Structural basis of adhesive binding by desmocollins and desmogleins Oliver J. Harrisona,b,1, Julia Braschc,1, Gorka Lassoa,d, Phinikoula S. Katsambaa,b, Goran Ahlsena,b, Barry Honiga,b,c,d,e,f,2, and Lawrence Shapiroa,c,f,2 aDepartment of Systems Biology, Columbia University, New York, NY 10032; bHoward Hughes Medical Institute, Columbia University, New York, NY 10032; cDepartment of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032; dCenter for Computational Biology and Bioinformatics, Columbia University, New York, NY 10032; eDepartment of Medicine, Columbia University, New York, NY 10032; and fZuckerman Mind Brain Behavior Institute, Columbia University, New York, NY 10032 Contributed by Barry Honig, April 23, 2016 (sent for review January 21, 2016; reviewed by Steven C. Almo and Dimitar B. Nikolov) Desmosomes are intercellular adhesive junctions that impart dense midline, consistent with a strand-swap mode of interaction first strength to vertebrate tissues. Their dense, ordered intercellular characterized for classical cadherins (19, 20). Nevertheless, the attachments are formed by desmogleins (Dsgs) and desmocollins identity of Dscs and Dsgs in these tomographic reconstructions could (Dscs), but the nature of trans-cellular interactions between these not be determined, and atomic-resolution structures of desmosomal specialized cadherins is unclear. Here, using solution biophysics and cadherins have not been available, with the exception of an NMR coated-bead aggregation experiments, we demonstrate family-wise structure of a monomeric EC1 fragment of mouse Dsg2 with an heterophilic specificity: All Dsgs form adhesive dimers with all Dscs, artificially extended N terminus (PDB ID code 2YQG). In addition, a with affinities characteristic of each Dsg:Dsc pair. Crystal structures small-angle X-ray scattering (SAXS) study showed a classical cad- of ectodomains from Dsg2 and Dsg3 and from Dsc1 and Dsc2 show herin-like monomeric molecular envelope for mouse Dsg2, but with binding through a strand-swap mechanism similar to that of homo- additional flexibility in the membrane-proximal region (21). Here we philic classical cadherins. However, conserved charged amino acids present crystal structures of ectodomains from human Dsgs and Dscs, inhibit Dsg:Dsg and Dsc:Dsc interactions by same-charge repulsion along with solution biophysical analysis and coated-bead aggregation and promote heterophilic Dsg:Dsc interactions through opposite- studies. Our results identify the central importance of heterophilic charge attraction. These findings show that Dsg:Dsc heterodimers binding between Dscs and Dsgs and reveal the structural basis of represent the fundamental adhesive unit of desmosomes and provide trans-cellular adhesive binding between desmosomal cadherins. a structural framework for understanding desmosome assembly. Results Heterophilic Adhesive Binding Between Dsgs and Dscs. Using a desmosome | cell adhesion | cadherin | heterophilic binding transient-transfection HEK-293 cell protein expression system (Ma- terials and Methods), we produced full-length ectodomains for all esmosomes are spot-weld-like intercellular adhesive junc- seven human desmosomal cadherins Dsg1–Dsg4 and Dsc1–Dsc3. We Dtions that link the intermediate filament networks of adja- first assessed the homo-association state of each of these proteins by cent cells to impart strength to the solid tissues of vertebrates (1–3). sedimentation-equilibrium analytical ultracentrifugation (AUC) (SI Dysfunction of desmosomes in inherited and acquired human diseases as well as in mouse genetic ablation studies causes Significance characteristic defects in heart muscle and skin (3–5), demon- strating their importance in tissues that undergo mechanical stress. Desmosomes are crucial for the integrity of tissues that undergo In electron micrographs, the hallmarks of mature desmosomes mechanical stress. Their intercellular attachments are assembled include a constant intermembrane distance of 280–340 Å, and from desmogleins (Dsgs) and desmocollins (Dscs), two families of apparently ordered electron-density in the intercellular space, specialized cadherins whose structures and interactions have often with a discrete midline connected by periodic cross-bridges remained uncharacterized. Our study demonstrates family-wise to the cell membranes (6–9). The intercellular attachments of heterophilic interactions between these proteins, with all Dsgs desmosomes are composed of transmembrane proteins from two forming adhesive dimers with all Dscs. Crystal structures of ecto- specialized cadherin subfamilies: desmocollins (Dscs) and des- domains from Dsg2 and Dsg3 and from Dsc1 and Dsc2 show mogleins (Dsgs). The human genome encodes three Dsc (Dsc1– binding through a strand-swap mechanism similar to that of clas- Dsc3) and four Dsg (Dsg1–Dsg4) proteins, which share an overall sical cadherins, which we show underlie heterophilic interactions. domain organization comprising four to five extracellular cadherin Conserved compatibly charged amino acids in the interfaces pro- (EC) domains, a single-pass transmembrane region, and an in- mote heterophilic Dsg:Dsc interactions. We show that Dsg:Dsc tracellular domain that binds to intermediate filaments via adap- heterodimers represent the fundamental adhesive unit of desmo- tor proteins desmoplakin and plakoglobin (1). Individual Dsgs and somes and provide a structural framework for understanding the Dscs show differential expression patterns: Dsg2 and Dsc2 are extracellular assembly of desmosomes. expressed widely in all desmosome-forming tissues (1), whereas other desmosomal cadherins are expressed specifically in stratified Author contributions: O.J.H., J.B., B.H., and L.S. designed research; O.J.H., J.B., G.L., P.S.K., epithelia with graded, overlapping patterns (1, 10). Notably, both and G.A. performed research; O.J.H., J.B., G.L., P.S.K., G.A., B.H., and L.S. analyzed data; Dscs and Dsgs appear necessary for adhesion in transfected cells and O.J.H., J.B., B.H., and L.S. wrote the paper. (1, 11–13), and loss of either in genetic experiments causes loss of Reviewers: S.C.A., Albert Einstein College of Medicine; and D.B.N., Memorial Sloan-Kettering normal desmosomal adhesion (5, 14, 15). Cancer Center. Although the ultrastructure of desmosomes is well characterized, a The authors declare no conflict of interest. molecular-level understanding of the binding interactions between Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, desmosomal cadherin extracellular regions that assemble these junc- www.pdb.org (PDB ID codes 5ERD, 5EQX, 5IRY, 5ERP, and 5J5J). tions has remained elusive. In particular, whether desmosomal cad- 1O.J.H. and J.B. contributed equally to this work. herins have homophilic preferences or whether interactions occur 2To whom correspondence may be addressed. Email: [email protected] or between heterophilic pairs has been a matter of dispute (1, 11–13, 16– [email protected]. 18). Electron tomography studies of native desmosomes (7, 8) have This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. revealed cadherins binding through their EC1 domains at the central 1073/pnas.1606272113/-/DCSupplemental. 7160–7165 | PNAS | June 28, 2016 | vol. 113 | no. 26 www.pnas.org/cgi/doi/10.1073/pnas.1606272113 Downloaded by guest on September 27, 2021 Appendix,TableS1). Each of the four Dsgs and Dsc2 and Dsc3 To assess their adhesive properties on apposed surfaces, we appeared to be either monomers or very weak dimers in solution with coupled C-terminally biotinylated Dsg or Dsc ectodomains to KD values higher than 400 μM, weaker than the weakest-binding NeutrAvidin-coated beads and monitored their aggregation by classical cadherin (E-cadherin, KD = 156 μM; ref. 22). Only Dsc1 fluorescence microscopy (Fig. 2). As expected from the SPR results showed appreciable homodimerization, but this appeared to be a (Fig. 1A), robust aggregation was observed when beads coated with result of technical issues involving the formation of nonnative inter- Dsgs were mixed with beads coated with Dscs, and no aggregation molecular disulfides and was not observed in other binding assays was observed for any Dsg:Dsg or Dsc:Dsc pair. Because Dscs and (see following text and Materials and Methods). Dsgs were immobilized on separate surfaces, the bead aggregation We then used surface plasmon resonance (SPR) to measure all assays confirm that Dsg:Dsc heterophilic interactions can form in a pairwise homophilic and heterophilic interactions among the seven trans-orientation between apposed surfaces analogous to apposed – cell membranes in desmosomes. Aggregation was dependent on the human desmosomal cadherins (Fig. 1 A C). Consistent with the 2+ AUC results, no significant homophilic interactions were observed, presence of Ca , and identical results were obtained when Dsg2 and and in addition, no significant binding within families (Dsg:Dsg or Dsc2 were coated together on the same beads (SI Appendix,Fig.S3). Dsc:Dsc) was detected (Fig. 1A). In contrast, all experiments in which Together, the AUC, SPR, and bead aggregation data show that a Dsg was passed over a Dsc surface or a Dsc was passed over a Dsg Dsgs and Dscs form strong strand-swapped adhesive trans interac- surface showed binding responses indicative of strong heterophilic tions with family-wise heterophilic specificity, which are likely to interaction (Fig. 1A). Optimal fits of SPR data were