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

The SARS-CoV-2 spike employs mobile receptor-binding domains (RBDs) to engage the human ACE2 receptor and to facilitate virus entry, which can occur through low pH-endosomal pathways. To understand how ACE2 binding and low pH impact spike conformation, we determined cryo-EM structures –at serological and endosomal pH– delineating spike recognition of up to three ACE2 molecules. RBDs freely adopted ‘up’ conformations required for ACE2 interaction, primarily through RBD movement combined with smaller alterations in neighboring domains. In the absence of ACE2, cryo-EM structures revealed single-RBD-up conformations to dominate at pH 5.5, resolving into a solitary all-down conformation at lower pH. Notably, a pH-dependent refolding region (residues 824-858) at the spike-interdomain interface displayed dramatic structural rearrangements and mediated RBD positioning through coordinated movements of the entire trimer apex. These findings provide insight into how receptor interactions and endosomal pH alter RBD positioning and potentially facilitate immune evasion from RBD-up binding antibody.

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.

No abstract

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 ...

Desmosomes are intercellular adhesive junctions that impart strength to vertebrate tissues. Their dense, ordered intercellular attachments are formed by desmogleins (Dsgs) and desmocollins (Dscs), but the nature of trans-cellular interactions between these specialized cadherins is unclear. Here, using solution biophysics and coated-bead aggregation experiments, we demonstrate family-wise heterophilic specificity: All Dsgs form adhesive dimers with all Dscs, with affinities characteristic of each Dsg:Dsc pair. Crystal structures of ectodomains from Dsg2 and Dsg3 and from Dsc1 and Dsc2 show binding through a strand-swap mechanism similar to that of homophilic classical cadherins. However, conserved charged amino acids inhibit Dsg:Dsg and Dsc:Dsc interactions by same-charge repulsion and promote heterophilic Dsg:Dsc interactions through oppositecharge attraction. These findings show that Dsg:Dsc heterodimers represent the fundamental adhesive unit of desmosomes and provide a structural framework for understanding desmosome assembly. Dysfunction of desmosomes in inherited and acquired human diseases as well as in mouse genetic ablation studies causes characteristic defects in heart muscle and skin (3-5), demonstrating their importance in tissues that undergo mechanical stress. In electron micrographs, the hallmarks of mature desmosomes include a constant intermembrane distance of 280-340 Å, and apparently ordered electron-density in the intercellular space, often with a discrete midline connected by periodic cross-bridges to the cell membranes (6-9). The intercellular attachments of desmosomes are composed of transmembrane proteins from two specialized cadherin subfamilies: desmocollins (Dscs) and desmogleins (Dsgs). The human genome encodes three Dsc (Dsc1-Dsc3) and four Dsg (Dsg1-Dsg4) proteins, which share an overall domain organization comprising four to five extracellular cadherin (EC) domains, a single-pass transmembrane region, and an intracellular domain that binds to intermediate filaments via adaptor proteins desmoplakin and plakoglobin (1). Individual Dsgs and Dscs show differential expression patterns: Dsg2 and Dsc2 are expressed widely in all desmosome-forming tissues (1), whereas other desmosomal cadherins are expressed specifically in stratified epithelia with graded, overlapping patterns (1, 10). Notably, both Dscs and Dsgs appear necessary for adhesion in transfected cells (1,(11)(12)(13), and loss of either in genetic experiments causes loss of normal desmosomal adhesion (5,14,15).Although the ultrastructure of desmosomes is well characterized, a molecular-level understanding of the binding interactions between desmosomal cadherin extracellular regions that assemble these junctions has remained elusive. In particular, whether desmosomal cadherins have homophilic preferences or whether interactions occur between heterophilic pairs has been a matter of dispute (1,(11)(12)(13)(16)(17)(18). Electron tomography studies of native desmosomes (7, 8) have revealed cadherins binding through their EC1 domains...

SUMMARY Binding between DIP and Dpr neuronal-recognition proteins has been proposed to regulate synaptic connections between lamina and medulla neurons in the Drosophila visual system. Each lamina neuron was previously shown to express many Dprs. Here, we demonstrate, by contrast, that their synaptic partners typically express one or two DIPs, with binding specificities matched to the lamina neuron-expressed Dprs. A deeper understanding of the molecular logic of DIP/Dpr interaction requires quantitative studies on the properties of these proteins. We thus generated a quantitative affinity-based DIP/Dpr interactome for all DIP/Dpr protein family members. This revealed a broad range of affinities and identified homophilic binding for some DIPs and some Dprs. These data, along with full-length ectodomain DIP/Dpr and DIP/DIP crystal structures, led to the identification of molecular determinants of DIP/Dpr specificity. This structural knowledge, along with a comprehensive set of quantitative binding affinities, provides new tools for functional studies in vivo.

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.