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Alpha-helical coiled-coil oligomerization domains are almost ubiquitous in

the collagen superfamily*

Audrey McAlinden1, Thomasin A. Smith2, Linda J. Sandell1, Damien Ficheux3, David

A.D. Parry2 and David J.S. Hulmes3

1Department of Orthopedic Surgery, Washington University School of Medicine, Barnes-

Jewish Hospital, St Louis, MI 63110, USA; 2Institute of Fundamental Sciences, Massey

University, Palmerston North, New Zealand; 3Institut de Biologie et Chimie des Protéines,

CNRS UMR 5086, Université Claude Bernard Lyon 1, 69367 Lyon cedex 7, France.

Corresponding author :

Dr D.J.S. Hulmes, IBCP, 7 passage du Vercors, 69367 Lyon cedex 07, France.

Tel : (33) (0)4 72 72 26 67; Fax : (33) (0)4 72 72 26 04; e-mail : d.hulmes@ibcp.fr

Running Title : Coiled-coil domains in the collagen superfamily

*This work was supported by the CNRS (Programme “Protéomique et Ingeniérie des

Protéines”) and National Institutes of Health Grants AR48250-01 (to A.M.) and AR-36994 (to

L.S.).

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on August 14, 2003 as Manuscript M302429200 by guest on January 17, 2020

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Summary

Alpha-helical coiled-coils are widely occurring protein oligomerization motifs. Here we show

that most members of the collagen superfamily contain short, repeating heptad sequences

typical of coiled coils. Such sequences are found at the N-terminal ends of the C-propeptide

domains in all fibrillar procollagens. When fused C-terminal to a reporter molecule containing

a collagen-like sequence that does not spontaneously trimerize, the C-propeptide heptad

repeats induced trimerization. C-terminal heptad repeats were also found in the

oligomerization domains of the multiplexins (collagens XV and XVIII). N-terminal heptad

repeats are known to drive trimerization in transmembrane collagens, while fibril associated

collagens with interrupted triple helices (FACITs), as well as collagens VII, XIII, XXIII and

XXV, were found to contain heptad repeats between collagen domains. Finally, heptad repeats

were found in the von Willebrand factor A domains known to be involved in trimerization of

collagen VI, as well as in collagen VII. These observations suggest that coiled-coil

oligomerization domains are widely used in the assembly of collagens and collagen-like

proteins.

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Introduction

The mechanisms controlling chain oligomerization in extracellular matrix and related

proteins are currently topics of active research (1, 2). In the case of the collagen superfamily,

which includes both collagens and collagen-like proteins (3-5), a number of structural features

have been identified that are essential for bringing together component polypeptide chains

with the correct stoichiometry, thus leading to folding of the triple-helix. Early studies on the

procollagen precursors of the fibrillar collagens (types I, II, III, V and XI) (6) showed that the

C-propeptide regions are necessary to direct correct chain association, which is followed by

zipper-like folding of the triple-helix in the C- to N-terminal direction (7). This concept was

subsequently extended to basement membrane collagen type IV (8-10), microfibril forming

collagen VI (11, 12), members of the C1q family (13) including collagens VIII and X (14-17)

and the emilins (18), and the FACITs1 (19-21).

Alpha-helical coiled coils have been shown to be oligomerization domains in both

collagens and collagen-like proteins. The paradigm here is the collectin family (22), which

includes the lung surfactant proteins (SP-A, SP-D), mannan binding proteins (MBP-A, MBP-

C), conglutinin and collectin-43. Collectins are trimeric molecules in which each chain

contains a collagen-like domain followed by an α-helical coiled-coil region and then a

carbohydrate recognition domain (CRD). The amino acid sequence of the coiled-coil domain

is characterized by up to four heptad repeats (a-b-c-d-e-f-g) in which hydrophobic amino acid

residues occur at positions a and d (23). Three dimensional structures of the coiled-coil and

CRD regions in MBP and SP-D show that chains within the trimer are associated via a three-

stranded coiled coil, with the three CRDs arranged as distinct lobes (24-26). Several studies

have shown that the coiled-coil region is both necessary and sufficient for trimerization of SP-

D (27, 28). In a recent study (29), the first two heptad repeats of SP-D were shown to be

1 The abbreviations used are: CHO, chinese hamster ovary; CPII, C-propeptide region of procollagen II; CRD,carbohydrate recognition domain; FACITs, fibril associated collagens with interrupted triple-helices; SP-D,

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sufficient to drive trimerization and folding when fused C-terminal to the N-propeptide region

of procollagen IIA, which also contains a collagen triple-helix but which alone does not

spontaneously trimerize.

Coiled-coil oligomerization domains have also been shown to be essential for

trimerization and folding of the membrane associated collagens XIII (30, 31) and XVII (32).

Similar domains occur in collagen XXIII (33), the Alzheimer amyloid plaque component

precursor CLAC-P/collagen XXV (34) and the collagen-like membrane proteins MARCO and

ectodysplasin-A1 (31). In all of these proteins, the transmembrane domain is found close to

the N-terminus and is followed immediately by a heptad repeat region at the beginning of the

large ectodomain. Once trimerized via the coiled-coil domains, folding of the C-terminal

collagen helix then proceeds in the N- to C-terminal direction. N-terminal coiled-coil

oligomerization domains are thought to play similar roles in the assembly of macrophage

scavenger receptor proteins (35), which also contain collagen-like regions. Several coiled-coil

domains, as well as collagen-like regions, are also present in the emilins (36).

Here we show that putative α-helical coiled-coil oligomerization domains are present

in most members of the collagen superfamily, located either before, after or between collagen-

like regions, suggesting a general role in triple-helical assembly.

Experimental Procedures

Sequence analysis - All sequences were obtained from SWISSPROT, SPTREMBL or

REMTREMBL databases. Potential coiled-coil sequences in all polypeptide chains of the

collagen superfamily were searched initially using the program COILS2 (37). Since the

reliability of this computational approach is very limited, particularly for short (14 residue)

sequences, all "hits" (regardless of score) were also examined by informed eye in order to

surfactant protein-D.

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assess the likelihood of coiled coil formation. Experience has shown that this approach can

more easily recognize heptad discontinuities and individual or small groups of residues that

are generally disruptive to coiled-coil formation, hence allowing elimination of some regions

from further consideration.

Although sequences were analyzed for their potential to form either two or three-

stranded helices using SCORER (38), their very short lengths (2-3 heptads in general)

rendered the results statistically uncertain. The presence of three-chain structures in the

proteins reported here would nonetheless strongly favor the formation of three-stranded rather

two-stranded coiled-coils.

To test the plausibility of a three-stranded coiled-coil geometry for the heptad

containing region of type I procollagen C-propeptides, a model three-stranded coiled-coil was

built based on the homotrimeric GCN4-pII leucine zipper structure (PDB code 1gcm).

Appropriate residue replacements were made, placing the proα1 chains onto chains A and B

and the proα2 chain onto chain C of GCN4-pII, in such a way as to ensure the heptad repeat

patterns were in phase. The packing of the core residues was then checked manually using

Turbo-Frodo 5.5 (http://afmb.cnrs-mrs.fr/TURBO_FRODO), noting particularly the positions

of the a and d residues relative to those in the GCN4-pII structure. Automated energy

minimization was done in a multi-step process using conjugate gradients followed by

simulated annealing with CNSsolve 0.5 (39). In the conjugate gradient minimization, the

main-chain atoms were first held fixed followed by harmonically restraining them to their

initial positions with an energy constant of 10 kcal/mol.A2. Annealing using torsion dynamics

was employed with the main-chain atoms again harmonically restrained.

Synthesis of chimeric IIA N-pro/CPII cDNA fusion constructs - cDNA fusion constructs

encoding the full-length human type IIA procollagen N-propeptide linked to 18, 25, or 31

amino acids of the coiled-coil domain present at the beginning of the type II procollagen C-

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propeptide (IIA N-pro/CPII; Fig. 3) were synthesized by overlap extension PCR.

Oligonucleotide primers used for PCR and overlap extension PCR procedures were

synthesized by Invitrogen. One PCR was carried out to amplify type IIA N-propeptide cDNA

using primers 1 and 2 (Table II) for 30 cycles (95°C, 30s; 55°C, 30s; 72°C, 50s). Here, IIA N-

pro/SP-D cDNA in pGEM3Z was used as a substrate (28). Another set of PCRs was done to

amplify cDNA encoding the 18, 25 or 31 amino acid sequence of the C-propeptide (plus the

terminal alanine residue of the telopeptide at the procollagen C-proteinase cleavage site)

corresponding to amino acid numbers 1172-1189, 1172-1196 or 1172-1202, respectively

(Gene Bank Accession number X16468). Table II shows the sequence of the PCR primer

pairs used to amplify the cDNA fragments encoding each of the three C-propeptide regions.

Each of these PCRs was carried out for 30 cycles (95°C, 30s; 55°C, 30s; 72°C, 25s) and

oligonucleotides encoding amino acid residues 1172-1189, 1172-1196 or 1172-1202 of the C-

propeptide were used as substrates. Overlap extension PCRs were then prepared by mixing

cDNA encoding the IIA N-propeptide with cDNA encoding one of the three C-propeptide

cDNA fragments. To promote overlap extension, a preliminary PCR was carried out for 5

cycles in the absence of primers (95°C, 30s; 52°C, 30s; 72°C, 45s). Primer pairs were then

added to each PCR (Table II) and IIA N-pro/CPII cDNA fusion constructs were amplified for

30 cycles (95°C, 30s; 55°C, 30s; 72°C, 55s). cDNA fusion constructs were purified from 1%

agarose gels, digested with EcoRI and subcloned into pcDNA3 (Invitrogen). DNA

sequencing using the T7 and sp6 promoter primers confirmed correct orientation of the

inserted cDNA fragments.

Transient transfections - Chinese Hamster Ovary (CHO) cells were plated overnight in

6-well tissue culture dishes at a density of 3x105 cells/ml. Cells were transiently transfected

with pcDNA3 alone or pcDNA3 containing each of the three cDNA fusion constructs (IIA N-

pro/CPII aa 1172-1189/1196/1202) using FuGENE 6 reagent (Roche Molecular

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Biochemicals), in the presence of 50 µg/ml ascorbate. Cells transfected to synthesize full-

length IIA N-pro/SP-D fusion protein (28) or just the type IIA N-propeptide were included as

controls: IIA N-pro/SP-D forms trimers in solution while IIA N-pro alone remains

monomeric. Conditioned medium (12-36 ml per fusion construct) was harvested after 2 days

in culture and clarified by centrifugation. PMSF (1mM) was added to the medium and

proteins were precipitated overnight at 4°C with 33% ammonium sulfate. Precipitated

proteins were collected by centrifugation and washed three times in saturated ammonium

sulfate. Protein pellets were re-suspended in PBS (1-3 ml) and dialyzed overnight against

PBS at 4°C.

Chemical crosslinking and detection of chimeric fusion proteins - Covalent

crosslinking of fusion proteins was done using bis-(sulfosuccinimidyl)suberate (BS3; Pierce).

Increasing amounts of BS3 (0, 0.1, 0.5, 1 or 2mM final concentration) prepared in 5mM

sodium citrate, pH 5, were added to dialyzed conditioned medium for 1h at room temperature.

Addition of SDS-PAGE loading buffer containing Tris-HCl (0.5M) inhibited the reaction.

Samples were boiled for 5 min prior to SDS-PAGE which was carried out without sulfydryl

reduction. Proteins were transferred to nitrocellulose membranes by Western blotting and the

presence of cross-linked dimers or trimers was identified by immunolocalization using anti-

IIA polyclonal antisera (40).

Peptide analysis - Peptides corresponding to GCN4-pII (RMKQIEDKIEEILSKIYHI-

ENEIARIKKLIGER-NH2) and residues 1172-1189 (ADQAAGGLRQHDAEVDAT-NH2),

1172-1196 (ADQAAGGLRQHDAEVDATLKSLNNQ-NH2) and 1172-1202 (ADQAAGG-

LRQHDAEVDATLKSLNNQIESIRS-NH2) from human procollagen II were synthesized on

a Milligan 9050 instrument with Fmoc/DIC/HOBt chemistry. They were then cleaved using

TFA with classical scavengers and precipitated in diethyl ether, followed by centrifugation.

Pellets were then dissolved in water and lyophilized. The crude peptides were then re-

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dissolved in water and purified on a Vydac column (C18, 5 µm, 25 x 1 cm) with an

appropriate gradient of acetonitrile in 0.1 % TFA. They were then characterized by

electrospray mass spectrometry (SCIEX API 165) and by HPLC (HP1100) using an analytical

Vydac C18 column with a 30 min gradient from 7 % to 63 % acetonitrile.

Far UV (180 - 260 nm) circular dichroism measurements were carried out using

thermostated 0.2 mm path length quartz cells in a Jobin-Yvon CD6 instrument, calibrated with

aqueous d-10-camphorsulfonic acid. Peptides (200 µM - 400 µM) were analyzed at 25 0C in

20 mM KH2PO4/NaOH, 150 mM NaF, pH 7.2, in the presence or absence of 50 % TFE (41).

Spectra were measured with wavelength increment 0.2 nm, integration time 1 sec and

bandpass 2 nm. For cross-linking analysis with BS3, conditions were the same as those used

for the fusion proteins (see above), except that peptide concentrations were as for the circular

dichroism.

Results

Coiled coils in fibrillar procollagen C-propeptides - Fig. 1A shows an amino acid

sequence alignment of the C-propeptide domains of the human fibrillar procollagens. From

the pattern of repeating hydrophobic residues at positions a and d, and the frequent occurrence

of charged residues at positions e and g, it can be seen that there are up to four heptad repeats,

indicative of coiled-coils, beginning soon after the procollagen C-proteinase cleavage site. In

all chains, the g position at the end of the fourth heptad repeat is a proline which therefore

defines the end of the α-helical region. The extent of the heptad repeat pattern is similar to

that found in the collectins (Fig. 1B), again C-terminal to the collagenous region. This

suggests the existence of a coiled-coil structure near the N-terminus of all fibrillar C-

propeptide trimers. Putative coiled-coil regions were also found C-terminal to the triple helix

in the fibrillar procollagens XXIV (42) and XXVII (43, 44) (Fig. 1A).

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When the C-propeptide heptad repeats were further analyzed using SCORER (38), a

preference for three-stranded, as opposed to two-stranded, coiled coils was evident (data not

shown). Modeling of the three-dimensional structure of the type I procollagen C-propeptide

di-heptad sequence spanning the d positions of heptads two and four (Fig. 1A), based on that

adopted by the mutated form of the GCN4 leucine zipper (GCN4-pII, in which residues at the

a and d positions are isoleucines) (45) was consistent with a three-stranded coiled-coil

conformation free of steric clashes (Fig. 2). Possible ionic interactions stabilizing the structure

are listed in Table I. Based on this analysis, and in view of the recent observation that a di-

heptad repeat in SP-D is sufficient for trimerization (29), this suggests that the heptad repeats

in the fibrillar procollagen C-propeptides could also act as oligomerization domains.

Coiled coils in procollagens as oligomerization domains - In order to test whether the

heptad repeats in the procollagen C-propeptides can act as oligomerization domains, we

adopted the approach described by McAlinden et al (29) in which putative trimerization

domains were fused C-terminal to the type IIA procollagen N-propeptide and expressed in

CHO cells. The IIA N-propeptide contains a short, interrupted (GXY)n region (25 triplets)

which alone does not spontaneously trimerize. When fused to at least the first two heptad

repeats of the neck region of surfactant protein-D (SP-D), however, trimerization ensues, as

revealed by electrophoretic analysis after stabilization of trimers by cross-linking. Using the

same IIA N-propeptide as a trimerization reporter molecule, we made fusion constructs using

sequences from the C-propeptide of human type II procollagen, beginning at the alanine

residue in the procollagen C-proteinase cleavage site, up to the end of the second, third or

fourth heptad repeats (Fig. 3). As shown in Fig. 4A, positive controls with the IIA N-

propeptide fused to the entire C-terminal region of SP-D (beginning at the first heptad repeat)

readily formed dimers and trimers, as expected (29), while in the absence of an

oligomerization domain, only monomers were observed (Fig. 4B). In contrast, when the IIA

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N-propeptide was fused to the N-terminal sequence of the type II procollagen C-propeptide,

up to position f in the fourth heptad repeat, cross-linkable dimers and trimers were again

observed (Fig. 4C). Similar results were obtained with fusion constructs containing only the

first three (Fig. 4D) or two (Fig. 4E) heptad repeats.

The data obtained with the fusion constructs show that sequences from the N-terminal

region of the procollagen II C-propeptide can trimerize when linked to the IIA N-propeptide.

To determine whether these sequences are capable of trimerizing independently, the

corresponding peptides were synthesized along with GCN4-pII as a positive control. When

analyzed by circular dichroism (Fig. 5A), GCN4-pII gave a spectrum characteristic of an

almost fully α-helical coiled coil, as shown by the positions and heights of the peaks at 192,

208 and 222 nm, with a ratio of mean residue ellipticities [θ]220/[θ]208 of 1.039. In the presence

of 50 % TFE (Fig. 5B), which disrupts coiled coils and stabilizes α-helices (46), this ratio

decreased to 0.84, due mainly to changes in the peak at 208 nm, as expected. In contrast, all

three peptides from the N-terminal region of the procollagen II C-propeptide yielded spectra

characteristic of random coils, in the absence of TFE. Therefore these peptides do not form

stable coiled coils independently. In the presence of 50 % TFE, spectra clearly characteristic

of α-helices were obtained with the two longer peptides (residies 1172-1196 and 1172-1202),

while the shorter peptide (residues 1172-1189) showed some signs of α-helical structure.

Therefore all three peptides are capable of adopting an α-helical fold.

The results obtained by circular dichroism were confirmed by cross-linking analysis

using BS3, followed by SDS-PAGE, which showed trimerization of GCN4-pII but not of a

similar length peptide (residues 1172-1202) from the human procollagen II C-propeptide (data

not shown).

C-terminal coiled coils in other members of the collagen superfamily - Alpha-helical

coiled-coil heptad repeats were also found in the C-propeptides of known invertebrate fibrillar

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procollagens, including the sponge Ephydatia muelleri, hydra, annelids (alvinella, arenicola,

riftia), mollusc (abalone) and sea urchin (Fig. 1C) (47, 48). Both the length and distance of the

heptad repeats from the end of the collagen triple-helical region were similar to those in the

vertebrate fibrillar collagens (Fig. 1A), though in hydra and annelid fibrillar procollagens the

intervening sequence was relatively long (Fig. 1C). Similar C-terminally located heptad

sequences were also found in some bacteriophage tail fiber collagen-like proteins (data not

shown).

As pointed out by Beck and Brodsky (49), a possible coiled-coil oligomerization

domain C-terminal to the triple helix is also found in the collagen-like tail subunit of

acetylcholinesterase (ColQ) (50). The sequence (Fig. 1D) consists of 2.5 heptad repeats and is

found about 30 residues C-terminal to the last GPP triplet, similar to that in the procollagen C-

propeptides. Probable trimerization domains have also been identified in the multiplexins

(collagens XV and XVIII) (51, 52), immediately after the most C-terminal triple-helical

domain. Examination of the corresponding sequences showed that these also contain heptad

repeats indicative of coiled coils, albeit with a relative paucity of charged residues in positions

e and g (Fig. 1D). Finally, sequence analysis of the recently described collagen XXVI (53)

also revealed a putative coiled-coil region (31) in the C-terminal non triple-helical domain

(Fig. 1D). This consists of 3 perfect heptad repeats and is located a similar distance from the

end of the collagen triple-helical regions to those in ColQ and collagens XV and XVIII.

N-terminal coiled coils - Coiled-coil motifs N-terminal to collagen sequences have

been shown to be necessary for trimerization of the transmembrane collagens XIII (30, 31)

and XVII (32). These are shown in Fig. 1E, along with homologous sequences in collagen

XXIII (33), CLAC-P/collagen XXV (34), MARCO and ectodysplasin-A. Following the

suggestion of Balding et al (54), the heptad repeat is shown extending into the transmembrane

region.

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Coiled coils N-terminal to collagen triple-helices have also been described in the

scavenger receptors (35, 55) and the emilins (36). To add to this sub-group, sequence analysis

revealed a perfect 3 heptad substructure (31) about 45 residues N-terminal to the first triple-

helical domain in the recently described collagen XXVI (Fig. 1F) (53). Finally, N-terminally

located heptad repeats were found in c. elegans cuticle collagens as well as streptococcus and

bacteriophage tail fiber collagen-like proteins (data not shown).

Inter triple-helical domain coiled coils - Putative coiled-coil oligomerization domains

were also found in the fibril associated collagens with interrupted triple-helices (FACITs).

Unlike the previous examples, however, these were found between (Gly-X-Y)n regions, in the

NC2 domain, between triple-helical domains COL1 and COL2 (Fig. 1G). A further

distinguishing feature is that the coiled-coil regions in FACITs are discontinuous, consisting

of two partially overlapping di-heptad repeats.

Coiled-coil sequences separating triple-helical regions were also found in collagens

VII, XIII, XXIII and XXV (Fig. 1H,I). The NC3 domains of collagens XIII and XXV, which

are relatively highly conserved, consist of 2.5 heptad repeats, while that of collagen XXIII is

much shorter (31). In collagen VII, the non triple-helical interruption in the long collagen

domain contains 3 heptad repeats.

Intra-domain coiled coils - Finally, putative coiled-coil motifs were also found within

von Willebrand factor A (VWA) domains. These occur N-terminal to the triple-helical region

in collagen VII, and C-terminal to the triple-helical regions in all three chains of collagen VI,

in the latter in domains known be required for trimerization (11, 12). The corresponding

sequence alignment is shown in Fig. 1J. Secondary structure prediction on the NPS@ server

(56); www.ibcp.fr) indicated an α-helical conformation for all these sequences. Since all

known three-dimensional structures of VWA domains (57) conform to a Rossman type fold

with internal β-sheets and exposed α-helices, we predict that these putative coiled-coil

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sequences in collagens VI and VII would also be surface located. In this way, three adjacent

VWA domains might trimerize via coiled-coil interactions.

Discussion

Here we show that most members of the collagen superfamily, including collagen

triple-helix containing proteins not normally classified as collagens, contain short, 2-4 heptad

repeat α-helical coiled-coil domains. These include all fibrillar procollagens (types I, II, III, V,

XI, XXIV and XXVII), all FACITs (collagens IX, XII, XIV, XIX, XX and XXIII), all

transmembrane collagens (types XIII, XVII, XXIII and XXV, MARCO, ectodysplasin,

scavenger receptors), the collectins, the multiplexins (collagens XV and XVIII), the emilins,

the collagen-like tail subunit of acetylcholinesterase, and collagens VI, VII and XXVI. The

only exceptions were basement membrane collagen IV, collagen XXII, the ficolins (58) and

non-emilin members of the C1q family. The widespread occurrence of these domains suggests

a general role in the assembly of collagen triple helices.

Cooperativity between collagen-like and coiled-coil domains - The observation that the

heptad repeat containing peptides from the N-terminal region of the procollagen II C-

propeptide do not form α-helical coiled coils independently shows that trimerization is a

cooperative process involving both coiled-coil and collagen-like regions. The absence of

coiled-coil formation with the isolated peptides is not surprising, as it is generally agreed that

the minimum length for the formation of a coiled-coil as an autonomously folding unit is three

heptad repeats or 21-23 residues (46). Even the longest peptide examined (residues 1172-

1202) contains only two perfect heptad repeats, given the presence of the aspartate residue at

position a in heptad repeat 2 (Fig. 1A). In addition to the collagen-like region, cooperativity

probably also exists between folding of the coiled-coil region and of the rest of the C-

propeptide (see below).

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Implications for the structures of procollagen C-propeptide regions - These

observations give insights into the three-dimensional structure of the procollagen C-

propeptides. Recent studies using low angle X-ray scattering have shown that the low-

resolution structure of the isolated C-propeptide trimer from type III procollagen consists of

three major lobes together with one minor lobe (59). We hypothesized that each major lobe

corresponds to the loop containing the intra-chain disulfide bonds in each of the three

component chains, while the minor lobe corresponds to a putative junction region. Here we

show that the junction region most likely begins with a three-stranded coiled coil. The three-

dimensional structure of the C-propeptide trimer therefore shows some similarities to the C-

terminal regions of the collectins (24-26), where the CRD domains of each of the three chains

also appear as independent lobes, connected by a coiled-coil junction region.

Coiled-coils and procollagen assembly - What is the role of the coiled-coil region in

fibrillar procollagen chain trimerization? The C-propeptides of all the fibrillar procollagen

chains (each ~250 residues in length) are highly conserved throughout most of their length (4,

60). Early observations on naturally occurring mutants of type I procollagen have shown the

importance of the C-propeptides in trimerization (61). Frameshift mutations resulting in

deletion and replacement of sequences C-terminal to the coiled-coil regions in both the

proα1(I) and proα2(I) chains either prevent or delay trimerization (62-64). More recently, by

site directed mutagenesis, it has been shown (65) that the presence of the last ten amino acids

in the C-propeptide of the proα2(I) chain is essential for formation of the [proα1(I)]2proα2(I)

heterotrimer. These results and the experiments of Bulleid and colleagues which resulted in

the identification of the “molecular recognition sequence” (66) approximately half way

through the C-propeptide sequence suggest that the coiled-coil region is not sufficient for

trimerization.

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In contrast, the results reported here, using the procollagen IIA N-propeptide fused to

the beginning of the procollagen II C-propeptide domain, show that the heptad repeat

containing region of the latter is indeed sufficient to induce trimerization. Previous results

with IIA N-pro fused to similar heptad repeats in SP-D have shown that these are also

sufficient to induce correct folding of the IIA N-pro triple helix (29). These results are

analogous to those of Bulleid et al (67) who showed that procollagen III molecules continue to

trimerize when the C-propeptides are replaced by the 30-residue transmembrane domain of

influenza virus haemagglutinin. We therefore suggest that the C-propeptide coiled-coil region

alone would be sufficient to drive trimerization of fibrillar procollagen molecules lacking the

rest of the C-propeptide. When followed by the remainder of the C-propeptide sequence,

however, the coiled coil might be de-stabilized by unfavorable interactions associated with

incorrect chain stoichiometry or the presence of mutations. Only when chains assemble with

the correct stoichiometry, and in the absence of unfavorable mutations, would formation of the

coiled-coil region be possible, leading to nucleation and folding of the adjacent collagen-triple

helix. We predict that mutations within the coiled-coil region would prevent trimerization,

especially if these were to occur at the a and d, or possibly e and g, positions. No such

mutations have been described.

Heterotrimerization versus homotrimerization of fibrillar collagen α2 chains -

Detailed examination of the beginning of the heptad repeat region of the procollagen C-

propeptides reveals a possible explanation for the known tendency of α2 chains (in types I, V

and XII collagens) not to form heterotrimers. As shown in Fig. 1A, these sequences are

distinguished from all fibrillar procollagen α1 chains by the presence of proline residues

within the first or second heptad repeat. Since proline residues cannot be incorporated into α-

helices, other than in the first turn, the presence of prolines is likely to impede homotrimer

assembly of α2 chains, thus favoring the assembly of heterotrimers in the presence of α1

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chains. The fact that Lees et al (66) were able to obtain homotrimerization of proα2(I) chains,

after having replaced their "molecular recognition sequence" by that of the proα1(III) chain,

shows however that homotrimerization of α2(I) chains is possible, when α1(I) chains are

absent.

Inter triple-helical coiled coils - The presence of discontinuous heptad repeats in the

inter triple-helical NC2 domain of FACITs was surprising and unusual. Skips and stutters,

corresponding to an insertion of one residue and a deletion of three residues, respectively, in

an otherwise continuous heptad repeat have been described in a number of coiled-coil

structures (68). In this case, however, there is an effective deletion of two residues between

the overlapping heptad repeats. This is uncommon, and suggests either that there is a major

perturbation of the structure at the site of the two-residue deletion, or that one of the heptad

sequences dominates at the expense of the other. We suggest that these coiled-coil regions in

the NC2 domains of the FACITs may be involved in trimerization and subsequent folding of

the adjacent collagenous domains, in addition to the NC1 domains and adjoining

hydroxyproline rich (GXY)n repeats (19-21). A similar conclusion has recently been reached

in the case of the coiled-coil region in the NC3 domain of collagen XIII (31), where it was

shown that the adjacent COL2 and COL3 regions can fold even when the N-terminal coiled

coil is absent.

Conclusion - Heptad repeat sequences were not found in collagen IV nor in the non-

emilin members of the C1q family which includes collagens VIII and X. In the case of type IV

collagen, chain association is assured by the C-terminal non-triple helical domains (NC1,

~230 residues), each of which consists of two homologous sub-domains. These associate to

form trimers, consisting predominantly of β–strands, with a novel protein fold (9, 10). In

members of the C1q family, trimerization is assured by the C-terminal C1q domain (~160

residues), which assembles to form a tightly packed β-sandwich structure related to the tumor

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necrosis factor fold (17). Thus different members of the collagen superfamily appear to have

evolved three principal structural mechanisms to ensure chain trimerization in molecular

assembly, either the type IV collagen NC1 domain, the C1q domain or, as suggested here, the

α-helical coiled coil.

Acknowledgements - We thank E. Crouch, J.-Y. Exposito and F. Ruggiero for

discussions, and M. Gordon for sharing the sequences of collagen XXIII and XIV prior to

publication. The paper describing coiled-coil sequences in collagens XIII, XXIII and XXVI

(31) was published while this manuscript was in the process of revision.

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Figure Legends

Figure 1. Putative α-helical coiled-coil domains in members of the collagen superfamily.

In all sequences, hydrophobic residues in the a and d positions of the coiled-coil heptad

repeats are highlighted. (A) Alignment of the fibrillar procollagen chains, starting with the

final GPP triplet near the end of the main triple-helical region (shown in bold) and including

the first ~40 residues of the C-propeptides. Known procollagen C-proteinase cleavage sites are

underlined. Heptad repeats 1-4 are indicated (above), as are proline residues (white on black)

which serve to demarcate the coiled-coil region. Also shown is the sequence of GCN4-pII,

showing the numbering system used in Table I. (B) Alignment of the collectins, showing ~50

residues beginning near the end of the collagen-like region. (C) Alignment of invertebrate

fibrillar procollagen chains, starting with the final GPP triplet near the end of the main triple-

helical region and including the beginning of the C-propeptides. (D) Partial sequence of the

human collagen-like tail subunit of acetylcholinesterase (ColQ) beginning near the end of the

collagen-like region. Also shown is an alignment of the putative trimerization domains in

collagens XV and XVIII, beginning near the end of the most C-terminal (GXY)n region, as

well as an equivalent region in collagen XXVI. (E) Alignment of transmembrane collagens in

the region of the putative transmembrane domain (outlined). The (GXY)n regions begin at

least 57 residues C-terminal to the transmembrane regions. (F) Sequence of collagen XXVI N-

terminal to the first (GXY)n region (COL1), part of which is shown (in bold). (G) Alignment

of NC2 domains in the FACITs, showing also four GXY triplets in adjacent COL1 and COL2

domains. The discontinuous heptad repeat is indicated. (H) Alignment of NC3 domains in

collagens XIII, XXIII and XXV, showing also four GXY triplets in adjacent COL2 and COL3

domains. (I) The triple-helical interruption in collagen VII, with four triplets from adjacent

(GXY)n regions in bold. (J) Heptad repeats in von Willebrand factor A domains in collagens

VII (N-terminal to the (GXY)n region) and VI (C-terminal to the (GXY)n region). Sequences

are identified by name or collagen type/alpha chain, followed by species (ab=abalone,

al=alvinella, ar=arenicola, rf=riftia, b=bovine, c=chicken, h=human, m=mouse, hy=hydra,

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sp=sponge, su=sea urchin) then residue positions from the start of translation (where

available) .

Figure 2. Structural model of the heptad containing region of the type I procollagen C-

propeptide trimer based on the GCN4-pII leucine zipper structure. The three-stranded

coiled coil is shown in stereo view, with residues in positions a and d in red. The structure

includes two α1(I) chains and one α2(I) chain spanning the region between the d positions in

heptads two and four (Fig. 1A).

Figure 3. Amino acid sequences of coiled-coil regions attached to the full-length type

IIA procollagen N-propeptide. Fusion proteins were synthesised containing the human IIA

N-propeptide fused to 18, 25 or 31 amino acids of the coiled-coil region from the human type

II procollagen C-propeptide (IIA N-pro/CPII amino acids 1172-1189; 1172-1196 or 1172-

1202, respectively). As a positive control to demonstrate trimerization, IIA N-pro/SP-D

fusion protein was also synthesised which consists of the IIA N-propeptide attached to the

coiled-coil neck domain and carbohydrate recognition domain (CRD) of rat surfactant protein

D. IIA N-propeptide alone was also synthesised as a negative control. The C-terminal

sequence of IIA N-pro is shown, beginning at the end of the short triple-helical region which

contains 25 GXY repeats.

Figure 4. Trimerization of the type IIA procollagen N-propeptide (IIA N-pro) by coiled-

coil regions of the type II procollagen C-propeptide (CPII). Chimeric fusion proteins were

precipitated from conditioned CHO cell culture media after transient transfections, then re-

suspended and chemically cross-linked with increasing concentrations (0-2 mM) of BS3.

Monomers (M) and cross-linked dimers (D) and trimers (T) were detected by SDS-PAGE,

Western blotting and immunolabelling using anti-IIA polyclonal antisera. (A) IIA N-pro/SP-D

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(IIA N-pro attached to the coiled-coil neck domain and carbohydrate recognition domain

(CRD) of lung surfactant protein-D) (29), positive trimerization control. (B) IIA N-pro alone,

negative control. (C-E) IIA N-pro fused to sequences at the beginning of CPII (plus the

terminal alanine residue in the telopeptide) containing approximately (C) four (aa 1172-1202)

(D) three (aa 1172-1196) or (E) two (aa 1172-1189) heptad repeats (Figs. 1A and 3).

Figure 5. Analysis of peptides by circular dichroism. Peptides corresponding to GCN4-pII,

and residues 1172-1189, 1172-1196 and 1172-1202 in human procollagen II were analyzed at

25 °C in 20 mM KH2PO4/NaOH, 150 mM NaF, pH 7.2, in the concentration range 200 to 400

µM, in (A) the absence and (B) the presence of 50 % TFE. Data are expressed as mean residue

ellipticities [θ]MRW.

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Table I. Possible interactions between charged amino acid residues in the putative coiled

regions of the human fibrillar procollagen C-propeptides. Both inter-chain (between positions

g and e) and intra-chain (separated by 3 or 4 residues) interactions are indicated. Residues are

identified according to the GCN4-pII sequence shown in Fig. 1A.

i → i+3 i → i+4 g → eproα1(I) E15 - R18 D4 - K8proα2(I) D4 - K8proα1(II) E15 - R18 D4 - K8proα1(III)proα1(V) E15 - K18 K11 - E15

E15 - R19E13 - K18

proα2(V) E15 - R18proα1(XI) E15 - K18 K11 - E15 D13 - K18proα2(XI) D8 - R11

E15 - R18R12 - E16E15 - R19

E13 - R18

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Table II. Oligonucleotide primers used for amplification of cDNA encoding the type IIA N-

propeptide (IIA N-pro) or three regions of the type II procollagen C-propeptide domain

(CPII). The three CPII fragments amplified encode the coiled-coil region corresponding to

amino acid numbers 1172-1198, 1172-1196 or 1172-1202 (amino acid numbers were

obtained from the published sequence: Gene Bank Accession number X16468). Primer

pairs used for overlap extension PCR to synthesise each of the three fusion constructs (IIA N-

pro/CPII aa 1172-1189/1196/1202) are shown. F=forward primer, R=reverse primer. EcoRI

sites are highlighted in bold. Double underlined sequence in primer 2 corresponds to the most

5’ region of the CPII coiled-coil domain. Underlined sequence in primer 3 corresponds to the

most 3’ region of the IIA N-propeptide.

cDNA Primer pairs for PCR (5’-3’)

IIA N-propeptide F 1: ggtacgaattcatgattcgcctcgggR 2: tgcctggtcggccattggtccttgcat

CPII (aa 1172-1189) F 3: aggaccaatggccgaccaggcagcagccR 4: catgggaattctcatcatgtggcatccacctc

CPII (aa 1172-1196) F 3: aggaccaatggccgaccaggcagcagccR 5: catgggaattctcatcactggttgttgaggga

CPII (aa 1172-1202) F 3: aggaccaatggccgaccaggcagcagccR 6: catgggaattctcatcagctgcggatgctctc

Fusion cDNA construct Primer pairs for overlap extension PCR

IIA N-pro/CPII (aa 1172-1189) 1 + 4IIA N-pro/CPII (aa 1172-1196) 1 + 5IIA N-pro/CPII (aa 1172-1202) 1 + 6

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| hep1 | hep2 | hep3 | hep4Ia1_h(1190-1259) GPPSAGFD-FSFLP-QPPQEKAHDGGRYYRADD------ NV RDRDLE DTT KS SQQ EN RS EGSRKNPARTCIIa1_h(1143-1214) GPPGPGID-MSAFAGLGPREKGPDPLQYMRADQA----- GG RQHDAE DAT KS NNQ ES RS EGSRKNPARTCIa2_h(1099-1163) GPPGVSGGG---------YDFGYDG-DFYRADQPRS---A SLR KDYE DAT KS NNQ ET LT EGSRKNPARTCIIIa1_h(1191-1262) GAPGPCCGGVGAAAIAGIGGEKAGGFAPYYGDE------ MD KINTDE MTS KS NGQ ES IS DGSRKNPARNCVa2_h(1221-1293) GPPGHLTAALGDIM-GHYDESMPDPLPEFTEDQAA---- DDKNKTD G HAT KS SSQ ET RS DGSKKHPARTCVa1_h(1567-1639) GPPGEVIQP-LPIQASR-TRRNIDASQLLDDG---NGEN VD ADG EE FGS NS KLE EQ KR LGTQQNPARTCXIa1_h(1537-1607) GPPGEVIQP-LPILSSKKTRRHTEGMQADAD------DN LD SDG EE FGS NS KQD EH KF MGTQTNPARTCXIa2_h(1495-1571) GPPGEVIQP-LPIQMPKKTRRSVDGSRLMQEDEAIPTGGA GS GG EE FGS DS REE EQ RR TGTQDSPARTCXXIVa1_h(1471-1545) GPPGAPGPRKQMDINAAIQALIESNTALQMESYQNT---EVT IDHSEE FKT NY SNL HS KN LGTRDNPARICXXVIIa1_h(1616-1859) GPPGGPIQLQQDDLGAAFQTWMDTSGALRPESYSYP---DRL LDQGGE FKT HY SNL QS KT LGTKENPARVC abcdefgabcdefgabcdefgabcdefgabcdefgaGCN4-pII R KQ EDK EE LSK YH ENE AR KKL GER | | | | | 0 7 14 21 28

B conglutinin_b(178-220) ---GLAE NA KQR TI DGH RR QNA SQ KKA LFP-DGQAVGEcollectin43_b(160-205) GETSVLE DT RQR RN EGE QR QNI TQ RKA LFP-DGQAVGESP-D_h(220-262) ---GLPD AS RQQ EA QGQ QH QAA SQ KKVELFP-NGQSVGEMBP_h(98-136) -------GKSPDGDSSL ASERKA QTE AR KKW TFS-LGKQVGNSP-A_h(98-136 --------GLPAHLDEE QAT HD RHQ LQTRGA SLQGSIMTVGECL-L1_h(116-157) --GTVCDCGRYRKF GQ DIS AR KTS KF KNV AGI---RETEE abcdefgabcdefgabcdefgabcdefga

C a1_su(1155-1231) GPPGQVQSSYGVRYPSFQSGG-KGQGSSPYGYAYRDDSKNDA KIQDTE LGA SA GQQ EL KAPQGKAKTNPARSCa2_su(2929-3009) GPPGEVSMAAMPRMPQQQ----QSKGPSQYSHYYRDEIPKTVEQLDRTQ QIY AK ESE LS IEPLG-SRDQPIRSChd1a_ab(1126-1187) GPPG-----YGPVYSPQPSWN-KGP-----DPYQYDEPE------GGMA YEN NR REA VR GHSRLGSRTSPGKNChd2a_ab(1170-1238) GPPGES--VYGRAMTGWATGS-KGPGYMGDVPSAEGEPE------E RN IKA KD EEE KK RDPTG-TKDAPGRTCemf1_sp GPPGPT--GGGIILVPVNDQN-PTRSPVSGSVFYRGQAE---ETDVNLGSVAD IE HKK QH KSPTG-TKDSPARSCfam1a_ar GPPGSS--SYGGDY..(19 aa)..MGDDPKA--TGRVRSKEDLKKDEN FEA VE GDA EA KNPTG-THAAPARTCfap1a_al GPPGMM----QEMN..(19 aa)..YGDDPNAGKTSKRYTKEDLKKDET FEA VE GEA EA KNPTG-TRAAPARTCfcol_rf GPPGNS--DYGAAP..(19 aa)..MGDDPNR-----MDT-KDEKPEXG WAS AQ QA KNPTG-TKDMPARTCfcol_hy(1107-1207) GPPGPAMLPPWSGG..(34 aa)..YQVYRYYSSNKTKT DE TEIENN NNR KI KSS EA KKPNG-SKEFPARTC abcdefgabcdefgabcdefgabcdefg

D ColQ_h(286-353) GPPGRCLCGPTMNVNNPSYGESVYGPSSPRVPVIF VNNQEE ER NTQNAI FRRDQRSLYFKDSLGXVa1_h(1124-1191) GQPGLPGSRNLVTAFSNMDDMLQKAHLVIEGTFIY RDSTEF IR RDG KK QLGELIPIPADSPPPXVIIIa1_h(1195-1263) GPPGTMGASSGVRLWATRQAMLGQVHEVPEGWLIF AEQEEL VR QNG RK QLE RTPLPRGTDNEXXVIa1_m(324-393) GAPGSQGLVDERVVARPSGEPSVKEEEDKASAAEGEG QQ REA KI AER LI EHM GVHDPLASPEG abcdefgabcdefgabcdefga

E transmembrane region

XIIIa1_h(39-123) LPSPGSCG LTL LCSLALSLL HFRTAE QAR LR EAERGEQQMETAILGRVNQLLDE..(22 aa)..GPPXXIIIa1_h(32-123) GSRAVSALCLLLSVGSAAACLL GVQ AA QGR AA EEEREL RRAGPPGALDAWAEPH..(29 aa)..GPPXXVa1_h(30-125) TMPPC VL ALLSVV VVSCLY GYKTND QAR AA ESAKGAPSIHLLPDTLDHLKTMV..(33 aa)..GPPXVIIa1_h(466-569) KWLLG LLTWLL LG LFG IA AEE RK KAR DE ERIRRS LPYGDSMDRIEKDRLQ..(41 aa)..GSPMARCO_h(41-150) GVNFS AV VIY IL TAG GL VVQ LN QAR RV EMY LNDTLAAEDSPSFSLLQSA..(47 aa)..GEQEDA_h(35-182) GEGNSCIL LGF GLSLALHLLTLCC LE RRERGAESR GGSGTPGTSGTLSSLG..(85 aa)..GPP abcdefgabcdefgabcdefgabcdefgabcdefgabcd

F XXVIa1(131-202) CTR SD SER TT EAK LL EAAEQPSGPDNDLPPPQSTPPTWNEDFLPDAIPIAHPGPRRRRPTGPAGPP abcdefgabcdefgabcd

G COL2 NC2 COL1

IXa1I_h(745-798) GLPGVQGPPGR-------APTDQH KQ C R QEH AE AAS KRP-DS-----------GATGLPGRPGPPIXa2_h(508-561) GQPGRQGVEGR-------DATDQH VD A K QEQ AE AVS KRE-AL-----------GAVGMMGPPGPPXVIa1_h(1421-1483) GLPGVPGSMGD---MVNYDE KRF RQEI K DER AY TSR QFP-MEMAAAP------GRPGPPGKDGAPXIXa1_h(998-1066) GPPGSPGIPGIPADAVSFEE KKY NQEV R EER AV LSQ KLP-AAMLAAQA----YGRPGPPGKDGLPIXa3_h(508-562) GITGKPGVPGK-------EASEQR RE CGG SEQ AQ AAH RKPLAP-----------GSIGRPGPAGPPXXIa1_h(776-836) GPPGLDGKPGR-------EFSEQF RQ CTD RAQ PV GSPGIPGPPGPIXIIa1_h(2887-2953) GLKGEKGDRGD---IASQNM RAV RQ CEQ SGQ NR NQM NQIPNDYQSS---RNQPGPPGPPGPPGSAXIVa1_h GAKGERGERGD---LQSQAM RSV RQ CEQ QSH AR TAI NQIPSHSSSI---RTVQGPPGEPGRPGSPXXa1_c(1297-1366) GPKGERGEKGE---PQS AT YQL SQ CER QSH LK DSF HEHARKPVPVWEGRLKPGEPGSPGPPGPP abcdefgabcdefga abcdefgabcdefga

H COL2 NC3 COL3

XIIIa1_h(430-475) GPKGSKGEPGKGE VD NGN NE LQE RT ALMGPPGLPGQIGPPXXVa1_h(415-460) GPPGQKGDQGATK ID NGN HE LQR TT TVTGPPGPPGPQGLQXXIIIa1_h(384-423) GADGLKGEKGESASDS QES AQ IVE------PGPPGPPGPPGPM abcdefgabcdefgabcd

I VIIa1_h(1928-1990) GERGLRGEPGSVPN DRL ET GIK SA REI ET DESSGSFLPVPERRRGPKGDSGEQGPP defgabcdefgabcdefgabcd

J VIIa1_h(1044-1259) QTPVCPRGLADVVFLPHATQDN HR EATRRV ERL LA GPLGPQAVQVGLLSYSHRP..(151 aa)..GQKVIa1_h(662-720) GPD..(69 aa)..MQEHVSLRSPSIRN QE KEA KS QWM GGTFTGEALQYTRDQLLPPSPNVIa2_h(587-719) GLT..(71 aa)..TFEAIQLDDEHIDS SS KEA KN EWI GGTWTPSALKFAYDRLIKESRRVia3_h(2371-2505) GDS..(73 aa)..TTEIRFADSKRKSV LDKIKN QV LTSKQQSLETAMSFVARNTFKRVRNG abcdefgabcdefgabcd

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N

C

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IIA N-pro/SP-D

IIA N-pro

IIA N-pro/CPII (aa 1172-1189)

IIA N-pro/CPII (aa 1172-1196)

IIA N-pro/CPII (aa 1172-1202)

SRL M L L L F Y...GPPGLGGNFAAQMAGGFDEKAGGAQLGVMQGPM/DSAA RQQ EA NGK QR EAA KKAALFPDG - CRD

A L V...GPPGLGGNFAAQMAGGFDEKAGGAQLGVMQGPM/ADQ AGG RQHDAE DAT

A L V L L...GPPGLGGNFAAQMAGGFDEKAGGAQLGVMQGPM/ADQ AGG RQHDAE DAT KS NNQ

A L V L L I I...GPPGLGGNFAAQMAGGFDEKAGGAQLGVMQGPM/ADQ AGG RQHDAE DAT KS NNQ ES RS

...GPPGLGGNFAAQMAGGFDEKAGGAQLGVMQGPM

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18311481644937

26

TD

18311481644937

26

TD

M

M

TD

M

18311481644937

26

TD

M

0 0.1 1 2 mM BS3

M

0 0.1 1 2 mM BS3

0 0.1 1 2 mM BS3

IIA N-pro

IIA N-pro/CPII (1172-1189)

IIA N-pro/CPII (1172-1202) IIA N-pro/CPII (1172-1196)

0 0.1 1 2 mM BS3

0 0.1 1 2 mM BS3

A B

C D

E

IIA N-pro/SP-D

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[θ] M

RW

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-40

-20

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wavelength (nm)

1172-11891172-11961172-1202

GCN4-pII

[θ] M

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wavelength (nm)

GCN4-pII

1172-11891172-11961172-1202

A

B

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D. Parry and David J. S. HulmesAudrey McAlinden, Thomasin A. Smith, Linda J. Sandell, Damien Ficheux, David A.

collagen superfamilyAlpha-helical coiled-coil oligomerization domains are almost ubiquitous in the

published online August 14, 2003J. Biol. Chem. 

  10.1074/jbc.M302429200Access the most updated version of this article at doi:

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