Structure of the Discoidin Domain Receptor 1 Extracellular Region Bound to an Inhibitory Fab Fragment Reveals Features Important for Signaling

Abstract

SummaryThe 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.

Low Stability and a ConservedN-Glycosylation Site Are Associated with Regulation of the Discoidin Domain Receptor Family by GlucoseviaPost-TranslationalN-Glycosylation

“…This is supported by the results of an X-ray crystallographic study, which suggested that asparagine 211 of DDR1 is one of the residues that interacts with calcium in one of the two proposed calcium-binding sites in the discoidin-like domain. 5) In addition, we observed that depletion of calcium impaired the generation of high-molecular-mass forms of DDR1 and DDR2, similarly to the effect of mutations in asparagines 211 and 213 of DDR1 and DDR2 respectively (Wong et al, unpublished results).…”

“…In fact, an X-ray crystallographic study of DDR1 found that asparagine 211 is one of two sites occupied by N-glycan. 5) In addition, our in silico analysis predicted asparagine 213 of DDR2 and asparagine 211 of DDR1 as the sites of highest Nglycosylation potential among the sites in them (Supplemental Table S3). Another possibility is that this site and/or its N-glycan is associated with the binding of calcium in the discoidin-like domain, and that complex formation with calcium is required for the proper posttranslational maturation of DDRs.…”

“…These seven RTKs consisted of ones having numbers, of N-glycosylation consensus sites from a low ( 5) to a high number (>5) and different degrees, of predicted N-glycosylation potential in their extracellular domains. For example, DDR1, DDR2, EphA2, and EphB4 contain a relatively small number of N-glycosylation consensus sites ( 5) with low N-glycosylation potential. By contrast, EGFR, IGF-1R, and c-Met belong to a group of RTKs that contain in total more than five sites for N-glycosylation and have high potential for N-glycosylation (Supplemental Table S2 and ref.…”

“…6,7) Based on an X-ray crystallographic study of DDR1, Carafoli et al suggested that the DS-like domain also plays a role in activating receptors upon ligand binding. 5) Furthermore, their study suggested that the DSlike domain contains two possible calcium ion-binding sites and two N-glycans. One of these was found at asparagine 211, an N-glycosylation site conserved in all vertebrate DDR family proteins (Supplemental Fig.…”

Defective Ca2+ binding in a conserved binding site causes incomplete N-glycan processing and endoplasmic reticulum trapping of discoidin domain receptors

“…17,18) In addition, an X-ray crystallographic study has characterized another structural unit in DDR proteins, called a DS-like domain, which was named owing to a structural similarity to the DS domain. 19) It was suggested that this domain forms a specific contact with the DS domain and is necessary for the activation of the receptors. This study also demonstrated that the DS-like domain contains a calcium-binding site and two N-glycosylation sites.…”