Structure of the discoidin domain receptor 1 extracellular region bound to an inhibitory Fab fragment reveals features important for signaling

Abstract

The discoidin domain receptors, DDR1 and DDR2, are constitutively dimeric receptor tyrosine kinases that are activated by triple-helical collagen. Aberrant DDR signaling contributes to several human pathologies, including many cancers. We have generated monoclonal antibodies (mAbs) that inhibit DDR1 signaling without interfering with collagen binding. The crystal structure of the monomeric DDR1 extracellular region bound to the Fab fragment of mAb 3E3 reveals that the collagen-binding discoidin (DS) domain is tightly associated with the following DS-like domain, which contains the epitopes of all mAbs. A conserved surface patch in the DS domain outside the collagen-binding site is shown to be required for signaling. Thus, the active conformation of the DDR1 dimer involves collagen-induced contacts between the DS domains, in addition to the previously identified association of transmembrane helices. The mAbs likely inhibit signaling by sterically blocking the extracellular association of DDR1 subunits.

Graphical abstract

Figure 1

The Anti-DDR1 mAbs Block Collagen-Induced DDR1 Phosphorylation DDR1b was transiently expressed in HEK293 cells, and the cells were stimulated with 10 μg/ml collagen I in the absence or presence of 10 μg/ml of the indicated anti-DDR1 mAbs. Aliquots of cell lysates were analyzed by SDS-PAGE and western blotting. The blots were probed with anti-phosphotyrosine (anti-PY) mAb 4G10 (upper blot) and reprobed with anti-DDR1 Ab (lower blot). Control, mouse IgG1 isotype control Ab. The experiment was performed three times with similar results. See also Figure S1.

Figure 2

The Anti-DDR1 mAbs Bind to the DS-Like Domain and Do Not Inhibit Ligand Binding (A) ELISA showing binding of the indicated anti-DDR1 mAbs to recombinant DDR proteins immobilized on 96-well plates. Shown is a representative of three independent experiments, each performed in duplicate. (B) Solid-phase binding assay with recombinant DDR1-Fc protein added to 96-well plates coated with either casein or collagen peptide III-23 (Xu et al., 2011). DDR1-Fc was preincubated with the indicated anti-DDR1 mAbs before addition to the wells. Bound DDR1-Fc was detected with anti-human Fc Ab and was measured as absorbance at 492 nm. Shown is a representative of three independent experiments, each performed in triplicate. The error bars indicate the sample standard deviation (n = 3).

Figure 3

Crystal Structure of the DDR1-3E3 Fab Complex (A) Overall structure. The 3E3 Fab fragment is shown as a surface (tan, light chain; gray, heavy chain), and DDR1 is shown as a cartoon (cyan, DS domain; green, DS-like domain; and red, collagen-binding loops; Carafoli et al., 2009; orange, disulphide bridges). A calcium ion is shown as a magenta sphere and the two N-linked glycans are shown as light blue sticks. The N and C termini of the DDR1 construct are indicated. The β strands of the jelly roll in the DS and DS-like domains are numbered 1–8, and the extra β strands in the DS-like domain are labeled a–e. (B) Superposition of the DS domain (cyan) and the DS-like domain (green) of DDR1. (C) Detailed structure of the interface between the DS domain (cyan) and the DS-like domain (green) in DDR1. Selected residues are shown in atomic detail and labeled. Hydrogen bonds are indicated by dashed lines. See also Figures S2 and S3.

Figure 4

A Conserved Patch in the DS Domain Is Required for DDR1 Signaling (A) The lattice contact resulting in a symmetric DDR1 dimer (see text). The DDR1 molecule on the left is in cyan (DS domain) and green (DS-like domain); the DDR1 molecule on the right is in gray, with the collagen-binding loops (Carafoli et al., 2009) in red. The 2-fold symmetry axis is vertical. Selected residues are shown in atomic detail (pink, conserved surface patch in the DS domain). (B) Cell surface expression of mutants. Wild-type DDR1b or the indicated mutants were transiently expressed in HEK293 cells. The cells were stained on ice with 10 μg/ml of anti-DDR1 mAb 7A9 (filled gray histograms) or mouse IgG1 isotype control Ab (black lines) followed by FITC-conjugated goat-anti mouse IgG and analysis by flow cytometry. The experiment was performed twice with similar results. (C) Collagen-induced activation of mutants. Wild-type DDR1b or the indicated mutants were transiently expressed in HEK293 cells. The cells were stimulated with collagen I at the indicated concentrations (in μg/ml). Aliquots of cell lysates were analyzed by SDS-PAGE and western blotting. The blots were probed with anti-phosphotyrosine (anti-PY) mAb 4G10 (upper blot) and reprobed with anti-DDR1 Abs (lower blot). The experiment was performed three times with similar results. (D) Solid-phase binding assay with recombinant DDR1-Fc protein (filled circles, wild-type; open circles, R32E mutant) added to 96-well plates coated with collagen peptide III-23 (Xu et al., 2011). Bound DDR1-Fc was detected with anti-human Fc Ab and was measured as absorbance at 492 nm. Shown is a representative of two independent experiments, each performed in duplicate.

Figure 5

Detailed Structure of the DDR1-3E3 Fab Interface The 3E3 Fab fragment is shown as a semitransparent surface (tan, light chain; gray, heavy chain), and the DDR1 region interacting with the Fab is shown as a green cartoon. Selected interface residues are shown in atomic detail and labeled. Hydrogen bonds are indicated by dashed lines.

Figure 6

Epitope Mapping of Anti-DDR1 mAbs (A) Wild-type DDR1b or the indicated mutants were transiently expressed in HEK293 cells. The cells were stained on ice with 10 μg/ml of the indicated anti-DDR1 mAbs or mouse IgG1 isotype control Ab, followed by FITC-conjugated goat-anti mouse IgG and analysis by flow cytometry. Binding of isotype control Ab is shown by the filled gray histograms. Shown are representative data of at least three experiments for each DDR1 mutant. (B) Surface representation of DDR1 structure showing the location of mAb epitopes determined by mutation (mut1, blue: 3G10, 3H10, 7A9; mut6, purple: 1F7, 1F10). The 3E3 footprint from the crystal structure is shown in light orange, and mut5 is shown in dark orange. The DS and DS-like domains are in cyan and green, respectively. The collagen-binding site and conserved surface patch (Arg32, Leu99, Leu152, and Tyr183) are shown in red and light pink, respectively. The C terminus is indicated. See also Figure S4.