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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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Essential Role of Trp52 in DDR2 Function(A) Solid-phase binding assay with recombinant wild-type or W52A DDR2-Fc proteins added for 3 hr at room temperature to 96-well plates coated with either collagen I or BSA. Shown is a representative of three independent experiments, each performed in duplicate.(B) Full-length wild-type or W52A DDR2 was transiently expressed in HEK293 cells. After stimulation for 90 min with collagen I (Coll), aliquots of cell lysates were analyzed by SDS-PAGE and Western blotting. The blots were probed with anti-phosphotyrosine (anti-PY) monoclonal antibody 4G10 (upper blot) or polyclonal anti-DDR2 antibodies (lower panel). The positions of molecular markers (in kilodaltons) are indicated. Collagen I was used at different concentrations as indicated (in μg/ml). The experiment was performed three times with very similar results.
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fig6: Essential Role of Trp52 in DDR2 Function(A) Solid-phase binding assay with recombinant wild-type or W52A DDR2-Fc proteins added for 3 hr at room temperature to 96-well plates coated with either collagen I or BSA. Shown is a representative of three independent experiments, each performed in duplicate.(B) Full-length wild-type or W52A DDR2 was transiently expressed in HEK293 cells. After stimulation for 90 min with collagen I (Coll), aliquots of cell lysates were analyzed by SDS-PAGE and Western blotting. The blots were probed with anti-phosphotyrosine (anti-PY) monoclonal antibody 4G10 (upper blot) or polyclonal anti-DDR2 antibodies (lower panel). The positions of molecular markers (in kilodaltons) are indicated. Collagen I was used at different concentrations as indicated (in μg/ml). The experiment was performed three times with very similar results.

Mentions: Abdulhussein et al. (2004) examined a number of DDR1 point mutants for receptor activation by collagen I. They found that mutation of Arg105 or Ser175 to alanine abolished DDR1 activation, in agreement with our structure. Intriguingly, however, mutation of Trp53 (corresponding to Trp52 in DDR2) did not have an effect on DDR1 activation, and deletion of several residues in loop L1 was required to abolish DDR1 activation by collagen I (Abdulhussein et al., 2004). These findings are difficult to reconcile with the critical role of Trp52 in collagen recognition by DDR2 (Figure 2C). To support our interpretation of the structure, we tested DDR2 W52A mutant constructs for collagen I binding and receptor activation. A soluble ectodomain construct with the W52A mutation was secreted at similar levels as the corresponding wild-type protein, but failed to bind to collagen in an established solid-phase assay (Figure 6A). Likewise, full-length DDR2 W52A expressed in 293 cells could not be activated by collagen (Figure 6B). We conclude that Trp52 is indispensable for collagen recognition and signaling by DDR2. Given that the GVMGFO motif is also the major binding site for DDR1 (authors' unpublished data), we find it difficult to believe that the corresponding tryptophan in DDR1, Trp53, is not required for receptor activation.


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)

Essential Role of Trp52 in DDR2 Function(A) Solid-phase binding assay with recombinant wild-type or W52A DDR2-Fc proteins added for 3 hr at room temperature to 96-well plates coated with either collagen I or BSA. Shown is a representative of three independent experiments, each performed in duplicate.(B) Full-length wild-type or W52A DDR2 was transiently expressed in HEK293 cells. After stimulation for 90 min with collagen I (Coll), aliquots of cell lysates were analyzed by SDS-PAGE and Western blotting. The blots were probed with anti-phosphotyrosine (anti-PY) monoclonal antibody 4G10 (upper blot) or polyclonal anti-DDR2 antibodies (lower panel). The positions of molecular markers (in kilodaltons) are indicated. Collagen I was used at different concentrations as indicated (in μg/ml). The experiment was performed three times with very similar results.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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fig6: Essential Role of Trp52 in DDR2 Function(A) Solid-phase binding assay with recombinant wild-type or W52A DDR2-Fc proteins added for 3 hr at room temperature to 96-well plates coated with either collagen I or BSA. Shown is a representative of three independent experiments, each performed in duplicate.(B) Full-length wild-type or W52A DDR2 was transiently expressed in HEK293 cells. After stimulation for 90 min with collagen I (Coll), aliquots of cell lysates were analyzed by SDS-PAGE and Western blotting. The blots were probed with anti-phosphotyrosine (anti-PY) monoclonal antibody 4G10 (upper blot) or polyclonal anti-DDR2 antibodies (lower panel). The positions of molecular markers (in kilodaltons) are indicated. Collagen I was used at different concentrations as indicated (in μg/ml). The experiment was performed three times with very similar results.
Mentions: Abdulhussein et al. (2004) examined a number of DDR1 point mutants for receptor activation by collagen I. They found that mutation of Arg105 or Ser175 to alanine abolished DDR1 activation, in agreement with our structure. Intriguingly, however, mutation of Trp53 (corresponding to Trp52 in DDR2) did not have an effect on DDR1 activation, and deletion of several residues in loop L1 was required to abolish DDR1 activation by collagen I (Abdulhussein et al., 2004). These findings are difficult to reconcile with the critical role of Trp52 in collagen recognition by DDR2 (Figure 2C). To support our interpretation of the structure, we tested DDR2 W52A mutant constructs for collagen I binding and receptor activation. A soluble ectodomain construct with the W52A mutation was secreted at similar levels as the corresponding wild-type protein, but failed to bind to collagen in an established solid-phase assay (Figure 6A). Likewise, full-length DDR2 W52A expressed in 293 cells could not be activated by collagen (Figure 6B). We conclude that Trp52 is indispensable for collagen recognition and signaling by DDR2. Given that the GVMGFO motif is also the major binding site for DDR1 (authors' unpublished data), we find it difficult to believe that the corresponding tryptophan in DDR1, Trp53, is not required for receptor activation.

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.

Show MeSH