biochem 1981;120:203–11. Uitto J, Christiano AM. Molecular basis for the dystrophic forms of epidermolysis bullosa: mutations in the type VII collagen gene. Arch Dermatol Res 1994;287:16–22. Vanacore R, Ham AJ, Voehler M, Sanders CR, Conrads TP, Veenstra TD, et al. A sulfilimine bond identified in collagen IV. Science 2009;325:1230–4. Vanacore RM, Friedman DB, Ham AJ, Sundaramoorthy M, Hudson BG. Identification of S-hydroxylysyl-methionine as the covalent cross-link of the noncollagenous (NC1) hexamer of the alpha1alpha1alpha2 collagen IV network: a role for the post-translational modification of lysine 211 to hydroxylysine 211 in hexamer assembly. J Biol Chem 2005;280:29300–10. Verdone G, Corazza A, Colebrooke SA, Cicero D, Eliseo T, Boyd J, et al. NMR-based homology model for the solution structure of the C-terminal globular domain of EMILIN1. J Biomol NMR 2009;43:79–96. Verdone G, Doliana R, Corazza A, Colebrooke SA, Spessotto P, Bot S, et al. The solution structure of EMILIN1 globular C1q domain reveals a disordered insertion necessary for interaction with the alpha4beta1 integrin. J Biol Chem 2008;283:18947–56. von der Mark K, Kirsch T, Nerlich A, Kuss A, Weseloh G, Gluckert K, et al. Type X collagen synthesis in human osteoarthritic cartilage. Indication of chondrocyte hypertrophy. Arthritis Rheum 1992;35:806–11. Weber S, Dölz R, Timpl R, Fessler JH, Engel J. Reductive cleavage and reformation of the interchain and intrachain disulfide bonds in the globular hexameric domain NC1 involved in network assembly of basement membrane collagen (type IV). Eur J Biochem 1988;175:229–36. Wilson R, Freddi S, Chan D, Cheah KS, Bateman JF. Misfolding of collagen X chains harboring Schmid metaphyseal chondrodysplasia mutations results in aberrant disulfide bond formation, intracellular retention, and activation of the unfolded protein response. J Biol Chem 2005;280:15544–52. Wirz JA, Boudko SP, Lerch TF, Chapman MS, Bächinger HP. Crystal structure of the human collagen XV trimerization domain: a potent trimerizing unit common to multiplexin collagens. Matrix Biol 2011;30:9–15. Woolfson DN. Building fibrous biomaterials from alpha-helical and collagen-like coiled-coil peptides. Biopolymers 2010;94:118–27. Yurchenco PD, Schittny JC. Molecular architecture of basement membranes. FASEB J 1990;4:1577–90. Zhang Y, Chen Q. The noncollagenous domain 1 of type X collagen. A novel motif for trimer and higher order multimer formation without a triple helix. J Biol Chem 1999;274:22409–13.