Identification and Characterization of the X-Dimer of Human P-Cadherin: Implications for Homophilic Cell Adhesion

Abstract

Cell adhesion mediated by cadherins depends critically on the homophilic trans-dimerization of cadherin monomers from apposing cells, generating the so-called strand-swap dimer (ss-dimer). Recent evidence indicates that the ss-dimer is preceded by an intermediate species known as the X-dimer. Until now, the stabilized form of the X-dimer had only been observed in E-cadherin among the classical type I cadherins. Herein, we report the isolation and characterization of the analogous X-dimer of human P-cadherin. Small-angle X-ray scattering (SAXS) and site-directed mutagenesis data indicates that the overall architecture of the X-dimer of human P-cadherin is similar to that of E-cadherin. The X-dimerization is triggered by Ca(2+) and governed by specific protein-protein interactions. The attachment of three molecules of Ca(2+) with high affinity (Kd = 9 μM) stabilizes the monomeric conformation of P-cadherin (ΔTm = 17 °C). The Ca(2+)-stabilized monomer subsequently dimerizes in the X-configuration by establishing protein-protein interactions that require the first two extracellular domains of the cadherin. The homophilic X-dimerization is very specific, as the presence of the highly homologous E-cadherin does not interfere with the self-recognition of P-cadherin. These data suggest that the X-dimer could play a key role in the specific cell-cell adhesion mediated by human P-cadherin.

Disruption of cell adhesion by an antibody targeting the cell-adhesive intermediate (X-dimer) of human P-cadherin

“…The position of the peak depends on the self-affinity and shape of the dimer, as well as the exchange rate between the monomeric and dimeric species (Fig. 3a) and are consistent with previous data1519. The importance of the exchange kinetics is manifested by the slightly greater elution volume of EC12 K14E (slow exchange) with respect to EC12 (fast exchange), although we note that in both constructs the strand-swap dimer is the major component.…”

“…1a)15161718. In particular, the mechanism of cell adhesion by human P-cadherin has also been elucidated at the molecular and cellular levels192021. In these studies we have showed that the so-called strand-swap dimer is the active cell adhesive species, in agreement with what has been reported for other classical cadherins.…”

“…Because P-cadherin achieves cell adhesion by forming a strand-swap dimer via the kinetic and thermodynamic intermediate state termed X-dimer21, we employed several constructs each affecting the dimerization process in a predictable manner (Fig. 3a, Supplementary Table 1)21: (i) EC12 samples the three molecular states (monomer, X-dimer, and strand-swap dimer) in a fast exchange of substates, (ii) EC12 K14E is characterized by the slow exchange between monomer and strand-swap dimer and the absence of the intermediate state X-dimer, (iii) MEC12 is distinguished by a fast exchange between the monomer and X-dimer conformations1921, and (iv) MEC12 K14E is unable to dimerize, thus remaining as a monomer at all times.…”

“…Overexpression of cadherin constructs has been described previously19. In brief, each cadherin construct was expressed as a fusion protein with a SUMO tag in Rosetta2 (DE3) cells.…”

“…The X-dimer is stabilized by intermolecular interactions at the hinge region between EC1 and EC2, and can be stably produced by the addition of a residue, generally a Met residue, prior to the N-terminus of the amino acid sequence151922. The X-dimer thus functions as a thermodynamic and kinetic intermediate platform facilitating the conformational changes from the monomer state to the strand-swap dimer state.…”