PMC

Heterophilic binding between Dsg- and Dsc-coated beads. Bead aggregation assay showing mixtures of green fluorescent Dsg- or Dsc-coated beads (columns) with similarly coated red fluorescent beads (rows) after 1 h of aggregation. (Scale bar: 0.5 mm.)

Overall structures of Dsg and Dsc extracellular regions. Crystal structures shown in ribbon representation of human Dsg2, Dsg3, Dsc1, Dsc2ΔEC1, and chimera Dsc2_EC1Dsg2_EC2-5 ectodomains (left to right). Ca²⁺ ions are shown as green spheres, and N-linked and O-linked glycans are shown in wheat and violet, respectively. Electron density for EC5 of Dsg3 was not observed, and its likely position is indicated with a dashed line. Predicted intermembrane distances derived from measurements between C-termini are indicated by gray arrows for the complete homodimer structures Dsg2 and Dsc1.

Adhesive interfaces of desmosomal cadherins. (A and B) EC1 domains of strand-swapped homodimer (Dsg2, Dsc1, Dsc2) or self-docked monomer (Dsg3) configurations shown in ribbon representation. Selected interface residues are shown as cyan sticks (see Results), N-glycans are shown in wheat, and clusters of aromatic residues in Dscs are shown as green sticks. (C) Superposition of Dsgs, Dscs, and N-cadherin over EC1, showing differences in the position of the AB-loop. (D) Electrostatic surfaces showing the interface regions in EC1 for each desmosomal cadherin structure. (Scale bar: red, −5 kT; blue, +5 kT.)

Heterophilic interactions between Dsgs and Dscs in SPR. (A) Wild-type Dsg1–Dsg4, Dsc1–Dsc3, or (B) double-mutant Dsg2 W2A A82I and Dsc2 W2A A80I analytes (columns) were tested at concentrations of 3, 6, and 12 µM (12 µM omitted for Dsg4) over surfaces of wild-type Dsg2 (top row), Dsg3, Dsc1, Dsc2, and Dsc3 (bottom row). Responses were normalized for analyte molecular weight differences and are drawn to the same scale across rows. (C) Heterophilic binding affinities (K_D) from fitting of SPR data to 1:1 interaction models (). ±Errors of the fit.

Calcium coordination in the EC3–EC4 linker of desmosomal cadherins. (A) Ribbon representation of EC3–EC4 linkers of desmosomal cadherins and classical N-cadherin. Side chains or backbone carbonyls coordinating Ca²⁺ ions (green spheres) are shown as sticks. Residues that fail to coordinate Ca²⁺ in desmosomal cadherins are highlighted with magenta text, and corresponding Ca²⁺-coordinating residues in classical cadherins are underlined. A Dsc-specific disulfide bond in EC4 is shown as sticks. Violet spheres represent O-linked glycans, and gray spheres represent water. (B) Bijvoet anomalous difference maps, contoured at 3 σ (green mesh), showing Ca²⁺ ion positions in EC3–EC4 for desmosomal cadherins, as in A. (C) Multiple sequence alignment of EC3–EC4 linkers of human Dsgs, Dscs, and N-cadherin. Conserved and nonconserved Ca²⁺-binding residues are highlighted green and red; gray lines indicate Ca²⁺ coordination. (D) Superposition of Cα-traces over domain EC3, colored according to A.

Dependence of Dsg2:Dsc2 binding on charged interface residues. (A and B) SPR affinity measurements of (A) Dsg2 or (B) Dsc2 analytes injected over surfaces captured with Dsc2 in 150, 300, or 600 mM NaCl. Black traces: experimental data at five concentrations from 12 to 0.75 μM. Red traces: fits to 1:1 interaction models. Kinetic constants: K_a 8.3(3) × 10³ M⁻¹⋅s⁻¹, K_d 0.29(1)s⁻¹ (150 mM NaCl); K_a 4(1) × 10³ M⁻¹⋅s⁻¹, K_d 0.2 (5)s⁻¹ (300 mM NaCl); K_a 1.4(4) × 10³ M⁻¹⋅s⁻¹, K_d 0.10(2)s⁻¹ (600 mM NaCl). Errors of last digit in parentheses. (C) Putative Dsg2:Dsc2 EC1 heterodimer model showing interface residues mutated in D–G. (D) Binding of wild-type or mutant Dsg2 over captured Dsc2 surfaces. (E) Binding of wild-type or mutant Dsc2 over Dsg2 surfaces. (F) Dsc2 E99Q E101Q mutant over a Dsc2 surface. (G) Dsg2 K17Q K18Q mutant over a Dsg2 surface. Proteins in D–G tested at 12, 6, and 3 μM; scale on D and E, Left.