Structural Basis of Type VI Collagen Dimer Formation*

Stephen Ball, Jordi Bella, Cay Kielty, and Adrian ShuttleworthDagger

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains, the formation of dimers is determined principally by interactions of the alpha 2(VI) chain. Key components of this interaction are the metal ion-dependent adhesion site (MIDAS) motif of the alpha 2C2 A-domain and the GER sequence in the helical domain of another alpha 2(VI) chain. Replacement of the alpha 2(VI) C2 domain with the alpha 3(VI) domain abolished dimer formation, whereas alterations in the alpha 2(VI) C1 domain did not disrupt dimer formation. When the helical sequences were investigated, replacement of the alpha 2(VI) sequence GSPGERGDQ with the alpha 3(VI) sequence GEKGERGDV abolished dimer formation. Mutating the Pro-108 to a Lys-108 in this alpha 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 alpha 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, alpha 1(VI), alpha 2(VI), and alpha 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 alpha 1(VI), alpha 2(VI), and alpha 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).


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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 alpha I-domains) and several collagens, and appear to act as homologous adhesion domains. The crystal structures of several alpha I-domains reveal a Rossman-type fold, with a six-stranded parallel beta -sheet flanked by seven amphipathic alpha -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 alpha 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 alpha 2beta 1 I-domain has been shown to be a critical determinant of collagen binding (14).

Integrin heterodimers alpha 1beta 1, alpha 2beta 1, alpha 3beta 1, alpha 10beta 1, and alpha 11beta 1 can all bind collagen (15-18). Their alpha I-domains contain an additional helix (alpha C-helix) protruding from the MIDAS site, which creates a groove centered on the metal ion (19). Residues on the upper surface of the alpha 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 alpha 2beta 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 alpha 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 alpha 1- and alpha 2 I-domains. When a GFOGER containing collagen peptide was co-crystallized in complex with a alpha 2 I-domain (14), the collagen glutamate was shown to coordinate the metal ion, while the arginine and phenylalanine residues made contact with the alpha 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 alpha 1- and alpha 2 I-domains each contain a conserved MIDAS motif, small structural differences may explain why the alpha 1I-domain has a higher binding affinity to type IV collagen (23, 26).

We have previously shown that the alpha 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 alpha 1 and alpha 2 chains, but not alpha 3(VI). We have investigated the role of the alpha 2C2 MIDAS site in dimer formation to define the molecular recognition sequences that determine intracellular type VI collagen assembly.

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ABSTRACT
INTRODUCTION
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Generation of alpha 2(VI) Chimeric Construct-- A chimeric construct consisting of a signal peptide sequence, the N1, helix, and C1 domains of the alpha 2(VI) chain and C2 domain of the alpha 3(VI) chain was generated by overlap extension PCR. Clone pGEMalpha 2C1, described previously (27), was utilized to amplify the signal peptide sequence and alpha 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 alpha 2(VI) C1 domain and a 27-base overhang (bases 7411-7437) (2) to the 5' end of the alpha 3(VI) C2 domain. Clone pGEMalpha 3C2, described previously (27), was used to generate an alpha 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 alpha 3(VI) C2 domain and a 23-base overhang (bases 2456-2478) (1) to the 3' end of the alpha 2(VI) C1 domain and alpha 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 alpha 2C1 alpha 2(VI) chain (1 ng) and alpha 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 alpha 2C1 and alpha 3C2 overlap regions. Signal peptide forward primer and alpha 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 alpha 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 alpha -Chains-- A QuikChangeTM site-directed mutagenesis kit (Strategene, Cambridge, UK) was used to introduce mutations in type VI collagen alpha -chain constructs cloned into vector pGEM (Promega). A reaction mixture consisted of alpha -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.

alpha 2(VI) C2 Domain MIDAS Mutations-- Using clone pGEMalpha 2C2, described previously (27), the alpha 2(VI) C2 domain MIDAS motif DGSER (residues 818-822) (1) was mutated to the aligned alpha 3(VI) C2 domain sequence DSAET (residues 2392-2396) (2) (alpha 2C2/1M) (Fig. 2), using two complementary oligonucleotides (5'-CGTCTTCCTGCTGGACAGCGCTGAGACCCTGGGTGAGC-3') (bases 2520-2557) (2) containing the mutations shown underlined. The alpha 2(VI) C2 domain MIDAS motif DGSER (residues 818-822) (1) was mutated to DGSES (alpha 2C2/3M) (Fig. 2), using two complimentary oligonucleotides (5'-CCTGCTGGACGGCTCCGAGAGCCTGGGTGAGCAGAACTTCCAC-3') (bases 2526-2568) (1) containing the mutations shown underlined. The alpha 2(VI) C2 domain MIDAS motif TDG (residues 927-929) (1) was mutated to TTG (alpha 2C2/2M) (Fig. 2), using two complimentary oligonucleotides (5'-GCTGTCCTTCGTGTTCCTCACGACCGGCGTCACGGGCAACGAC-3') (bases 2841-2883) (1) containing the mutations shown underlined.


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Fig. 2.   Mutated alpha 2(VI) and alpha 3(VI) chain COOH-terminal constructs. Each alpha -chain construct contains the same signal peptide sequence, designated SP. Amino- and carboxyl-terminal domains are shown as shaded boxes, while the helical region is represented by a box with diagonal lines. The approximate position of normal and mutated residues is shown, together with the estimated molecular size of the translated product. Designated nomenclatures for the mutants shown here are used in Figs. 4 and 5.

alpha 2(VI) Helix Mutations-- The alpha 2(VI) helix amino-terminal GER triplet (residues 109-111) (28) was mutated to GDR (alpha 2C2/1H) (Fig. 3), using two complementary oligonucleotides (5'-GAAGCAGGGAGTCCAGGGGACCGAGGAGACCAAGGCGCA-3') (bases 310-348) (1), containing the mutation shown underlined. A alpha 2(VI) helix sequence GSPGERGDQ (residues 106-114) (1) incorporating the amino-terminal GER triplet was mutated to the corresponding alpha 3(VI) helix sequence GEKGERGDV (residues 106-114) (1) (alpha 2C2/2H) (Fig. 3) of identical position, using two complimentary oligonucleotides (5'-GGGGAAGCAGGAGAGAAAGGAGAAAGAGGAGATGTTGGCGCAAGGGGG-3') (bases 307-354) (1), containing the mutations shown underlined. The alpha 2(VI) helix sequence PGER (residues 108-111) (1) was mutated to KGER (alpha 2C2/4H) (Fig. 3), using two complementary oligonucleotides (5'-CCGGGGGAAGCAGGGAGTAAAGGGGAGCGAGGAGACCAA-3') (bases 304-342) (1), incorporating the mutations shown underlined. The alpha 2(VI) helix sequence GSPGER (residues 106-111) (1) was mutated to GEPGER (alpha 2C2/3H) (Fig. 3), using two complementary oligonucleotides (5'-TACCCGGGGGAAGCAGGGGAGCCAGGGGAGCGAGGAGAC-3') (bases 301-339) (1), incorporating the mutations shown underlined. The alpha 2(VI) helix sequence GERGDQ (residues 109-114) (1) was mutated to GERGDV (alpha 2C2/5H) (Fig. 3), using two complementary oligonucleotides (5'-CCAGGGGAGCGAGGAGACGTTGGCGCAAGGGGGACC-3') (bases 322-357) (1), incorporating the mutations shown underlined.


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Fig. 3.   Mutated alpha 2(VI) and alpha 3(VI) chain triple-helical constructs. Each alpha -chain construct contains the same signal peptide sequence, designated SP. Amino- and carboxyl-terminal domains are shown as shaded boxes, while the helical region is represented by a box with diagonal lines. The approximate position of normal and mutated residues is shown, together with the estimated molecular size of the translated product. Designated nomenclatures for the mutants shown here are used in Fig. 6.

alpha 2(VI) Chimera Mutation-- Using clone pGEMalpha 2(VI) chimera, the alpha 3(VI) C2 domain sequence DSAET (residues 2392-2396) (2) was mutated to the alpha 2(VI) C2 domain MIDAS motif DGSER (residues 818-822) (2), (alpha 2-chimera; Fig. 2), using two complementary oligonucleotides (5'-GGCTTTCATCTTAGACGGCTCCGAGCGGACCACCCTGTTCCAG-3') (bases 7491-7533) (2) incorporating the mutations shown underlined.

alpha 3(VI) C2 Domain Mutation-- Clone pGEMalpha 3C2, described previously (27), alpha 3(VI) C2 domain sequence DSAET (residues 2392-2396) (2) was mutated to the alpha 2(VI) C2 domain MIDAS motif DGSER (residues 818-822) (1) (alpha 3C2/M) (Fig. 2), using two complementary oligonucleotides (5'-GGCTTTCATCTTAGACGGCTCCGAGCGGACCACCCTGTTCCAG-3') (bases 7491-7533) (2) incorporating the mutations shown underlined.

alpha 3(VI) Helix Mutation-- The alpha 3(VI) helix sequence, GEKGERGDV (residues 106-114) (28), contained in the above alpha 3(VI) C2 domain-mutated construct (Fig. 2, alpha 3C2/M mutant), was mutated to the corresponding alpha 2(VI) helix sequence GSPGERGDQ (residues 106-114) (28) (Fig. 3, alpha 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 alpha -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 alpha -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% beta -mercaptoethanol. Type VI collagen alpha -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 alpha 2(VI) C2 domain were built based on the crystal structure coordinates of the complex between the A-domain of alpha 2beta 1 integrin and a collagen peptide containing the GFOGER sequence (14). First, a homology model for alpha 2(VI) C2 was built based on the alignment of its sequence to both von Willebrand factor A-domain (27), and the alpha A-domain of alpha 2beta 1 integrin, using the coordinates of the ligand-bound form of the alpha 2beta 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-alpha 2(VI) C2 interaction were then assembled using collagen triple-helical models with the following sequences: (EAGSOGERGDQGARG)3 from alpha 2(VI), (PRGEKGERGDVGIRG)3 from alpha 3(VI), and a model with sequence ((PKGDOGAFGLKGEKG) (EAGSOGERGDQGARG) (PRGEKGERGDVGIRG)) to represent an alpha 1alpha 2alpha 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 alpha 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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INTRODUCTION
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All DNAs encoding the mutated alpha -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 alpha -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 alpha 2(VI) chain was constructed in which the alpha 2(VI) C2 domain was replaced with the alpha 3(VI) C2 domain (see Fig. 2, alpha 2-chimera-normal). When these alpha 2(VI) wild type and alpha 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 alpha 2(VI) chain produced only low levels of monomers (Fig. 4, lanes 1 and 3). The alpha 3(VI) chain was also unable to form dimers (Fig. 4, lane 2). Replacement of the alpha 2(VI) C2 domain with the alpha 3(VI) C2 domain thus abolished dimer formation.


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Fig. 4.   Analysis of alpha 2(VI) and alpha 3(VI) COOH-terminal mutations on dimer assembly. Normal or mutated alpha 2(VI) and alpha 3(VI) chain RNA were translated for 4 h and the products immunoprecipitated using polyclonal antisera VIA and then separated using a 0.5% agarose 3% polyacrylamide composite gel under non-reducing conditions. Lane 1, alpha 2(VI) chain (normal); lane 2, alpha 3(VI) chain (normal); lane 3, alpha 2-chimera-normal; lane 4, alpha 2(VI) DSAET mutant (alpha 2C2/1M mutant); lane 5, alpha 3(VI) DGSER mutant (alpha 3C2/M mutant); lane 6, alpha 2-chimera (mutant). Translation products representing lower monomer, higher monomer, dimer, and tetramer are as shown.

Manipulation of the MIDAS domain within the alpha 2(VI) C2 domain was also found to abolish dimer formation. Thus, when the alpha 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 alpha 3(VI) chain (27) (see Fig. 2, alpha 2C2/1M mutant), no dimers were formed (Fig. 4, lane 4); compare with wild type alpha 2(VI) chain after 4 h translation (see Fig. 4, lane 1). Upon introduction of the alpha 2(VI) chain C2 domain sequence (DGSER) into the alpha 3(VI) chain to replace DSAET (see Fig. 2, alpha 3C2/M mutant), no dimers were formed (Fig. 4, lane 5). In the case of the alpha 2-chimera containing (DSAET), dimer and tetramer formation could be re-instated by re-introducing the MIDAS motif DGSER to replace DSAET (alpha 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 alpha 3(VI) chain C2 domain has a conserved threonine but lacks a conserved aspartate in a position constituting the MIDAS motif, whereas the alpha 2(VI) chain C2 domain contains a conserved aspartate (residue 928) in the coordinating position. We examined whether the conserved aspartate residue within the alpha 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 alpha 3(VI) chain. The Asp-928 right-arrow Thr-928-mutated alpha 2(VI) chain (see Fig. 2, alpha 2C2/2M mutant) produced monomer, dimer, and tetramer assemblies at a slightly lower level to non-mutated alpha 2(VI) chain (Fig. 5, lanes 1 and 2).


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Fig. 5.   Effects of alpha 2(VI) C2 domain MIDAS motif mutations on dimer assembly. Normal or mutated alpha 2(VI) chain RNA were translated for 4 h, the products immunoprecipitated using polyclonal antisera VIA, then separated using a 0.5% agarose 3% polyacrylamide composite gel under non-reducing conditions. Lane 1, alpha 2(VI) chain (normal); lane 2, alpha 2(VI) Thr-928 (alpha 2C2/2M mutant); lane 3, alpha 2(VI) Ser-822 (alpha 2C2/3M mutant). Translation products representing lower monomer, higher monomer, dimer, and tetramer are as shown.

The potential MIDAS motif within the alpha 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 alpha 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, alpha 2C2/3M mutant). After 4 h of translation the Arg-822 right-arrow Ser-822-mutated alpha 2(VI) chain produced a much lower level of monomer, dimer, and tetramer formation when compared with non-mutated alpha 2(VI) chain (Fig. 5, lanes 1 and 3).

Changing the potential MIDAS site in the C1 domain of the alpha 2(VI) chain with sequences in the alpha 3(VI) C1 and C2 domains did not prevent dimer and tetramer formation (see Fig. 2, alpha 2C2/4M and alpha 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 alpha 1 and alpha 2 I-domains (24). A GER triplet is present in the alpha 2(VI) helix sequence at residues 109 to 111. An Glu-110 right-arrow Asp-110-mutated alpha 2(VI) chain (see Fig. 3, alpha 2C2/1H mutant) produced only low levels of monomer assembly (Fig. 6, lane 2). The alpha 3(VI) helix sequence also contains a GER triplet (residues 109-111) in exactly the same position as that found in the alpha 2(VI) helix sequence (residues 109-111). Residues 106-114 (GSPGERGDQ) in the alpha 2(VI) helix were substituted for residues 106-114 (GEKGERGDV) in the alpha 3(VI) helix by site-directed mutagenesis (see Fig. 3, alpha 2C2/2H mutant). The mutated alpha 2 chain produced only low levels of monomer after 4-h translation Fig. 6, lane 3).


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Fig. 6.   Analysis of alpha 2(VI) triple-helical mutations on dimer assembly. Normal or mutated alpha 2(VI) chain RNA was translated for 4 h and the products immunoprecipitated using polyclonal antisera VIA and then separated using a 0.5% agarose 3% polyacrylamide composite gel under non-reducing conditions. Lane 1, alpha  2(VI) chain (normal); lane 2, alpha 2(VI) Asp-110 (alpha 2C2/1H mutant); lane 3, alpha 2(VI) GEKGERGDV (106-114) (alpha 2C2/2H mutant); lane 4, alpha 2(VI) Glu-107 (alpha 2C2/3H mutant); lane 5, alpha 2(VI) Lys-108 (alpha 2C2/4H mutant); lane 6, addition of 0.5 mM alpha ,alpha '-dipyridyl to the reaction; lane 7, alpha  2(VI) Val-114 (alpha 2C2/5H mutant). Translation products representing lower monomer, higher monomer, dimer, and tetramer are as shown.

To delineate which residues within the alpha 2(VI) helix sequence GSPGERGDQ (residues 106-114) are important to type VI collagen assembly, individual constituents were substituted for identically positioned alpha 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 right-arrow Lys-108-mutated alpha 2(VI) chain (see Fig. 3, alpha 2C2/4H mutant) produced monomer, dimer, and tetramer assemblies at a similar level to a non-mutated alpha 2(VI) chain (Fig. 6, lane 5, compare with lane 1). Similarly, an Ser-107 right-arrow Glu-107-mutated alpha 2(VI) chain (see Fig. 3, alpha 2C2/3H mutant) produced monomer, dimer, and tetramer assemblies at a comparable level to non-mutated alpha 2(VI) chain (Fig. 6, lane 4). However, a Gln-114 right-arrow Val-114-mutated alpha 2(VI) chain (see Fig. 3, alpha 2C2/5H mutant) produced only monomers (Fig. 6, lane 7).

The alpha 3(VI) chain did not form dimers and tetramers even when the alpha 2(VI) helical and C2 domains were inserted (see Fig. 3, alpha 3C2/MH mutant) (data not shown).

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ABSTRACT
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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 alpha 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 alpha 2(VI) chain that determine dimer formation. The alpha 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 alpha 1 and alpha 2 I-domains (24). Studies on the interaction between the integrin alpha 2beta 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 alpha 1 chains of types I and IV collagen, GxOGER may account for the ability of several loci in collagens to bind to alpha 2beta 1. The collagenous sequence of the alpha 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 alpha 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 alpha 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 alpha 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 alpha 2(VI) C2 A-domain (A chain) with the triple-helical region of an alpha 2(VI) homotrimer. As in the collagen-integrin alpha 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 alpha 3-alpha 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 alpha 2(VI) C2 (residue Gly-932 in the beta D-alpha 5 loop). A completely equivalent interaction is seen in the collagen-integrin alpha I-domain structure between His-258 in the beta D-alpha 5 loop from the alpha 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.


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Fig. 7.   Ribbon diagram of a model for the interaction between alpha 2(VI) C2 domain in complex with a triple helix of collagen VI alpha 2 residues 104-118. The three loops at the top of the C2 domain, labeled D, S, and R in the beta A-alpha 1 loop, are part of the D main types of secondary structure seen for the DGSER motif that is part of the MIDAS site. A bound metal ion is represented as a blue sphere, with two water molecules participating in metal coordination shown as cyan spheres. Residue Glu-C112 from the middle strand of the triple helix (shown in red as E) completes the coordination of the metal ion. Some other residues shown in red and yellow in the triple helix are thought to be important for binding to the C2 domain (see "Discussion"). The figure was prepared using the program SETOR (35).

One of the distinctive features in the alpha 2(VI) C2 A-domain is the DGSER sequence of the amino acids from the beta A-alpha 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 alpha 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 beta A-alpha 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 beta A-alpha 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 alpha 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 alpha 2beta 1 integrin A-domain in the crystal structure of their complex (14). The model for the interaction between alpha 2(VI) C2 and a homotrimer triple helix made of three alpha 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, alpha 1(VI), alpha 2(VI), and alpha 3(VI). To preserve a distribution of residues similar to the homotrimer case, alpha 2 would obviously need to be the middle strand (main interaction with C2), with alpha 1 being the leading strand (no interaction with C2) and alpha 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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Fig. 8.   Distribution of the residues in collagen VI triple-helical segments that interact with the A-domain type structures. Highlighted in yellow are residues that participate in interactions through their side chains (conformation- and sequence-dependent). Highlighted in cyan are residues that participate in interactions through their main chains (only conformation-dependent). Interactions in a have been observed in crystal structure. Homology models suggest interactions in b and c.


    FOOTNOTES

* 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.

Dagger 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

    ABBREVIATIONS

The abbreviation used is: MIDAS, metal ion-dependent adhesion site.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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