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Crystallographic insight into collagen recognition by discoidin domain receptor 2.

Carafoli F, Bihan D, Stathopoulos S, Konitsiotis AD, Kvansakul M, Farndale RW, Leitinger B, Hohenester E - Structure (2009)

Bottom Line: We have determined the crystal structure of the discoidin domain of human DDR2 bound to a triple-helical collagen peptide.Collagen binding results in structural changes of DDR2 surface loops that may be linked to the process of receptor activation.A comparison of the GVMGFO-binding sites of DDR2 and SPARC reveals a striking case of convergent evolution in collagen recognition.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Imperial College London, London SW7 2AZ, UK.

ABSTRACT
The discoidin domain receptors, DDR1 and DDR2, are widely expressed receptor tyrosine kinases that are activated by triple-helical collagen. They control important aspects of cell behavior and are dysregulated in several human diseases. The major DDR2-binding site in collagens I-III is a GVMGFO motif (O is hydroxyproline) that also binds the matricellular protein SPARC. We have determined the crystal structure of the discoidin domain of human DDR2 bound to a triple-helical collagen peptide. The GVMGFO motifs of two collagen chains are recognized by an amphiphilic pocket delimited by a functionally critical tryptophan residue and a buried salt bridge. Collagen binding results in structural changes of DDR2 surface loops that may be linked to the process of receptor activation. A comparison of the GVMGFO-binding sites of DDR2 and SPARC reveals a striking case of convergent evolution in collagen recognition.

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Collagen Peptide Binding by the DDR2 DS Domain(A) Solid-phase binding assay with recombinant DS2-Fc protein (Leitinger, 2003) added to 96-well plates coated with triple-helical collagen peptides at 10 μg/ml: GPC-(GPP)5-GPRGQOGVXGFO-(GPP)5-GPC-NH2, where X is either methionine or norleucine. Shown is a representative of three independent experiments, each performed in duplicate.(B) Analytical size exclusion chromatograms of the free DDR2 DS domain and its complex with the triple-helical collagen peptide Ac-GPOGPOGPOGPR-GQOGVNleGFOGPOGPOG-NH2. The DS domain and peptide were mixed in the indicated molar ratios. A globular molecular mass standard of 29 kDa, carbonic anhydrase, elutes at 12.3 ml from this column.
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fig1: Collagen Peptide Binding by the DDR2 DS Domain(A) Solid-phase binding assay with recombinant DS2-Fc protein (Leitinger, 2003) added to 96-well plates coated with triple-helical collagen peptides at 10 μg/ml: GPC-(GPP)5-GPRGQOGVXGFO-(GPP)5-GPC-NH2, where X is either methionine or norleucine. Shown is a representative of three independent experiments, each performed in duplicate.(B) Analytical size exclusion chromatograms of the free DDR2 DS domain and its complex with the triple-helical collagen peptide Ac-GPOGPOGPOGPR-GQOGVNleGFOGPOGPOG-NH2. The DS domain and peptide were mixed in the indicated molar ratios. A globular molecular mass standard of 29 kDa, carbonic anhydrase, elutes at 12.3 ml from this column.

Mentions: During the course of our previous study (Konitsiotis et al., 2008), we discovered that substitution of methionine in GVMGFO by the isosteric amino acid norleucine (Nle) increases DDR2 binding in a solid-phase assay ∼10-fold (Figure 1A). We synthesized a number of short triple-helical peptides for co-crystallization with the DDR2 DS domain. The peptides contained the DDR2-binding sequence, GPRGQOGVNleGFO, flanked by 2–3 GPO repeats at either end; the GPRGQO sequence was included because it is required for DDR2 activation in cells (Konitsiotis et al., 2008). Since we obtained crystals with the first peptide tested, Ac-GPOGPOGPOGPRGQOGVNleGFOGPOGPOG-NH2, we did not perform a systematic analysis of the remaining peptides. We used analytical size exclusion chromatography to demonstrate peptide binding to the DDR2 DS domain in solution (Figure 1B). The free DS domain (molecular mass, 20.1 kDa) eluted as a single peak at 12.7 ml, corresponding to a monomer. When the triple-helical collagen peptide Ac-GPOGPOGPOGPRGQOGVNleGFOGPOGPOG-NH2 (molecular mass, 7.9 kDa) was added in a two-fold molar excess, a protein-peptide complex was formed that eluted at 12.0 ml (note that the peptide does not contribute to the absorption at 280 nm). This elution volume is consistent with a complex of 1:1 stoichiometry. Unlike the free DS domain, which is not very soluble, the DS-collagen peptide complex could be concentrated to 10 mg/ml and crystallized. Diffraction data to 1.6 Å resolution were collected using synchrotron radiation and the structure of the DDR2 DS-collagen peptide complex was solved by molecular replacement (Figure 2; Table 1).


Crystallographic insight into collagen recognition by discoidin domain receptor 2.

Carafoli F, Bihan D, Stathopoulos S, Konitsiotis AD, Kvansakul M, Farndale RW, Leitinger B, Hohenester E - Structure (2009)

Collagen Peptide Binding by the DDR2 DS Domain(A) Solid-phase binding assay with recombinant DS2-Fc protein (Leitinger, 2003) added to 96-well plates coated with triple-helical collagen peptides at 10 μg/ml: GPC-(GPP)5-GPRGQOGVXGFO-(GPP)5-GPC-NH2, where X is either methionine or norleucine. Shown is a representative of three independent experiments, each performed in duplicate.(B) Analytical size exclusion chromatograms of the free DDR2 DS domain and its complex with the triple-helical collagen peptide Ac-GPOGPOGPOGPR-GQOGVNleGFOGPOGPOG-NH2. The DS domain and peptide were mixed in the indicated molar ratios. A globular molecular mass standard of 29 kDa, carbonic anhydrase, elutes at 12.3 ml from this column.
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fig1: Collagen Peptide Binding by the DDR2 DS Domain(A) Solid-phase binding assay with recombinant DS2-Fc protein (Leitinger, 2003) added to 96-well plates coated with triple-helical collagen peptides at 10 μg/ml: GPC-(GPP)5-GPRGQOGVXGFO-(GPP)5-GPC-NH2, where X is either methionine or norleucine. Shown is a representative of three independent experiments, each performed in duplicate.(B) Analytical size exclusion chromatograms of the free DDR2 DS domain and its complex with the triple-helical collagen peptide Ac-GPOGPOGPOGPR-GQOGVNleGFOGPOGPOG-NH2. The DS domain and peptide were mixed in the indicated molar ratios. A globular molecular mass standard of 29 kDa, carbonic anhydrase, elutes at 12.3 ml from this column.
Mentions: During the course of our previous study (Konitsiotis et al., 2008), we discovered that substitution of methionine in GVMGFO by the isosteric amino acid norleucine (Nle) increases DDR2 binding in a solid-phase assay ∼10-fold (Figure 1A). We synthesized a number of short triple-helical peptides for co-crystallization with the DDR2 DS domain. The peptides contained the DDR2-binding sequence, GPRGQOGVNleGFO, flanked by 2–3 GPO repeats at either end; the GPRGQO sequence was included because it is required for DDR2 activation in cells (Konitsiotis et al., 2008). Since we obtained crystals with the first peptide tested, Ac-GPOGPOGPOGPRGQOGVNleGFOGPOGPOG-NH2, we did not perform a systematic analysis of the remaining peptides. We used analytical size exclusion chromatography to demonstrate peptide binding to the DDR2 DS domain in solution (Figure 1B). The free DS domain (molecular mass, 20.1 kDa) eluted as a single peak at 12.7 ml, corresponding to a monomer. When the triple-helical collagen peptide Ac-GPOGPOGPOGPRGQOGVNleGFOGPOGPOG-NH2 (molecular mass, 7.9 kDa) was added in a two-fold molar excess, a protein-peptide complex was formed that eluted at 12.0 ml (note that the peptide does not contribute to the absorption at 280 nm). This elution volume is consistent with a complex of 1:1 stoichiometry. Unlike the free DS domain, which is not very soluble, the DS-collagen peptide complex could be concentrated to 10 mg/ml and crystallized. Diffraction data to 1.6 Å resolution were collected using synchrotron radiation and the structure of the DDR2 DS-collagen peptide complex was solved by molecular replacement (Figure 2; Table 1).

Bottom Line: We have determined the crystal structure of the discoidin domain of human DDR2 bound to a triple-helical collagen peptide.Collagen binding results in structural changes of DDR2 surface loops that may be linked to the process of receptor activation.A comparison of the GVMGFO-binding sites of DDR2 and SPARC reveals a striking case of convergent evolution in collagen recognition.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Imperial College London, London SW7 2AZ, UK.

ABSTRACT
The discoidin domain receptors, DDR1 and DDR2, are widely expressed receptor tyrosine kinases that are activated by triple-helical collagen. They control important aspects of cell behavior and are dysregulated in several human diseases. The major DDR2-binding site in collagens I-III is a GVMGFO motif (O is hydroxyproline) that also binds the matricellular protein SPARC. We have determined the crystal structure of the discoidin domain of human DDR2 bound to a triple-helical collagen peptide. The GVMGFO motifs of two collagen chains are recognized by an amphiphilic pocket delimited by a functionally critical tryptophan residue and a buried salt bridge. Collagen binding results in structural changes of DDR2 surface loops that may be linked to the process of receptor activation. A comparison of the GVMGFO-binding sites of DDR2 and SPARC reveals a striking case of convergent evolution in collagen recognition.

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