Figure 1. Desmoglein-2 adhesion is mediated via tryptophan swap at position 2.

(A) Predicted interaction model of desmoglein-2 (DSG2) extracellular domains via exchange of tryptophan residue at position 2 into a hydrophobic pocket of the opposing molecule. Cartoon 3D presentation of PDB entry 5ERD⁹, tryptophan-2 is highlighted by ball and stick presentation. (B) Schematic of single molecule force spectroscopy experiments. Recombinant extracellular domains (EC) of DSG2-WT or DSG2-W2A protein were coupled to a mica surface and AFM cantilever via a human Fc-tag (hFc) and a PEG₂₀-linker and probed as indicated. (C) Binding frequency of DSG2-W2A/DSG2-WT heterotypic and homotypic interactions at a pulling speed of 2 μm/s are shown. hFc served as control for unspecific binding. *P< 0.05, one-way ANOVA, Dunnett’s post hoc test. Each independent coating procedure with minimum 625 force curves is taken as biological replicate. (D) Histogram of binding forces distribution with peak fit at a pulling speed of 2 μm/s corresponding to data in C. (E) Determination of the bond half lifetime via Bell’s equation¹¹ of mean loading rates and binding forces analysed from data of pulling speeds at 0.5, 1, 2, 5, and 7.5 μm/s. Average of values from four independent coating procedures with minimum 625 force curves each. R = R squared, k_off = off rate constant, τ₀ = bond half lifetime under zero force. (F) Dissociation assays to determine cell-cell adhesion were performed in CaCo2 cells (WT or DSG2 KO background) expressing DSG2-WT-mGFP or DSG2-W2A-mGFP constructs. mGFP empty vector served as control. *P< 0.05, one-way ANOVA, Sidak’s post hoc test. Corresponding Western blot analysis confirmed effective expression of DSG2 constructs (*) vs. the endogenous protein (arrow) in CaCo2 cells. α-tubulin (TUBA) served as loading control. (G) displays representative images of monolayer fragmentation from experiments in F. (H) Dissociation assays in immortalized keratinocytes isolated from neonatal murine skin of the respective genotype. *P< 0.05 or as indicated, Welch’s ANOVA, Dunnett’s post hoc test. (I) Macroscopic cardiac phenotype of DSG2-W2A mut/mut mice at the age of 15 weeks. (J) Cardiac hypertrophy was analysed as mean cross-sectional area of cardiomyocytes in haematoxylin/eosin stained sections. Scale bar: 30 μm. *P< 0.05, unpaired Student’s t-test. (K) Representative images of ICDs acquired by TEM, 3 mice per genotype. Orange asterisks mark intercellular widening, orange circle marks a ruptured junction. Scale bar: 1 μm.