Cadherin

Cadherins (named for "calcium-dependent adhering proteins") constitute a large superfamily of transmembrane glycoproteins (~115 genes in the human genome) that mediate homophilic cell-cell adhesion, tissue sorting, planar cell polarity, and mechanical force transmission across all Metazoa. Originally discovered in the late 1970s and early 1980s by Masatoshi Takeichi, Rolf Kemler, and François Hyafil through the observation that vertebrate epithelial and embryonic cells dissociate in the absence of extracellular calcium ions (Ca²⁺) unless protected from proteolysis by ~1 to 2 mM Ca²⁺, classical cadherins form the core structural zipper of the adherens junction (zonula adhaerens) and desmosome (macula adhaerens).

Every member of the cadherin superfamily contains between 2 and 34 tandem copies of a folded ~110-amino-acid β-sandwich module called the Extracellular Cadherin (EC) domain. High-resolution X-ray crystallography of the full-length classical cadherin ectodomain (PDB: 3Q2V, Harrison et al., 2011) reveals how two biophysical switches govern cadherin function: (1) at each linker between consecutive EC domains (EC1–EC2, EC2–EC3, EC3–EC4, EC4–EC5), a conserved 12-residue sequence signature (DxNDN, DxD, and LDRE) coordinates three bridging Ca²⁺ ions (3×Ca2+) that transform the ectodomain from a floppy, protease-sensitive string into a stiff, curved crescent rod; and (2) at the N-terminal tip of EC1, two cadherins protruding from opposing cells undergo 3D domain swapping ("strand-swapping"), inserting a conserved tryptophan side chain (Trp2) into a hydrophobic acceptor pocket on the partner EC1 domain while transmitting piconewton tension across the plasma membrane through p120-catenin, β-catenin, and α-catenin to the cortical F-actin cytoskeleton.

Based on ectodomain repeat number, strand-swapping geometry, and cytoplasmic signaling motifs, the vertebrate cadherin superfamily is divided into four major families:

1. Classical Cadherins (Type I and Type II): Contain five extracellular repeats (EC1–EC5), a single transmembrane α-helix, and a highly conserved ~150-residue cytoplasmic tail. Type I classical cadherins—including E-cadherin (CDH1) in epithelia, N-cadherin (CDH2) in neurons and cardiomyocytes, P-cadherin (CDH3) in placenta/basal epidermis, and R-cadherin (CDH4) in retina—possess a single conserved Trp2 residue and a His-Ala-Val (HAV) pocket motif in EC1. Type II classical cadherins (e.g., VE-cadherin / CDH5 in vascular endothelium, CDH6–12) lack the HAV motif and utilize two conserved tryptophans (Trp2 and Trp4) to anchor a larger hydrophobic strand-swap interface.
2. Desmosomal Cadherins (Desmogleins DSG1–4 and Desmocollins DSC1–3): Contain five EC1–EC5 ectodomains that form hyper-adhesive heterophilic trans junctions across the desmosome, but possess divergent cytoplasmic tails that bind plakoglobin (γ-catenin) and plakophilins to anchor intermediate filaments (keratins in epidermis; desmin in cardiac intercalated discs) rather than F-actin.
3. Protocadherins (Clustered PCDHA, PCDHB, PCDHG and Non-Clustered): Possess six or seven EC repeats (EC1–EC6/7) and lack the EC1 Trp2 strand-swap mechanism; instead, clustered α-, β-, and γ-protocadherins (~53 tandem variable exons in humans) form antiparallel EC1–EC4 head-to-tail trans zippers that provide a combinatorial "molecular barcode" for neuronal self-avoidance in dendrites.
4. Atypical Giant Cadherins (Fat FAT1–4, Dachsous DCHS1–2, and Flamingo/CELSR): Span 27 to 34 EC repeats (or 9 EC repeats coupled to a 7-pass GPCR transmembrane domain in CELSR/Flamingo) and regulate tissue growth via the Hippo–YAP/TAZ pathway and planar cell polarity (PCP).

Crystal Structure PDB: 3Q2V and the 3×Ca2+ Mechanical Hinge

Each individual EC domain (~110 residues) folds into a seven-stranded Greek-key β-barrel (A through G β-strands) structurally related to immunoglobulin domains. However, unlike immunoglobulin domains—which lack interdomain metal coordination—every junction between adjacent cadherin repeats (EC1–EC2, EC2–EC3, EC3–EC4, and EC4–EC5) forms an obligate three-calcium (3×Ca2+) binding pocket designates sites Ca1, Ca2, and Ca3. Across all metazoans and choanoflagellates, these three Ca²⁺ ions are octahedrally coordinated by negatively charged aspartate (Asp/D), glutamate (Glu/E), and asparagine (Asn/N) side-chain oxygens contributed by three invariant sequence motifs:

• DxNDN (or DxNDNAPxF): The canonical 5-residue interdomain linker joining the C-terminus of the preceding EC domain to the N-terminus of the next EC domain.
• DxD (DADD / DQND): Located on the B–C loop of the upper EC domain.
• LDRE (LDFET / LDYE): Located on the E–F loop of the upper EC domain, whose conserved glutamate bridges two adjacent Ca²⁺ ions simultaneously.

Biophysically, the Ca1–Ca3 sites bind Ca²⁺ cooperatively with macroscopic dissociation constants of K_d ≈ 20 to 450 μM. Because physiological extracellular fluid contains 1.2 to 2.0 mM free Ca²⁺, all 12 calcium sites along the EC1–EC5 ectodomain remain fully saturated in vivo, locking the five EC domains into an elongated, ~22-nm crescent-shaped rigid rod curved by ~90° from EC1 to EC5. If extracellular Ca²⁺ drops below ~100 μM (or is chelated by EDTA/EGTA), the acidic aspartate/glutamate cages electrostatically repel one another, causing the ectodomain to collapse into a globular, disordered conformation that cannot bridge the ~25-nm intercellular synaptic/epithelial cleft.

Two-Step X-Dimer to Trp2 Strand-Swapped Trans-Dimerization

In the crystal lattice of full-length E-cadherin, N-cadherin, and C-cadherin (PDB: 3Q2V), adhesive cell-cell junctions assemble through a cooperative two-stage thermodynamic pathway combining orthogonal trans interactions (between opposing cells) and cis interactions (between cadherins on the same cell membrane):

1. Kinetic Intermediate (X-Dimer): Two curved ectodomains from opposing cells initially collide near their EC1–EC2 calcium-binding linker region (involving Lys14 and Asp138) to form a cross-shaped X-dimer. Although the X-dimer has modest equilibrium affinity (K_d ≈ 100–300 μM), it lowers the activation energy barrier for opening the N-terminal A* β-strand of EC1 and exhibits catch-bond behavior under tensile shear.
2. Mature Trp2 Strand-Swapped Trans-Dimer: The N-terminal A* strand (residues 1–10) of each EC1 domain undocks from its own monomer and swaps across the intercellular interface, inserting the bulky indole ring of Tryptophan-2 (Trp2) deep into a conserved hydrophobic acceptor pocket lined by Ala80, Ile92, and Glu89 (where Glu89 forms a buried salt bridge with the free N-terminal α-amino group of Asp1/Trp2). Mutating Trp2 to alanine (W2A) completely abolishes strand swapping and eliminates homophilic cell-cell adhesion.
3. 2D Parakrystalline Junction Lattice (Cis Packing): Once trans-dimers lock opposing membranes at a fixed ~25 nm separation, loss of rotational entropy promotes weak lateral cis interactions between the convex surface of EC1 on one molecule and the concave EC2–EC3 groove of its neighbor on the same cell, condensing hundreds of cadherin dimers into an ordered 2D hexagonal cluster (adherens junction plaque).

The JMD (p120-Catenin) and CBD (β-Catenin → α-Catenin) Mechanosensor

The ~150-amino-acid cytoplasmic tail of classical cadherins is intrinsically disordered in isolation but folds upon binding two armadillo-repeat proteins:

1. Juxtamembrane Domain (JMD) → p120-Catenin: Located immediately inside the plasma membrane, the JMD contains a conserved core motif (EED and GGG) that binds p120-catenin (CTNND1). By masking an endocytic dileucine motif on the cadherin tail, p120-catenin prevents Hakai-mediated ubiquitination and clathrin-dependent endocytosis, stabilizing cadherins at the cell surface while locally inhibiting RhoA and activating Rac1/Cdc42.
2. Catenin-Binding Domain (CBD) → β-Catenin → α-Catenin: The distal C-terminal CBD binds along the positively charged groove of the 12 armadillo repeats of β-catenin (CTNNB1) with picomolar affinity once phosphorylated by casein kinase II (CK2). The N-terminal domain of β-catenin in turn binds the monomeric form of α-catenin (CTNNA1).

Single-molecule optical tweezers experiments (Buckley et al., 2014) demonstrated that the cadherin–β-catenin–α-catenin complex acts as a two-state mechanical catch bond with cortical F-actin. At zero force, α-catenin adopts an auto-inhibited conformation that binds F-actin weakly; however, when actomyosin contraction applies 5 to 15 piconewtons (pN) of tensile force across the adherens junction, two simultaneous events occur: (1) the C-terminal actin-binding domain (ABD) of α-catenin locks tightly onto the F-actin filament (increasing bond lifetime >10-fold), and (2) the central M-domain of α-catenin unfolds, exposing a cryptic α-helical binding site that recruits vinculin to reinforce the junction with additional actin bundles.

The E-to-N Cadherin Switch and Epithelial-to-Mesenchymal Transition (EMT)

Because homophilic binding affinities differ between Type I cadherin paralogs (N-cadherin homodimers have a K_d ≈ 22 μM compared to E-cadherin K_d ≈ 100 μM, while heterophilic E:N dimers have intermediate affinity), mixed populations of cells expressing different cadherins spontaneously sort out by differential interfacial tension (Steinberg's Differential Adhesion Hypothesis). During embryonic gastrulation, neural crest delamination, and carcinoma invasion, transcription factors of the Snail (SNAI1/2), ZEB (ZEB1/2), and Twist (TWIST1) families bind E-box motifs (CANNTG) in the CDH1 promoter to repress E-cadherin expression while upregulating mesenchymal N-cadherin (CDH2)—a developmental program termed the Epithelial-to-Mesenchymal Transition (EMT). In human pathology, germline inactivating mutations in CDH1 cause Hereditary Diffuse Gastric Cancer (HDGC) and invasive lobular breast carcinoma, in which malignant "signet-ring" cells lose epithelial cohesion and infiltrate the submucosa as isolated single cells.

Why does treating cultured epithelial cells with trypsin and EDTA cause them to detach from one another into a single-cell suspension?

Standard cell-dissociation buffers combine EDTA (which chelates extracellular Ca²⁺ ions below the 20–450 μM K_d of the DxNDN/DxD/LDRE interdomain hinges) with the protease trypsin. As soon as EDTA strips the three Ca²⁺ ions from each EC linker, the cadherin ectodomain loses its rigid crescent structure (PDB: 3Q2V), undocks its Trp2 strand-swapped trans-dimers, and exposes previously buried peptide loops between the EC domains to rapid cleavage by trypsin. If 2 mM Ca²⁺ is kept present, classical cadherins remain completely resistant to trypsin digestion.

Why does inserting the first amino acid (proline or methionine) left uncleaved at the N-terminus of a classical cadherin destroy cell adhesion?

Classical cadherins are synthesized as inactive pro-cadherins containing an N-terminal pro-domain (~130 residues) that must be cleaved off in the trans-Golgi network by furin proteases at a conserved Arg-X-Lys-Arg site. Cleavage generates the exact mature N-terminus starting at Asp1–Trp2. In the strand-swapped trans-dimer, the positively charged α-amino group (-NH3+) of residue 1 forms a buried salt bridge with Glu89 inside the partner's hydrophobic pocket while Trp2 docks inside the cavity; if even two extra amino acids remain attached at the N-terminus, steric clash prevents Trp2 from entering the pocket and abolishes adhesion.

Did cadherins evolve before or after the origin of multicellular animals?

Cadherins evolved before animals. Unicellular and colonial choanoflagellates (Monosiga brevicollis and Salpingoeca rosetta) encode 23 to 29 cadherin genes containing genuine EC repeats and DxNDN Ca²⁺-binding hinges that localize to the actin feeding collar to capture bacterial prey. The key evolutionary innovation at the stem of Metazoa (present in sponges, placozoans, cnidarians, and bilaterians) was the fusion of these extracellular EC repeats to a cytoplasmic catenin-binding domain (CBD), physically coupling extracellular homophilic adhesion to the intracellular β-catenin/α-catenin/F-actin cytoskeleton.

1. Boggon, T. J., Murray, J., Chappuis-Flament, S., Wong, E., Gumbiner, B. M., & Shapiro, L. (2002). "C-cadherin ectodomain structure and implications for cell adhesion mechanisms." Science, 296(5571), 1308–1313. https://doi.org/10.1126/science.1071559
2. Harrison, O. J., Jin, X., Hong, S., Bahna, F., Ahlsen, G., Brasch, J., ... & Shapiro, L. (2011). "The extracellular architecture of adherens junctions revealed by crystal structures of type I cadherins." Structure, 19(2), 244–256. https://doi.org/10.1016/j.str.2010.11.016
3. Buckley, C. D., Tan, J., Anderson, K. L., Hanein, D., Volkmann, N., Weis, W. I., ... & Dunn, A. R. (2014). "The minimal cadherin-catenin complex binds to actin filaments under force." Science, 346(6209), 1254211. https://doi.org/10.1126/science.1254211
4. Abedin, M., & King, N. (2008). "The premetazoan ancestry of cadherins." Science, 319(5865), 946–948. https://doi.org/10.1126/science.1151084