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Home , Cadherin, Cell adhesion, Desmocollin, Desmoglein, Desmosome, DSC1

Structural basis of adhesive binding by and

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 , Columbia University, New York, NY 10032; bHoward Hughes Medical Institute, Columbia University, New York, NY 10032; cDepartment of 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 (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 and provide trans-cellular adhesive binding between desmosomal cadherins. a structural framework for understanding assembly. Results Heterophilic Adhesive Binding Between Dsgs and Dscs. Using a desmosome | adhesion | | heterophilic binding transient-transfection HEK-293 cell 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 networks of adja- first assessed the homo-association state of each of these 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 as well as in mouse genetic ablation studies causes Significance characteristic defects in heart muscle and (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 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 and (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 , 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 obtained with a underlie intercellular adhesion in desmosomes. 1:1 binding model (SI Appendix, Fig. S1), yielding heterophilic Overall Structures of Dsgs and Dscs. We determined crystal structures KDs ranging between 3.6 and 43.9 μM (Fig. 1C), similar to the affinities of other tight-binding molecules for full-length glycosylated ectodomains of human Dsg2, Dsg3, and SI Ap- (22–28). Interestingly, the strongest-binding pairs (Dsg1:Dsc1 Dsc1 (Fig. 3) with diffraction limits between 2.9 and 3.1Å ( pendix,TableS2). Diffracting crystals of human Dsc2 ectodomains and Dsg4:Dsc1) correspond to those expressed in the outermost were not obtained, but we determined crystal structures of an layers of human skin, and the weakest (Dsg3:Dsc3) to those restricted EC2−EC5 fragment (Dsc2ΔEC1) at 2.7 Å resolution and of the to basal layers (1, 10), consistent with a potential role for differential adhesive EC1 domain in the context of a chimeric protein affinities in maintenance of cell arrangement in stratified epithelia (1). Dsc2EC1Dsg2EC2–EC5 at 3.3Å resolution (Fig. 3 and SI Appendix, Dsgs and Dscs both conserve sequence features in EC1 critical to Table S2). Each desmosomal cadherin extracellular region has an the strand-swap mechanism of adhesive binding in classical cadherins overall structure comprising an elongated, curved arrangement of (20, 29), suggesting strand-swapping mediates heterophilic Dsg:Dsc tandem EC domains connected by calcium-binding linker regions interactions. Consistent with this mechanism, deletion of domain EC1 (Fig. 3). Strand-swapped homodimers, formed between EC1 do- in Dsgs and Dscs abolished all heterophilic interactions, whereas – mains, were observed for Dsg2, Dsc1, and Dsc2EC1Dsg2EC2-5 (Fig. 3). deletion of the C-terminal EC4 EC5 domains had little effect on Evidence of extensive N- and O-linked glycosylation was also ob- binding (SI Appendix,Fig.S2A). Furthermore, combined mutation of served in the electron density maps. Notably, there are conserved, Trp2 (W2A), whose indole side chain anchors the swapping subfamily-specific N-linked glycosylation sites in the adhesive EC1 N-terminal β-strand (the A*-strand), and Ala80/82 [A80I (Dsc2) domains, although none are involved in adhesive contacts, and or A82I (Dsg2)], whose small side chain permits Trp2 docking in contiguous regions of conserved O-linked glycosylation in EC4 (Figs. the dimer-partner acceptor pocket, ablated all heterophilic bind- 3–5andSI Appendix,Fig.S4), corresponding to the novel mono-O- ing of Dsg2 and Dsc2 (Fig. 1B). Interestingly, Dsg2 containing mannosylation sites previously identified in Dsc2 and other cadherins only a W2A mutation on the swapping strand could still mediate by mass spectrometry (30, 31). Because inherited mutations in Dsg2 strong heterophilic binding to Dscs, whereas an A82I acceptor- and Dsc2 are associated with weakened heart muscle desmosomes in pocket mutation ablated all binding (SI Appendix, Fig. S2B). This arrhythmogenic right-ventricular cardio-myopathy (4, 32), we mapped was reversed in Dsc2, where the W2A mutation was more severe. pathogenic missense mutations associated with the onto the These mutants suggest that Dsg:Dsc interactions are asymmetric, Dsg2 and Dsc2 ectodomain structures (SI Appendix,Fig.S5). The with docking of the Dsc A*-strand in the Dsg acceptor pocket mutations were found to target structural elements including cal- representing the dominant interaction. cium-binding residues, glycosylation sites, and disulfide bonds in all

A B C K D Dsg1 Dsg2 Dsg3 Dsg4 [ M] 3.55 11.52 8.07 7.9 Dsc1 ±0.01 ±0.02 ±0.03 ±0.1 21.9 29.0 37.0 22.5 Dsc2 ±0.2 ±0.2 ±0.2 ±0.2 18.1 34.4 43.9 32.5 Dsc3 ±0.3 ±0.6 ±0.6 ±0.4 BIOPHYSICS AND COMPUTATIONAL BIOLOGY

Fig. 1. 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 (KD) from fitting of SPR data to 1:1 interaction models (SI Appendix, Fig. S1). ±Errors of the fit.

Harrison et al. PNAS | June 28, 2016 | vol. 113 | no. 26 | 7161 Downloaded by guest on September 27, 2021 2+ Dsg1 Dsg2 Dsg3 Dsc1 Dsc2 Dsc3 the presence of all three canonical Ca ions in the EC3–EC4 + linker of Dsc1 and Dsc2, whereas only two Ca2 ions were ob- Dsg1 served in Dsg2 and Dsg3, which lacked Ca3 that is normally coordinated primarily by residues in EC4 (Fig. 4A). Sequence analyses (Fig. 4C and ref. 21) show that all Dsgs lack two of the 2+ Dsg2 canonical Ca -binding residues in EC4 corresponding to Asn333 and Gln371 (N-cadherin numbering), explaining the lack of Ca3 coordination and suggesting Dsg1 and Dsg4 will adopt similarly bent 2+ Dsg3 conformations. In Dscs, most Ca -binding ligands are conserved; however, residues equivalent to Gln371 and Asp420, which coordinate Ca3 in classical cadherins, are positioned far from Ca3 in the Dsc Dsc1 structures (Fig. 4 A–C). In addition to the structural role of the EC3– EC4 bend, it is possible that these sites represent loci of flexibility, consistent with recent SAXS analysis of mouse Dsg2 (21), which could Dsc2 be important for the mechanical resilience of desmosomes.

Homodimer Interfaces Reveal Determinants of Heterophilic Specificity. Dsc3 Three of the crystal structures reported here (Dsg2, Dsc1, and Dsc2EC1Dsg2EC2–EC5) reveal cadherins engaged in strand-swapped dimers typical of the adhesive bound-state of classical cadherins Fig. 2. Heterophilic binding between Dsg- and Dsc-coated beads. Bead (Fig. 5). In contrast, the Dsg3 ectodomain structure reveals a mo- aggregation assay showing mixtures of green fluorescent Dsg- or Dsc-coated nomeric auto-inhibited conformation in which the A*-strand of EC1 beads (columns) with similarly coated red fluorescent beads (rows) after 1 h is self-docked into its own domain (Fig. 5A, Right). Both of these of aggregation. (Scale bar: 0.5 mm.) conformations represent distinct states in the 3D-domain swap binding mechanism (24, 33, 34) of desmosomal and type 1 classical cadherins (24), and observation of both forms is consistent with weak EC domains in addition to strand-swap interface residues in EC1. homodimerization affinities observed in our biophysical assays. The Notably, EC3 of Dsg2 contains a cluster of mutation sites, including molecular interactions between the hydrophobic acceptor pocket and four affecting the EC2–EC3 calcium binding linker and three sur- swapped strand in each of the desmosomal cadherin homodimers face-exposed residues in EC3 whose function is currently unknown. and the self-docked monomer form of Dsg3 are highly conserved and The overall structures of each Dsg and Dsc are similar to those are similar to those described for type 1 classical cadherins (24) of classical cadherins, except that a sharp bend is observed be- (SI Appendix,Fig.S6). The docked Trp2 side chain forms hydro- tween domains EC3 and EC4 in Dsg2 and Dsg3, and to a lesser phobic interactions with residues Ile/Val24, Tyr36, Ala80, and degree in Dsc1 and Dsc2 (Figs. 3 and 4). Dsg2 and Dsg3 have Leu92 (numbering according to Dsc1) that line the hydrophobic EC3–EC4 bend angles of ∼103° and ∼99°, whereas Dsc1 and pocket, whereas the indole nitrogen engages in a hydrogen bond Dsc2 each show a bend of ∼124° compared with ∼145° for with the backbone carbonyl of residue 90. In addition, the amino classical N-cadherin (Fig. 4D). These conformational differences terminus of the swapped strand engages in ionic bonds with the + likely result from the incomplete EC3–EC4 Ca2 -coordination acidic side chains of Asp27 and Glu89 of the partner domain. No- that is observed in the structures of desmosomal cadherins tably, the dimer structures reported here represent homodimers, (Fig. 4A). We produced Bijvoet difference Fourier maps using which our binding data show to be of very low affinity compared with + low-energy X-rays to directly visualize bound Ca2 by anomalous Dsg:Dsc heterodimers (Figs. 1 and 2 and SI Appendix,TableS1), scattering (Fig. 4B). Anomalous density maps clearly indicated and are presumably observed as a result of the high cadherin

C EC5 C EC5 EC5 C EC4 EC4 N465 N412 EC4 N413 N465 T431/433/435 329 Å S424/T426 326 Å N413 T431/433/435 EC3 N260 EC3 EC3 T321 N260

N133 EC2 EC2 T223 EC2 N133 N31 N63 T203/205 N61 N EC1 EC1 N EC1 N N N N EC1 EC1 N EC1 N EC1 N63 T203/205 Asn31 T203/205 EC2 EC2 EC2 EC2 EC2 N133 N31 N133 N131 T223

N257 N260 EC3 N260 EC3 EC3 EC3 EC3 T423/425 T431/433/435 T431/433/435 T427/429/431 S424/T428 N411 N413 N413 N494 N465 N465 EC4 EC4 EC4 EC4 EC4 C C EC5 C EC5 EC5 EC5 C C

Dsg2 Dsg3 Dsc1 Dsc2 EC1 Dsc2EC1Dsg2EC2-5

Fig. 3. Overall structures of Dsg and Dsc extracellular regions. Crystal structures shown in ribbon representation of human Dsg2, Dsg3, Dsc1, Dsc2ΔEC1, and 2+ chimera Dsc2EC1Dsg2EC2-5 ectodomains (left to right). Ca 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.

7162 | www.pnas.org/cgi/doi/10.1073/pnas.1606272113 Harrison et al. Downloaded by guest on September 27, 2021 A

Fig. 4. 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 car- + bonyls coordinating Ca2 ions (green spheres) are B + shown as sticks. Residues that fail to coordinate Ca2 in desmosomal cadherins are highlighted with ma- D genta text, and corresponding Ca2+-coordinating residues in classical cadherins are underlined. A Dsc- specific disulfide bond in EC4 is shown as sticks. Vi- olet spheres represent O-linked glycans, and gray spheres represent water. (B) Bijvoet anomalous dif- C ference maps, contoured at 3 σ (green mesh), + showing Ca2 ion positions in EC3–EC4 for desmo- somal 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) Super- position of Cα-traces over domain EC3, colored ac- cording to A.

concentrations in the crystals. Heterophilic Dsg:Dsc interactions are interface in Dsgs and a smaller negative patch in Dscs (Fig. 5D). nonetheless likely to depend on the same binding mechanism, The large positive Dsg potentials arise primarily from Arg10, according to our mutational data (Fig. 1B and SI Appendix,Fig.S2). Lys17, Lys/Arg18, and Lys97/99 (Fig. 5A), which are conserved Structural features of the EC1 interface regions of Dsgs and specifically in Dsgs (SI Appendix, Fig. S4). Notably, Lys17 and Dscs suggest a molecular basis for their family-wise heterophilic Lys/Arg18 are positioned close to the interface in part as a result specificity. Electrostatic potentials mapped to the molecular of a Dsg-specific shift of the AB-loop of ∼10 Å in Cα position surfaces of the Dsg2, Dsg3, Dsc1, and Dsc2 EC1 domains reveal relative to Dscs and classical cadherins (Fig. 5C). In the Dsg:Dsg an electropositive patch near the base of the strand-swapped dimers, the electropositive residues are brought together at Cα–Cα

Dsg2 Dsg3 A A1 C W2 W2 E1 W2 W2 EC1 EC1 EC1 EC1 N63 N-glycan P20 K99 AB loop N19 P18 (Dsg2/Dsg3) N63 K97 AB loop N63 N-glycan K/R18 N-glycan K18 R18 (Dsc1/Dsc2/ F17 N63 K17 N-glycan R10 N-cad) P16 K17 G15 K17 K17 R10 N31 N31 N-glycan N-glycan N31 N-glycan Y86 N31 B Y86 Dsc1 Y86 Dsc2 Y86 R1 Y90 Y86 Y86 N-glycan Y32 R1 Y32 Y32 Y32 Y32 W2 Y32 Y79 EC1 Y79 EC1 EC1 R1 EC1 F35 F79 F79 F35 W2 Y35 F79 Y35 Y77 W2 Tyr77 Y35 Y35 Y52 Y52 Y52 Y52 Y52 Y61 Y61 F61 F61 Y71 Y71 Y71 Y71 Y61 Y71 F20 F20 Y71 F20 E99 E99 E99 E99

D101 Fig. 5. Adhesive interfaces of desmosomal cadherins.

D (A and B)EC1domainsofstrand-swappedhomodimer BIOPHYSICS AND -5kT (Dsg2, Dsc1, Dsc2) or self-docked monomer (Dsg3) con- COMPUTATIONAL BIOLOGY figurations shown in ribbon representation. Selected interface residues are shown as cyan sticks (see Re- sults), 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- +5kT loop. (D) Electrostatic surfaces showing the interface regions in EC1 for each desmosomal cadherin struc- Dsg2 Dsg3 Dsc1 Dsc2 ture. (Scale bar: red, −5 kT; blue, +5 kT.)

Harrison et al. PNAS | June 28, 2016 | vol. 113 | no. 26 | 7163 Downloaded by guest on September 27, 2021 ABC

D F

E G

Fig. 6. Dependence of Dsg2:Dsc2 binding on charged interface residues. (A and B) SPR affinity measurements of (A)Dsg2or(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. 3 −1 −1 −1 3 −1 −1 −1 3 −1 −1 −1 Kinetic constants: Ka 8.3(3) × 10 M ·s , Kd 0.29(1)s (150 mM NaCl); Ka 4(1) × 10 M ·s , Kd 0.2 (5)s (300 mM NaCl); Ka 1.4(4) × 10 M ·s , Kd 0.10(2)s (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.

distances of ∼10 Å, leading to moderate electrostatic repulsion G), suggesting, along with the salt-dependence data, that additional (Fig. 5A and SI Appendix, Fig. S7), the exact magnitude of which residues weaken within-family binding. would depend on side chain and EC1/EC1 orientation. Compa- rable repulsive interactions are also evident in Dsc:Dsc dimers, Discussion where they arise primarily from conserved Dsc-specific acidic The solution biophysics and bead aggregation data reported here residues Glu99 and Asp101 (Fig. 5B and SI Appendix,Fig.S7). demonstrate that all Dsgs form strong heterophilic interactions with We built models of putative Dsg:Dsc heterodimers by superposi- all Dscs, whereas little or no specific binding is observed within the tion over the homodimer structures (SI Appendix,Fig.S8). In each respective subfamilies (Figs. 1 and 2). The heterophilic Dsg:Dsc in- model, the same charged residues that repel in the homodimers (Fig. teractions show characteristics consistent with binding through the 5andSI Appendix,Fig.S7) were positioned to form stabilizing ion strand-swap mechanism (Fig. 1 and SI Appendix,Fig.S2)andcan pairs (Glu99 to Lys/Arg18; Asp/Glu101 to Lys17; SI Appendix, form in a trans orientation between apposed surfaces (Fig. 2). Ecto- Fig. S8). This structural analysis suggests that the proximity of like domain structures of representative Dsgs and Dscs show distinct charges in Dsg:Dsg and Dsc:Dsc dimers and opposite charges in Dsg: features in the conserved strand-swap interface that suggest hetero- Dsc heterodimers provide a likely explanation for the family-specific dimers may be favored in part by electrostatic compatibility between heterophilic binding preferences we observe. We have reported interface regions of Dscs and Dsgs (Fig. 5 and SI Appendix,Fig.S8), previously that the same principle accounts for heterophilic prefer- which is supported by mutational analysis and by the effects of ionic ences in the family of adhesion proteins (25). We tested this strength (Fig. 6). Together, these findings suggest that Dsg:Dsc trans mechanism by investigating the effects of ionic strength on Dsg2:Dsc2 heterodimers represent the basic adhesive unit of desmosomes, which binding, as high salt concentrations should decrease the affinity of is consistent with the observed coexpression of Dsgs and Dscs in interactions with favorable electrostatics (Dsg2:Dsc2) and increase tissues (1), their apparent colocalization on both apposed cell surfaces the affinity of interactions limited by unfavorable electrostatics in desmosomes (1, 11, 35, 36), the requirement for expression of both subtypes for adhesion between transfected cells (1, 11–13), and pre- (Dsg:Dsg and Dsc:Dsc). Consistent with favorable electrostatic in- vious evidence of Dsg2:Dsc2 interaction in solution (16, 17). Al- teractions in Dsg2:Dsc2 heterodimers, we observed that the affinity though desmosome formation by Dsc2 alone has been reported in a of Dsg2:Dsc2 binding in SPR decreased approximately twofold Dsg-depleted colon carcinoma cell line (37), the structure and in- when NaCl concentrations were increased to 600 mM (Fig. 6A). No tegrity of these junctions compared with wild-type desmosomes re- detectable increase in Dsc2 homophilic binding was observed (Fig. mains to be determined. 6B), however, indicating that additional factors may disfavor these Preferential heterophilic binding between Dscs and Dsgs has im- interactions. portant implications for the extracellular structure of desmosomes. In a more targeted approach, we introduced point mutations in Current models of the extracellular architecture of desmosomes are Dsg2 and Dsc2 ectodomains to neutralize or reverse the charges of derived from electron tomography reconstructions of native desmo- Lys17 and Lys18 in Dsg2 and Glu99 and Glu101 in Dsc2, as these somes in plastic embedded or vitrified sections of mouse (7) or hu- residues are predicted to form interacting pairs in our heterodimer man skin (8). Both studies showed cadherin dimers interacting models (Fig. 6C and SI Appendix,Fig.S8). Neutralization of Lys17 through their EC1 domains at the desmosome central dense midline. and Lys18 in Dsg2 by substitution with either glutamine (K17Q Desmosomes of mouse skin appeared dense, but structurally less K18Q) or alanine (K17A K18A) markedly decreased binding to Dsc2 ordered compared with those of human skin, which formed regular in SPR assays, and reversal of the charge by mutation to Glu (K17E 2D arrays of cadherin dimers in the intercellular space. Despite these K18E) further decreased the interaction to background levels (Fig. advances, the specific arrangement of Dsgs and Dscs in these struc- 6D). Similarly, in Dsc2, neutralization of residues Glu99 and Glu101 tures could not be derived from the data, as Dsgs and Dscs could not by substitution with glutamine (E99Q E101Q) or charge reversal be distinguished. However, the overall structures of desmosomal + by substitution with Lysine (E99K E101K) decreased or ablated cadherins we report here suggest that, as a result of differential Ca2 Dsg2:Dsc2 binding (Fig. 6E). The neutralizing mutations did not binding between domains EC3 and EC4, Dsgs and Dscs may adopt measurably affect homodimerization of Dsg2 or Dsc2 (Fig. 6 F and subtly different overall conformations that could potentially be

7164 | www.pnas.org/cgi/doi/10.1073/pnas.1606272113 Harrison et al. Downloaded by guest on September 27, 2021 distinguished in future studies to shed light on how trans hetero- Dsg:Dsc trans-heterodimers, with the likelihood of a close-packed dimers are arranged in the desmosome. arrangement consistent with electron tomography observations that It is of interest to contrast our structural understanding of des- may involve cis hetero-interactions between alternating Dsgs and mosomes to that of adherens junctions (24) formed by related type 1 Dscs on each membrane surface. A complete understanding of des- classical cadherins. First, end-to-end distances seen in Dsc and Dsg ∼ – mosome extracellular architecture will require additional studies to homodimer structures predict intermembrane distances of 326 determine whether extracellular interactions other than the adhesive 329 Å, if perpendicular to the membrane (Fig. 3), which are consistent interactions defined here play a role in desmosome assembly. with measurements of native desmosomes by electron microscopy – (280 340 Å; refs. 7 and 8). In contrast, structures of type 1 classical Materials and Methods cadherin dimers (24) bend less sharply between EC3 and EC4 and are thus longer than Dsgs and Dscs, but paradoxically mediate Recombinant Dsg and Dsc ectodomains were expressed in HEK293 cells and purified shorter intermembrane spacing as a result of the oblique angle at by nickel affinity chromatography and gel filtration. Biotinylated proteins con- which assembled classical cadherins protrude from the membrane in taining C-terminal AviTag sequences were cotransfected with biotin ligase. AUC adherens junctions (24). Second, adherens junctions are formed by a experiments were performed in a Beckman XL-A/I analytical ultracentrifuge at self-assembling 2D molecular layer mediated by cis and trans inter- 25 °C, using UV detection. For SPR, biotinylated proteins were captured over actions (24), and we have found that interplay between cis and trans NeutrAvidin-immobilized surfaces, and binding of analytes at 3, 6, and 12 μMwas interactions is also crucial to clustered function (38). assessed at 25 °C, using a Biacore T100 biosensor. For bead aggregation, biotinylated Given the highly ordered structures observed in cryo-EM tomograms cadherins were captured on fluorescent NeutrAvidin-labeled microspheres, and (8), it seems likely that cis interactions will also be important for aggregation was assessed by fluorescence microscopy after 1 h mixing. For struc- desmosomes. ture determination, protein crystals were grown by vapor diffusion in hanging We do not observe cis interactions in our crystal structures. drops, and X-ray diffraction data were collected from single crystals at 100 K, using However, if such interactions were formed between Dsgs and Dscs, a wavelength of 0.979 Å at the 24-ID-C/E beamlines at Argonne National Labo- they would not be expected to be observed in the homomeric ratory. Structures were solved by molecular replacement using models 3Q2W, structures reported here. Interestingly, the Dsc1 and Dsc2 EC1 do- 3Q2V, and 1L3W. See SI Appendix, Materials and Methods for details. mains contain nine and seven conserved aromatic residues distal from the adhesive interface, respectively, which protrude from the surface ACKNOWLEDGMENTS. We thank Surajit Banerjee, Igor Kourinov, Frank V. Murphy, to give the appearance of an aromatic “pin-cushion” (Fig. 5B and SI and Narayanasami Sukumar at APS for synchrotron data collection support. Appendix,Fig.S4). The function of these unusual surface-exposed cDNA for Dsc3 was provided by Peter Koch (University of Colorado) and Aimee S. Payne (University of Pennsylvania). This work was supported by NIH aromatic residues is unknown, but it is possible that they mediate cis Grants R01 GM062270 (to L.S.) and R01 GM030518 (to B.H.) and NSF grant interactions by binding Dsgs in desmosome assembly. Overall, our Grant MCB-1412472 (to B.H.). X-ray data were collected at the NECAT results suggest a desmosome extracellular architecture composed of beamlines, funded by NIH Grant P41 GM103403.

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