Highlights d Structures of seven NTD-directed neutralizing antibody complexes with spike or NTD d Structures define distinct recognition classes, one observed in multiple donors d Supersite is glycan free, electropositive, with mobile b-hairpin and flexible loops d Most potent NTD-directed neutralizing antibodies may target this supersite

Summary Adherens junctions, which play a central role in intercellular adhesion, comprise clusters of type I classical cadherins that bind via extracellular domains extended from opposing cell surfaces. We show that a molecular layer seen in crystal structures of E- and N-cadherin ectodomains reported here and in the C-cadherin structure corresponds to the extracellular architecture of adherens junctions. In all three ectodomain crystals, cadherins dimerize through a trans adhesive interface and are connected by a second, cis, interface. Assemblies formed by E-cadherin ectodomains coated on liposomes also appear to adopt this structure. Fluorescent imaging of junctions formed from wild-type and mutant E-cadherins in cultured cells confirm conclusions derived from structural evidence. Mutations that interfere with the trans interface ablate adhesion, whereas cis interface mutations disrupt stable junction formation. Our observations are consistent with a model for junction assembly involving strong trans and weak cis interactions localized in the ectodomain.

Summary Self-avoidance, a process preventing interactions of axons and dendrites from the same neuron during development, is mediated in vertebrates through the stochastic single-neuron expression of clustered protocadherin protein isoforms. Extracellular cadherin (EC) domains mediate isoform-specific homophilic binding between cells, conferring cell recognition through a poorly understood mechanism. Here, we report crystal structures for the EC1-EC3 domain regions from four protocadherin isoforms representing the α, β and γ subfamilies. All are rod-shaped and monomeric in solution. Biophysical measurements, cell aggregation assays, and computational docking reveal that trans binding between cells depends on the EC1-EC4 domains, which interact in an antiparallel orientation. We also show that the EC6 domains are required for the formation of cis-dimers. Overall, our results are consistent with a model in which protocadherin cis-dimers engage in a head-to-tail interaction between EC1-EC4 domains from apposed cell surfaces, possibly forming a zipper-like protein assembly thus providing a size-dependent self-recognition mechanism.

Crystal structures of classical cadherins have revealed two dimeric configurations: in the first, Nterminal β-strands of EC1 domains "swap" between partner molecules. The second configuration (the "X-dimer"), also observed for T-cadherin, is mediated by residues near the EC1-2 calcium binding sites, and N-terminal β-strands of partner EC1 domains, though held adjacent, do not swap. Here we show that strand swapping mutants of type I and II classical cadherins form X-dimers. Mutant cadherins impaired for X-dimer formation show no binding in short timeframe surface plasmon resonance assays but in long timeframe experiments, have homophilic binding affinities close to wild-type. Further experiments show that exchange between monomers and dimers is slowed in these mutants. These results reconcile apparently disparate results from prior structural studies, and suggest that X-dimers are binding intermediates that facilitate the formation of strand swapped dimers.

Type I and II classical cadherins help to determine the adhesive specificities of animal cells. Crystal-structure determination of ectodomain regions from three type II cadherins reveals adhesive dimers formed by exchange of N-terminal beta strands between partner extracellular cadherin-1 (EC1) domains. These interfaces have two conserved tryptophan side chains that anchor each swapped strand, compared with one in type I cadherins, and include large hydrophobic regions unique to type II interfaces. The EC1 domains of type I and type II cadherins appear to encode cell adhesive specificity in vitro. Moreover, perturbation of motor neuron segregation with chimeric cadherins depends on EC1 domain identity, suggesting that this region, which includes the structurally defined adhesive interface, encodes type II cadherin functional specificity in vivo.

Many cell-cell adhesive events are mediated by the dimerization of cadherin proteins presented on apposing cell surfaces. Cadherinmediated processes play a central role in the sorting of cells into separate tissues in vivo, but in vitro assays aimed at mimicking this behavior have yielded inconclusive results. In some cases, cells that express different cadherins exhibit homotypic cell sorting, forming separate cell aggregates, whereas in other cases, intermixed aggregates are formed. A third pattern is observed for mixtures of cells expressing either N-or E-cadherin, which form distinct homotypic aggregates that adhere to one another through a heterotypic interface. The molecular basis of cadherin-mediated cell patterning phenomena is poorly understood, in part because the relationship between cellular adhesive specificity and intermolecular binding free energies has not been established. To clarify this issue, we have measured the dimerization affinities of N-cadherin and E-cadherin. These proteins are similar in sequence and structure, yet are able to mediate homotypic cell patterning behavior in a variety of tissues. N-cadherin is found to form homodimers with higher affinity than does E-cadherin and, unexpectedly, the N/Ecadherin heterophilic binding affinity is intermediate in strength between the 2 homophilic affinities. We can account for observed cell aggregation behaviors by using a theoretical framework that establishes a connection between molecular affinities and cell-cell adhesive specificity. Our results illustrate how graded differences between different homophilic and heterophilic cadherin dimerizaton affinities can result in homotypic cell patterning and, more generally, show how proteins that are closely related can, nevertheless, be responsible for highly specific cellular adhesive behavior.binding affinities ͉ cadherins ͉ cell adhesion ͉ differential adhesion hypothesis ͉ surface plasmon resonance E xpression of different cadherins has been associated with the sorting of cells into distinct layers or compartments (1, 2). This behavior is often viewed as a manifestation of homotypic cell-sorting behavior-like cells adhere to one another. However, cell layers characterized by the expression of different cadherins sometimes remain in contact with one another, suggesting that heterotypic adhesion may also be of physiological relevance. Consistent with in vivo observations, in vitro aggregation assays have shown that cells expressing different classical cadherins can adhere to one another (3, 4). In some such instances, cells form distinct aggregates that possess a common interface, whereas in others, cells are completely mixed. Thus, cells expressing cadherins can exhibit homotypic and/or heterotypic adhesive properties, albeit for reasons that remain to be explained. Here, we probe the molecular basis of this behavior.Cadherins constitute a large family of cell surface adhesion receptors that can be grouped into numerous subfamilies (5). The type I and type II ''classical cadherins'' are found ...

No abstract

Vertebrate genomes encode nineteen "classical" cadherins and about a hundred non-classical cadherins. Adhesion by classical cadherins depends on binding interactions in their amino terminal EC1 domains, which swap N-terminal β-strands between partner molecules from apposing cells. However, strand swapping sequence signatures are absent from non-classical cadherins, raising the question of how these proteins function in adhesion. Here we show that T-cadherin, a GPI-anchored cadherin, forms dimers through an alternative non-swapped interface near the EC1-EC2 calcium binding sites. Mutations within this interface ablate the adhesive capacity of T-cadherin. These nonadhesive T-cadherin mutants also lose the ability to regulate neurite outgrowth from T-cadherin expressing neurons. Our findings reveal the likely molecular architecture of the T-cadherin homophilic interface, and reveal its requirement for axon outgrowth regulation. The adhesive binding mode employed by T-cadherin may also be used by other non-classical cadherins.