Family-wide Structural and Biophysical Analysis of Binding Interactions among Non-clustered δ-Protocadherins

Abstract

Graphical Abstract Highlights d d-pcdh adhesive interactions are preferentially homophilic with distinct affinities d d1-and d2-pcdhs form canonical adhesive dimers with divergent specificity regions d Ectodomain-mediated cis interactions are not observed, in contrast to clustered pcdhs SUMMARYNon-clustered d1-and d2-protocadherins, close relatives of clustered protocadherins, function in cell adhesion and motility and play essential roles in neural patterning. To understand the molecular interactions underlying these functions, we used solution biophysics to characterize binding of d1-and d2-protocadherins, determined crystal structures of ectodomain complexes from each family, and assessed ectodomain assembly in reconstituted intermembrane junctions by cryoelectron tomography (cryo-ET). Homophilic trans (cell-cell) interactions were preferred for all d-protocadherins, with additional weaker heterophilic interactions observed exclusively within each subfamily. As expected, d1-and d2-protocadherin trans dimers formed through antiparallel EC1-EC4 interfaces, like clustered protocadherins. However, no ectodomain-mediated cis (same-cell) interactions were detectable in solution; consistent with this, cryo-ET of reconstituted junctions revealed dense assemblies lacking the characteristic order observed for clustered protocadherins.Our results define non-clustered protocadherin binding properties and their structural basis, providing a foundation for interpreting their functional roles in neural patterning.

Protocadherins at the Crossroad of Signaling Pathways

“…δ-PCDH are also characterized by homophilic interactions in trans (Hoshina et al, 2013;Bisogni et al, 2018;Harrison et al, 2020). Structurally, the trans-dimers formed by cPcdhs and δ2-Pcdhs were proven to be quite similar.…”

“…However, recent structural evidence obtained from the investigation of δ-Pcdhs in solution was not able to substantiate any ECD-mediated cis interactions. Moreover, no conservation of cis-interface motifs characterizing cPcdh ECDs was found in δ-Pcdhs, thereby ruling out cPcdh-like cis-interactions through the ECD for δ-Pcdhs (Harrison et al, 2020). The observed cis interactions between Pcdh19 with other δ2-Pcdhs in K562 cells might therefore rely on transmembrane or ICD interactions (Figure 3).…”

“…Besides homophilic trans binding, cell aggregation assays, solution biophysical measurements and X-ray crystallography have shown that cPcdhs can engage in homotypic and heterotypic dimers on the same cell membrane via cis interactions, expanding the array of unique cell surface protein identities (Schreiner and Weiner, 2010;Thu et al, 2014;Rubinstein et al, 2015;Goodman et al, 2016a). Similar cis interactions through the extracellular domains have not been found for ncPcdhs (Harrison et al, 2020), but it cannot be excluded that other domains (transmembrane or intracellular) mediate cis interactions between these Pcdh members.…”

Proximity-dependent proteomics reveals extensive interactions of Protocadherin-19 with regulators of Rho GTPases and the microtubule cytoskeleton

“…Despite the clear importance of the δ-pcdhs to both development and disease, relatively little is understood of their molecular function. Like the classical cadherins, the protocadherins can mediate homophilic cell adhesion (Bisogni et al, 2018;Cooper et al, 2016;Harrison et al, 2020). However, in contrast to the classical cadherins, the protocadherins do not have conserved binding sites for the armadillo proteins p120-catenin or for β-catenin, which links cadherins to the cytoskeleton though α-catenin (Ozawa et al, 1989;Ozawa and Kemler, 1992).…”

δ-protocadherins control neural progenitor cell proliferation by antagonizing Ryk and Wnt/β-catenin signaling

“…The δ-protocadherins (δ-pcdhs) are homophilic cell adhesion molecules within the cadherin superfamily (Bisogni et al, 2018;Cooper et al, 2016;Harrison et al, 2020), that are subdivided into δ1 and δ2 subfamilies on the basis of the number of extracellular cadherin repeats (7 for δ1-pcdhs and 6 for δ2-pcdhs) and conserved sequence motifs in their intracellular domains (Hulpiau and van Roy, 2011;Vanhalst et al, 2005). In the zebrafish, there are five δ1pcdhs (pcdh1a, pcdh1b, pcdh7a, pcdh7b and pcdh9) and six δ2-pcdhs (pcdh10a, pcdh10b, pcdh17, pcdh18a, pcdh18b and pcdh19), as well as the δ2-like paraxial protocadherin.…”