From the Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, United Kingdom
Received for publication, September 30, 2002, and in revised form, December 4, 2002
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ABSTRACT |
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We have determined the interactive sites required
for dimer formation in type VI collagen. Despite the fact that type VI
collagen is a heterotrimer composed of Type VI collagen has a ubiquitous distribution throughout
connective tissues, and it is thought to link cells and other matrix components. It has three different chains, 1(VI),
2(VI), and
3(VI)
chains, the formation of dimers is determined principally by
interactions of the
2(VI) chain. Key components of this interaction
are the metal ion-dependent adhesion site (MIDAS) motif of
the
2C2 A-domain and the GER sequence in the helical domain of
another
2(VI) chain. Replacement of the
2(VI) C2 domain with the
3(VI) domain abolished dimer formation, whereas alterations in the
2(VI) C1 domain did not disrupt dimer formation. When the helical
sequences were investigated, replacement of the
2(VI) sequence
GSPGERGDQ with the
3(VI) sequence GEKGERGDV abolished dimer
formation. Mutating the Pro-108 to a Lys-108 in this
2(VI)
sequence did not influence dimer formation and suggests that, unlike
the integrin I-domain/triple-helix interaction, hydroxyproline is not
required in collagen VI A-domain/helix interaction. These results
demonstrate that the
2(VI) chain position in the assembled
triple-helical molecule is critical for antiparallel dimer formation
and identify the interacting collagenous and MIDAS sequences involved.
These interactions underpin the subsequent assembly of type VI collagen.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1(VI),
2(VI), and
3(VI), each containing a short triple-helical region and globular extensions at NH2 and COOH termini (1, 2). In contrast to most other collagens, type VI collagen undergoes some polymerization prior to secretion. Heterotrimeric association of the
1(VI),
2(VI), and
3(VI) chains constitutes a monomer with a short
triple-helical region (100 nm) encompassed by NH2- and
COOH-terminal globular regions (3). Dimers are assembled by a staggered
antiparallel alignment of two disulfide bonded monomers, resulting in a
75-nm overlap between the two triple helices and alignment of the
COOH-terminal globular region of one molecule with the helical domain
of the other. In dimers, the triple-helical regions become supercoiled in a left-handed superhelix of pitch 37.5 nm (4, 5). Dimers then align
with their ends in register to form tetramers, which are the
secreted form. Tetramers then align end-to-end in the extracellular
space to form type VI collagen microfibrils (4) (Fig.
1).
View larger version (14K):
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Fig. 1.
Schematic diagram of type VI collagen dimer
and tetramer assembly.
The majority of the type VI collagen globular regions comprise domains
that have homology to von Willebrand factor A-domains (6).
A-domains are found in a number of proteins, including integrins
(designated I-domains) and several collagens, and appear to act as
homologous adhesion domains. The crystal structures of several
I-domains reveal a Rossman-type fold, with a six-stranded parallel
-sheet flanked by seven amphipathic
-helices (7). Side chains
from three loops closely opposed on the upper surface of the domain
coordinate a Mg2+ or Mn2+ ion, to form a
three-dimensional metal ion-dependent adhesion site
(MIDAS),1 which is conserved
in all
I-domains. Loop 1 contains a contiguous DXSXS sequence, where D is aspartate,
X is any amino acid, and S is serine; loop 2 contains a
threonine residue, while loop 3 contains an aspartate residue, located
~70 and 100 residues from the DXSXS sequence,
respectively. Mutation of individual MIDAS residues disrupts metal
binding and significantly reduces or eliminates ligand binding activity
(8-13). Metal ion binding to a MIDAS sequence in the
2
1 I-domain has been shown to be a
critical determinant of collagen binding (14).
Integrin heterodimers 1
1,
2
1,
3
1,
10
1, and
11
1 can all bind collagen (15-18). Their
I-domains contain an additional helix (
C-helix) protruding from
the MIDAS site, which creates a groove centered on the metal ion (19).
Residues on the upper surface of the
I-domain surrounding the MIDAS
groove contribute to the affinity and specificity of collagen binding
(7, 20-23). Structural analysis has shown that the collagen
triple-helix fits into the
2
1 I-domain
binding groove, where a glutamate residue from the collagen coordinates
the metal ion of the MIDAS motif (14). Ligand binding induces a
conformational change, resulting in the
C-helix unwinding and
facilitating collagen adhesion (14). Two triple-helical sequences
within type I collagen, GFOGER (24) and GLOGER (25), where O represents
hydroxyproline, have been identified as binding motifs for
1- and
2 I-domains. When a GFOGER containing collagen peptide was
co-crystallized in complex with a
2 I-domain (14), the collagen
glutamate was shown to coordinate the metal ion, while the arginine and
phenylalanine residues made contact with the
I-domain. The
conservative substitution of the collagen glutamate to aspartate
eliminated binding, since the aspartate is too short to coordinate the
ion (24). While the
1- and
2 I-domains each contain a conserved
MIDAS motif, small structural differences may explain why the
1I-domain has a higher binding affinity to type IV collagen (23,
26).
We have previously shown that the 2C2 A-domain is critical for dimer
formation in type VI collagen (27). Consideration of the staggered
antiparallel alignment of type VI collagen monomers in dimers suggests
that a C2 domain of one monomer interacts with the helical domain of
another type VI collagen molecule. In type VI collagen, a MIDAS motif
is found in C1 and C2 of
1 and
2 chains, but not
3(VI). We
have investigated the role of the
2C2 MIDAS site in dimer formation
to define the molecular recognition sequences that determine
intracellular type VI collagen assembly.
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EXPERIMENTAL PROCEDURES |
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Generation of 2(VI) Chimeric Construct--
A chimeric
construct consisting of a signal peptide sequence, the N1, helix, and
C1 domains of the
2(VI) chain and C2 domain of the
3(VI) chain
was generated by overlap extension PCR. Clone pGEM
2C1, described
previously (27), was utilized to amplify the signal peptide sequence
and
2(VI) N1, helix, C1 domains, using the signal peptide
forward primer (5'-CGGGCACACTAAGAGTCAGC-3') and reverse primer
(5'-CCAACTGCCAAATCCACAGGATGGGTCTGTTTGGCAGGGAAGGTCTGGGC-3') incorporating a 23-base fragment (bases 2456-2478) (1) to the 3'
end of the
2(VI) C1 domain and a 27-base overhang (bases 7411-7437) (2) to the 5' end of the
3(VI) C2 domain. Clone pGEM
3C2, described previously (27), was used to generate an
3(VI) chain C2
domain, using forward primer
(5'-GCCCAGACCTTCCCTGCCAAACAGACCCATCCTGTGGATTTGGCAGTTGG-3') incorporating a 27-base fragment (bases 7411-7437) (2) to the 5'
end of the
3(VI) C2 domain and a 23-base overhang (bases 2456-2478) (1) to the 3' end of the
2(VI) C1 domain and
3C2 reverse primer
(5'-GGTTACAGGCTATGTTGTGGTGG-3') (bases 8282-8304) (2) containing a
stop codon. PCR was performed using an Expand high fidelity PCR kit
(Roche Diagnostics, Lewes, UK). Reaction mixtures (50 µl) consisted
of 1 ng of template cDNA, reaction buffer containing 1.5 mM MgCl2, 200 µM dNTPs, 50 pmol
of forward and reverse primers, and 2.6 units of DNA polymerase mixture
(thermostable Taq and Pwo). Reaction mixture was
incubated for 3 min at 94 °C, immediately followed by 1 min at
94 °C, 2 min at 60 °C, and 3 min at 72 °C for 25 cycles, then
7-min incubation at 72 °C. Amplified fragments corresponding to the
expected sizes were isolated and then purified using a silicon
matrix-based purification kit (Qiagen, Crawley, UK). Purified short
length
2C1
2(VI) chain (1 ng) and
3C2 domain (1 ng) PCR
products were combined in a second round overlap extension PCR. A
reaction was performed for 30 s at 94 °C, 2 min at 42 °C, and 1 min at 72 °C for six cycles, in the absence of primers to allow self-annealing of the
2C1 and
3C2 overlap regions. Signal peptide forward primer and
3C2 reverse primer were then added and
reaction mixture immediately incubated for 25 cycles of 30 s at
94 °C, 1 min at 60 °C, and 3 min at 72 °C, followed by 7-min incubation at 72 °C. The amplified
2(VI) chimeric construct was isolated, purified, and cloned into TA vector pGEM (Promega,
Southampton, UK), then the sequence identity and reading frame
integrity were confirmed by dye-terminator automated sequencing.
Mutation of -Chains--
A QuikChangeTM
site-directed mutagenesis kit (Strategene, Cambridge, UK) was used to
introduce mutations in type VI collagen
-chain constructs cloned
into vector pGEM (Promega). A reaction mixture consisted of
-chain
clone (10 ng), two complementary oligonucleotides containing the
desired mutation (125 ng), 200 µM dNTPs, 2.5 units of
Pfu DNA polymerase, and reaction buffer to a final volume of
50 µl. The reaction mixture was incubated for 30 s at 95 °C,
immediately followed by 30 s at 95 °C, 1 min at 55 °C, and
12 min 30 s at 68 °C for 18 cycles, cooled to 4 °C, then 10 units of DpnI restriction enzyme added and incubated for 90 min at 37 °C. Mutated DNA was transformed into Epicurian Coli
XL1-Blue competent cells, then colonies containing the correct mutation
were identified by dye-terminator automated sequencing.
2(VI) C2 Domain MIDAS Mutations--
Using clone pGEM
2C2,
described previously (27), the
2(VI) C2 domain MIDAS motif DGSER
(residues 818-822) (1) was mutated to the aligned
3(VI) C2 domain
sequence DSAET (residues 2392-2396) (2) (
2C2/1M) (Fig.
2), using two complementary
oligonucleotides (5'-CGTCTTCCTGCTGGACAGCGCTGAGACCCTGGGTGAGC-3')
(bases 2520-2557) (2) containing the mutations shown underlined. The
2(VI) C2 domain MIDAS motif DGSER (residues 818-822) (1) was
mutated to DGSES (
2C2/3M) (Fig. 2), using two complimentary
oligonucleotides (5'-CCTGCTGGACGGCTCCGAGAGCCTGGGTGAGCAGAACTTCCAC-3')
(bases 2526-2568) (1) containing the mutations shown underlined. The
2(VI) C2 domain MIDAS motif TDG (residues 927-929) (1) was mutated
to TTG (
2C2/2M) (Fig. 2), using two complimentary oligonucleotides (5'-GCTGTCCTTCGTGTTCCTCACGACCGGCGTCACGGGCAACGAC-3')
(bases 2841-2883) (1) containing the mutations shown
underlined.
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2(VI) Helix Mutations--
The
2(VI) helix amino-terminal
GER triplet (residues 109-111) (28) was mutated to GDR
(
2C2/1H) (Fig. 3), using two
complementary oligonucleotides
(5'-GAAGCAGGGAGTCCAGGGGACCGAGGAGACCAAGGCGCA-3') (bases
310-348) (1), containing the mutation shown underlined. A
2(VI)
helix sequence GSPGERGDQ (residues 106-114) (1) incorporating the
amino-terminal GER triplet was mutated to the corresponding
3(VI)
helix sequence GEKGERGDV (residues 106-114) (1) (
2C2/2H) (Fig. 3)
of identical position, using two complimentary oligonucleotides (5'-GGGGAAGCAGGAGAGAAAGGAGAAAGAGGAGATGTTGGCGCAAGGGGG-3')
(bases 307-354) (1), containing the mutations shown underlined. The
2(VI) helix sequence PGER (residues 108-111) (1) was mutated to
KGER (
2C2/4H) (Fig. 3), using two complementary oligonucleotides (5'-CCGGGGGAAGCAGGGAGTAAAGGGGAGCGAGGAGACCAA-3') (bases
304-342) (1), incorporating the mutations shown underlined. The
2(VI) helix sequence GSPGER (residues 106-111) (1) was mutated
to GEPGER (
2C2/3H) (Fig. 3), using two complementary
oligonucleotides (5'-TACCCGGGGGAAGCAGGGGAGCCAGGGGAGCGAGGAGAC-3') (bases
301-339) (1), incorporating the mutations shown underlined. The
2(VI) helix sequence GERGDQ (residues 109-114) (1) was mutated to GERGDV (
2C2/5H) (Fig. 3), using two complementary oligonucleotides (5'-CCAGGGGAGCGAGGAGACGTTGGCGCAAGGGGGACC-3') (bases
322-357) (1), incorporating the mutations shown underlined.
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2(VI) Chimera Mutation--
Using clone pGEM
2(VI) chimera,
the
3(VI) C2 domain sequence DSAET (residues 2392-2396) (2) was
mutated to the
2(VI) C2 domain MIDAS motif DGSER (residues 818-822)
(2), (
2-chimera; Fig. 2), using two complementary
oligonucleotides
(5'-GGCTTTCATCTTAGACGGCTCCGAGCGGACCACCCTGTTCCAG-3') (bases 7491-7533) (2) incorporating the mutations shown underlined.
3(VI) C2 Domain Mutation--
Clone pGEM
3C2, described
previously (27),
3(VI) C2 domain sequence DSAET (residues
2392-2396) (2) was mutated to the
2(VI) C2 domain MIDAS motif DGSER
(residues 818-822) (1) (
3C2/M) (Fig. 2), using two
complementary oligonucleotides
(5'-GGCTTTCATCTTAGACGGCTCCGAGCGGACCACCCTGTTCCAG-3') (bases 7491-7533) (2) incorporating the mutations shown underlined.
3(VI) Helix Mutation--
The
3(VI) helix sequence,
GEKGERGDV (residues 106-114) (28), contained in the above
3(VI) C2
domain-mutated construct (Fig. 2,
3C2/M mutant), was mutated to the
corresponding
2(VI) helix sequence GSPGERGDQ (residues 106-114)
(28) (Fig. 3,
3C2/MH mutant) using two complementary
oligonucleotides
(5'-GGTGATAAAGGACCTCGAGGGAGTCCAGGGGAGCGAGGAGACCAAGGGATTCGAGGGGACCCG-3') (bases 298-360) (28) containing the mutations shown underlined.
Transcription and in Vitro Translation--
Cloned -chain
cDNA (10 µg) was linearized downstream to the insert using
restriction enzyme SpeI, followed by phenol/chloroform extraction and ethanol precipitation. Transcription reactions contained
100 units of T7 RNA polymerase (Promega), 10 mM
dithiothreitol, 40 units of RNase inhibitor, 3 mM NTPs, and
transcription buffer and incubated for 4 h at 37 °C. RNA was
purified using spin columns (Qiagen) and eluted in RNase-free water
containing RNase inhibitor. Translation of RNA transcripts was
performed using a rabbit reticulocyte lysate system (Promega);
reactions consisted of 35 µl of lysate, 50 mM KCl, 20 µM amino acids minus methionine, 50 µg/ml sodium ascorbate, 25 µCi of [35S]methionine, 40 units of RNase
inhibitor, 2.5 µl of RNA transcript, and 4× 105
semipermeabilized HT-1080 cells, prepared as described previously (27).
Translation reactions were incubated for 2 or 4 h at 30 °C,
terminated by adding 1 mM cycloheximide, then digested with 250 µg/ml proteinase K (Sigma, Poole, UK) in the presence of 10 mM CaCl2 for 20 min at 4 °C. The reaction
mixture was incubated at 4 °C with 10 mM
phenylmethylsulfonyl fluoride (Sigma) for 5 min, 25 mM
N-ethylmaleimide (Sigma, Poole, UK) for 15 min, then the
cell pellet was washed three times in KHM buffer (100 mM
potassium acetate, 20 mM Hepes, pH 7.2, 2 mM
magnesium acetate). No added mRNA controls were used in these
studies (27); in the absence of added mRNA, no translation products
were detected. All experiments were shown to be reproducible after
repeating a minimum of three times.
Immunoprecipitation and Detection of -Chain
Assemblies--
Washed cells were incubated for 30 min at 4 °C in
NET buffer (50 mM Tris-HCl, pH 7.4, 150 mM
NaCl, 5 mM EDTA, 0.25% (w/v) gelatin, 0.05% (v/v) Nonidet
P-40, and 0.02% (w/v) sodium azide), the supernatant was precleared
with 10% (w/v) protein A-Sepharose for 1 h at 4 °C, then
incubated with type VI collagen polyclonal antibody VIA (kindly donated
by Dr. S. Ayad) (29) for 16 h at 4 °C. The supernatant was then
incubated with 10% (w/v) protein A-Sepharose for 2 h at 4 °C,
centrifuged at 800 × g for 3 min, and the
immunoprecipitates were washed three times in NET buffer minus Nonidet
P-40 and gelatin and then resuspended in SDS-PAGE sample buffer, with
or without 5%
-mercaptoethanol. Type VI collagen
-chains were
analyzed using reducing 8% (w/v) polyacrylamide gels, while monomers,
dimers, and tetramers were observed using non-reducing 3% (w/v)
polyacrylamide 0.4% (w/v) agarose composite gels (27), which were
fixed, dried, and imaged using a Fujix BAS2000 phosphorimager.
Homology Models--
Atomic models for the interaction of a
collagen triple helix with 2(VI) C2 domain were built based on the
crystal structure coordinates of the complex between the A-domain of
2
1 integrin and a collagen peptide
containing the GFOGER sequence (14). First, a homology model for
2(VI) C2 was built based on the alignment of its sequence to both
von Willebrand factor A-domain (27), and the
A-domain of
2
1 integrin, using the coordinates of the ligand-bound form of the
2
1 A-domain as
reference. The program LOOK (Molecular Applications Group, Palo Alto,
CA) was used for this homology modeling. Different models for the
collagen-
2(VI) C2 interaction were then assembled using collagen
triple-helical models with the following sequences:
(EAGSOGERGDQGARG)3 from
2(VI), (PRGEKGERGDVGIRG)3 from
3(VI), and a model with sequence
((PKGDOGAFGLKGEKG) (EAGSOGERGDQGARG) (PRGEKGERGDVGIRG)) to represent an
1
2
3 collagen VI heterotrimer (see "Discussion"). A metal
ion was always modeled at the MIDAS site, and a few water molecules
were also included in the models to complete the metal coordination
sites and to account for the water-mediated hydrogen bonds in the
collagen triple helices (30, 31). The complex models thus assembled were subject to energy minimization using the program CNS (32). By
analogy with the collagen-integrin A-domain structure, the
2(VI) C2
domain is identified as the "A" chain, and the three chains in the
collagen triple helix are identified as "B," "C," and "D," respectively.
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RESULTS |
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All DNAs encoding the mutated -chains were verified by
sequencing, then shown by reducing SDS-PAGE to produce a translation product of the expected size and similar abundance to the corresponding non-mutated
-chains (data not shown). Differences in the monomer intensities observed on the agarose gels reflect differences in monomer
assembly or stability.
MIDAS Sequence Involvement in Type VI Collagen Assembly--
To
delineate the residues of the C2 domain that determined type VI
collagen assembly, a chimeric 2(VI) chain was constructed in which
the
2(VI) C2 domain was replaced with the
3(VI) C2 domain (see
Fig. 2,
2-chimera-normal). When these
2(VI) wild type and
2-chimera chains were translated and analyzed using non-reducing
agarose/acrylamide composite gels, the wild type chain produced dimers
and tetramers, but the chimeric
2(VI) chain produced only low levels
of monomers (Fig. 4, lanes 1 and 3). The
3(VI) chain was also unable to form dimers
(Fig. 4, lane 2). Replacement of the
2(VI) C2 domain with
the
3(VI) C2 domain thus abolished dimer formation.
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Manipulation of the MIDAS domain within the 2(VI) C2 domain was also
found to abolish dimer formation. Thus, when the
2(VI) C2 domain
sequence DGSER (residues 818-822), homologous to the MIDAS consensus
sequence DXSXS, was substituted by site-directed mutagenesis for the
corresponding aligned sequence DSAET (residues 2392-2396) found in the
C2 domain of the
3(VI) chain (27) (see Fig. 2,
2C2/1M mutant), no
dimers were formed (Fig. 4, lane 4); compare with wild type
2(VI) chain after 4 h translation (see Fig. 4, lane
1). Upon introduction of the
2(VI) chain C2 domain sequence
(DGSER) into the
3(VI) chain to replace DSAET (see Fig. 2,
3C2/M
mutant), no dimers were formed (Fig. 4, lane 5). In the case
of the
2-chimera containing (DSAET), dimer and tetramer formation
could be re-instated by re-introducing the MIDAS motif DGSER to replace
DSAET (
2 chimera mutant) (Fig. 4, lane 6).
In the MIDAS motif of integrin I-domains, a metal ion is coordinated by
a conserved DXSXS sequence and threonine and
aspartate residues, which are not contiguous in sequence. The 3(VI)
chain C2 domain has a conserved threonine but lacks a conserved
aspartate in a position constituting the MIDAS motif, whereas
the
2(VI) chain C2 domain contains a conserved aspartate (residue
928) in the coordinating position. We examined whether the conserved
aspartate residue within the
2(VI) chain C2 domain contributes to
type VI collagen assembly by substituting it with a threonine residue found in the identical position of the
3(VI) chain. The Asp-928
Thr-928-mutated
2(VI) chain (see Fig. 2,
2C2/2M mutant) produced monomer, dimer, and tetramer assemblies at a slightly lower level to
non-mutated
2(VI) chain (Fig. 5,
lanes 1 and 2).
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The potential MIDAS motif within the 2(VI) C2 domain sequence
(DGSER) differs from the MIDAS consensus sequence
(DXSXS), in that a serine is replaced by arginine
(residue 822) in the
2(VI) C2 domain. To establish whether this
homology difference affects type VI collagen assembly, the arginine
(residue 822) was substituted for a serine (see Fig. 2,
2C2/3M
mutant). After 4 h of translation the Arg-822
Ser-822-mutated
2(VI) chain produced a much lower level of monomer, dimer, and
tetramer formation when compared with non-mutated
2(VI) chain (Fig.
5, lanes 1 and 3).
Changing the potential MIDAS site in the C1 domain of the 2(VI)
chain with sequences in the
3(VI) C1 and C2 domains did not prevent
dimer and tetramer formation (see Fig. 2,
2C2/4M and
2C2/5M)
(data not shown).
Helix Sequence Involvement in Type VI Collagen Assembly--
The
triple-helical sequence GFOGER (where O represents hydroxyproline)
within collagen type I has been identified as the minimum recognition
motif for integrin 1 and
2 I-domains (24). A GER triplet is
present in the
2(VI) helix sequence at residues 109 to 111. An
Glu-110
Asp-110-mutated
2(VI) chain (see Fig. 3,
2C2/1H
mutant) produced only low levels of monomer assembly (Fig. 6, lane 2). The
3(VI) helix
sequence also contains a GER triplet (residues 109-111) in exactly the
same position as that found in the
2(VI) helix sequence (residues
109-111). Residues 106-114 (GSPGERGDQ) in the
2(VI) helix were
substituted for residues 106-114 (GEKGERGDV) in the
3(VI) helix by
site-directed mutagenesis (see Fig. 3,
2C2/2H mutant). The mutated
2 chain produced only low levels of monomer after 4-h translation
Fig. 6, lane 3).
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To delineate which residues within the 2(VI) helix sequence
GSPGERGDQ (residues 106-114) are important to type VI collagen assembly, individual constituents were substituted for identically positioned
3(VI) helix residues within the sequence GEKGERGDV (residues 106-114). The proline (residue 108) is predicted to be
hydroxylated (28). Hydroxylation is an important determinant of helical
stability, as shown by the addition of dipyridyl to the translation
reaction when, after 4 h of translation, no assemblies corresponding to monomer, dimer, or tetramer were observed (Fig. 6,
lane 6). A Pro-108
Lys-108-mutated
2(VI) chain (see
Fig. 3,
2C2/4H mutant) produced monomer, dimer, and tetramer
assemblies at a similar level to a non-mutated
2(VI) chain (Fig. 6,
lane 5, compare with lane 1). Similarly, an
Ser-107
Glu-107-mutated
2(VI) chain (see Fig. 3,
2C2/3H
mutant) produced monomer, dimer, and tetramer assemblies at a
comparable level to non-mutated
2(VI) chain (Fig. 6, lane
4). However, a Gln-114
Val-114-mutated
2(VI) chain (see
Fig. 3,
2C2/5H mutant) produced only monomers (Fig. 6, lane
7).
The 3(VI) chain did not form dimers and tetramers even when the
2(VI) helical and C2 domains were inserted (see Fig. 3,
3C2/MH
mutant) (data not shown).
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DISCUSSION |
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The elaboration of type VI collagen microfibrils extracellularly
is critically dependent on the formation of stable tetramers intracellularly. Little is known about the formation and stabilization of these intracellular assemblies and how dimers form. The observed staggered antiparallel arrangement of monomers to form dimers shows
that the COOH-terminal domain must interact with the helical domain of
another molecule (4). We previously showed (27) that the C2 domain of
the 2(VI) chain is a critical determinant of dimer formation and
tetramer elaboration. We have now delineated the molecular basis of
this dimer formation by identifying the sequences within the
2(VI)
chain that determine dimer formation. The
2(VI) chain provides the
residues in the helical portion of one monomer and the MIDAS motif of
the C2 domain in another monomer, and these interact in a specific
manner to form antiparallel dimers.
Previous studies of A-domain interactions with fibrillar collagen have
identified the triple-helical sequence GFOGER (where O represents
hydroxyproline) within collagen type I as a recognition motif for
integrin 1 and
2 I-domains (24). Studies on the interaction
between the integrin
2
1 and fibrillar
collagen revealed that the critical residues in the A-domain lay on the
three loops that both co-ordinated the metal ions and made direct H
bonds and salt bridges to the collagen (14). The authors also suggested that, while the GFOGER motif occurs only in the
1 chains of types I
and IV collagen, GxOGER may account for the ability of several loci in
collagens to bind to
2
1. The collagenous
sequence of the
2(VI) chain contains the homologous sequence
GSPGERGDQ, which we have shown here by site-directed mutagenesis is
required for A-domain mediated dimer formation. Recently, the crystal
structure of the A3-domain of von Willebrand factor was determined, and site-directed mutagenesis showed the collagen binding site is close to
the bottom face of A3 and not in the top face with the vestigial MIDAS
motif (33). Moreover, site-directed mutagenesis of residues on the top
face displayed normal collagen binding (34). Homology alignment of the
2(VI) C2 A-domain and the von Willebrand A3-domain shows very little
conservation in the three short sequences involved in this bottom face
collagen binding.
The evidence from our site-directed mutagenesis experiments, the
conservation of all but one of the metal binding features on 2(VI)
C2 A-domain, and the presence of an essential GxOGER collagenous
sequence indicates that the C2-triple helix interaction resembles that
of collagen with A-domain integrins. To gain further insights into the
molecular interaction between
2(VI) C2 domain and the triple-helical
region of type VI collagen, we built atomic homology models based on
the collagen-integrin A-domain crystal structure (14) (see
"Experimental Procedures"). Fig. 7
shows a model of the interaction of
2(VI) C2 A-domain (A chain) with the triple-helical region of an
2(VI) homotrimer. As in the
collagen-integrin
I-domain crystal structure (14), this model
predicts that the middle strand (C chain in
red) accounts for the majority of the interactions with the
C2 domain, with fewer from the trailing strand (D chain in
yellow) and none from the leading strand (B chain
in purple). The middle strand glutamate Glu-C110 completes the coordination of the metal ion in the MIDAS site in a manner completely equivalent to the collagen-integrin interaction.
Nevertheless, the model suggests that there might be some significant
differences. For example, the next residue in the GER triplet, Arg-C111
(not shown in Fig. 7), does not seem to have a suitable interaction partner in the
3-
4 loop equivalent to an aspartate residue seen in the collagen-integrin A-domain structure (14). Our model places
Arg-C111 main chain in contact to Phe-893 side chain on the C2 domain,
but cannot predict if and how this arginine residue may be essential
for binding specificity. The same residue in the trailing
strand, Arg-D111 (in yellow in Fig. 7), appears sandwiched between Glu-C110 and Asp-C113 from the middle strand and might be
important in forming a tandem of salt bridges to maintain Glu-C110 in
an appropriate conformation for metal binding. Our model suggests that
Arg-D111 may also bind to one of the water molecules coordinating the
metal ion in the MIDAS site. Residue Gln-C114 from the middle strand is
critical for collagen VI dimer and tetramer formation. This is the
first time that the triplet COOH-terminal to the GER motif is shown
biochemically to be selective in the binding of an A-domain structure
to a collagen triple helix. Our model predicts the interaction of
Gln-C114 with the main strand of
2(VI) C2 (residue Gly-932 in the
D-
5 loop). A completely equivalent interaction is
seen in the collagen-integrin
I-domain structure between His-258 in
the
D-
5 loop from the
I-domain, and a Hyp residue
in the middle strand that is four positions COOH-terminal to the
glutamate that completes the metal coordination (14). Thus, our model suggests a novel coordination pattern and opens a new line of investigation into A-domain interactions.
|
One of the distinctive features in the 2(VI) C2 A-domain is the
DGSER sequence of the amino acids from the
A-
1 loop contributing to the MIDAS site. That sequence differs from the conserved
DXSXS motif seen at the equivalent position in
metal-binding A-domains. Yet, mutation of that arginine residue to a
typical serine reduces formation of dimers and tetramers, which
suggests a novel architecture for the
2(VI) C2 MIDAS site. The
orientation of the arginine side chain in our model is obviously
unreliable, but it is not clear how to change the conformation of the
A-
1 loop to force such arginine residue to participate in the
construction of the MIDAS site. Arginine side chains are not good for
direct metal coordination, which probably will be completed through the
carbonyl group from the residue preceding Arg-822 or perhaps through an additional water molecule.
The collagen-integrin A-domain crystal structure shows direct hydrogen
bonding interactions between the collagen main chain and the A-
1
loop in the A-domain (14). The conformation of this loop in our model
is less reliable, as it has an insertion of two residues compared with
the same loop in
2 integrin A-domain. Yet the model suggests an
interaction between the side chain of Glu-821 from the DGSER motif and
Ser-C107, again in the middle strand of the collagen triple helix (not
shown in Fig. 7).
Fig. 8a summarizes the
distribution of residues in the collagen triple helix seen to interact
with the 2
1 integrin A-domain in the
crystal structure of their complex (14). The model for the interaction
between
2(VI) C2 and a homotrimer triple helix made of three
2(VI) chains suggests a very similar distribution of residues (Fig.
8b), although the nature and specificity of some of the
interactions will certainly differ with respect to the integrin case.
There is evidence that physiologically assembled collagen VI is a
heterotrimer of three chains,
1(VI),
2(VI), and
3(VI). To
preserve a distribution of residues similar to the homotrimer case,
2 would obviously need to be the middle strand (main
interaction with C2), with
1 being the leading strand (no
interaction with C2) and
3 the trailing strand, as shown in Fig. 8c. In fact, the model built with a heterotrimer
sequence predicts even more favorable interactions, such as Lys-D108 in the trailing strand with Glu-821 from the DGSER motif (Fig.
8c). Thus, the need to provide binding specificity to the C2
domain seems to dictate the way in which the three chains of
collagen VI need to assemble together to form
multimerization-competent heterotrimers.
|
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FOOTNOTES |
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* This work was supported by the Medical Research Council and the Biotechnology and Biological Sciences Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 44-161-275-5079;
Fax: 44-161-275-5082; E-mail: ashuttle@fs1.scg.man.ac.uk.
Published, JBC Papers in Press, December 7, 2002, DOI 10.1074/jbc.M209977200
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ABBREVIATIONS |
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The abbreviation used is: MIDAS, metal ion-dependent adhesion site.
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