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The low-density lipoprotein (LDL) receptor-related protein 2 (LRP2 or megalin) is representative of the phylogenetically conserved subfamily of giant LDL receptor-related proteins, which function in endocytosis and are implicated in diseases of the kidney and brain. Here we report high-resolution cryo-electron microscopy structures of LRP2 isolated from mouse kidney, at extracellular and endosomal pH. The structures reveal LRP2 to be a molecular machine that adopts a conformation for ligand binding at the cell surface and for ligand shedding in the endosome. LRP2 forms a homodimer, the conformational transformation of which is governed by pH-sensitive sites at both homodimer and intra-protomer interfaces. A subset of LRP2 deleterious missense variants in humans appears to impair homodimer assembly. These observations lay the foundation for further understanding the function and mechanism of LDL receptors and implicate homodimerization as a conserved feature of the LRP receptor subfamily.

Increasing spread by SARS-CoV-2 Omicron variants challenges existing vaccines and broadly reactive neutralizing antibodies (bNAbs) against COVID-19. Here we determine the diversity, potency, breadth and structural insights of bNAbs derived from memory B cells of BNT162b2-vaccinee after homogeneous Omicron BA.1 breakthrough infection. The infection activates diverse memory B cell clonotypes for generating potent class I/II or III bNAbs with new epitopes mapped to receptor-binding domain (RBD). The top eight bNAbs neutralize wildtype and BA.1 potently but display divergent IgH/IgL sequences and neuralization profiles against other variants of concern (VOCs). Two of them (P2D9 and P3E6) belonging to class III NAbs display comparable potency against BA.4/BA.5, although structural analysis reveals distinct modes of action. P3E6 neutralizes all variants tested through a unique bivalent interaction with two RBDs. Our findings provide new insights into hybrid immunity on BNT162b2-induced diverse memory B cells in response to Omicron breakthrough infection for generating diverse bNAbs with distinct structural basis.

The strength of binding between human angiotensin converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of viral spike protein plays a role in the transmissibility of the SARS-CoV-2 virus. In this study we focus on a subset of RBD mutations that have been frequently observed in infected individuals and probe binding affinity changes to ACE2 using surface plasmon resonance (SPR) measurements and free energy perturbation (FEP) calculations. Our SPR results are largely in accord with previous studies but discrepancies do arise due to differences in experimental methods and to protocol differences even when a single method is used. Overall, we find that FEP performance is superior to that of other computational approaches examined as determined by agreement with experiment and, in particular, by its ability to identify stabilizing mutations. Moreover, the calculations successfully predict the observed cooperative stabilization of binding by the Q498R N501Y double mutant present in Omicron variants and offer a physical explanation for the underlying mechanism. Overall, our results suggest that despite the significant computational cost, FEP calculations may offer an effective strategy to understand the effects of interfacial mutations on protein-protein binding affinities and in practical applications such as the optimization of neutralizing antibodies.

The stochastic expression of fewer than 60 clustered protocadherin (cPcdh) isoforms provides diverse identities to individual vertebrate neurons and a molecular basis for self-/nonself-discrimination. cPcdhs form chains mediated by alternating cis and trans interactions between apposed membranes, which has been suggested to signal self-recognition. Such a mechanism requires that cPcdh cis dimers form promiscuously to generate diverse recognition units, and that trans interactions have precise specificity so that isoform mismatches terminate chain growth. However, the extent to which cPcdh interactions fulfill these requirements has not been definitively demonstrated. Here, we report biophysical experiments showing that cPcdh cis interactions are promiscuous, but with preferences favoring formation of heterologous cis dimers. Trans homophilic interactions are remarkably precise, with no evidence for heterophilic interactions between different isoforms. A new C-type cPcdh crystal structure and mutagenesis data help to explain these observations. Overall, the interaction characteristics we report for cPcdhs help explain their function in neuronal self-/nonself-discrimination.

Differential binding affinities among closely related protein family members underlie many biological phenomena, including cell-cell recognition. Drosophila DIP and Dpr proteins mediate neuronal targeting in the fly through highly specific protein-protein interactions. We show here that DIPs/Dprs segregate into seven specificity subgroups defined by binding preferences between their DIP and Dpr members. We then describe a sequence-, structure- and energy-based computational approach, combined with experimental binding affinity measurements, to reveal how specificity is coded on the canonical DIP/Dpr interface. We show that binding specificity of DIP/Dpr subgroups is controlled by “negative constraints”, which interfere with binding. To achieve specificity, each subgroup utilizes a different combination of negative constraints, which are broadly distributed and cover the majority of the protein-protein interface. We discuss the structural origins of negative constraints, and potential general implications for the evolutionary origins of binding specificity in multi-protein families., Dpr (Defective proboscis extension response) and DIP (Dpr Interacting Proteins) are immunoglobulin-like cell-cell adhesion proteins that form highly specific pairwise interactions, which control synaptic connectivity during Drosophila development. Here, the authors combine a computational approach with binding affinity measurements and find that DIP/Dpr binding specificity is controlled by negative constraints that interfere with non-cognate binding.

Accumulation of somatic hypermutation (SHM) is the primary mechanism to enhance the binding affinity of antibodies to antigens in vivo. However, the structural basis of the effects of many SHMs remains elusive. Here, we integrated atomistic molecular dynamics (MD) simulation and data mining to build a high-throughput structural bioinformatics pipeline to study the effects of individual and combination SHMs on antibody conformation, flexibility, stability, and affinity. By applying this pipeline, we characterized a common mechanism of modulation of heavy-light pairing orientation by frequent SHMs at framework positions 39H, 91H, 38L, and 87L through disruption of a conserved hydrogen-bond network. Q39LH alone and in combination with light chain framework 4 (FWR4L) insertions further modulated the elbow angle between variable and constant domains of many antibodies, resulting in improved binding affinity for a subset of anti-HIV-1 antibodies. Q39LH also alleviated aggregation induced by FWR4L insertion, suggesting remote epistasis between these SHMs. Altogether, this study provides tools and insights for understanding antibody affinity maturation and for engineering functionally improved antibodies.

SUMMARY Synaptic connectivity within adult circuits exhibits a remarkable degree of cellular and subcellular specificity. We report that the axon guidance receptor Robo2 plays a role in establishing synaptic specificity in hippocampal CA1. In vivo, Robo2 is present and required postsynaptically in CA1 pyramidal neurons (PNs) for the formation of excitatory (E) but not inhibitory (I) synapses, specifically in proximal but not distal dendritic compartments. In vitro approaches show that the synaptogenic activity of Robo2 involves a trans-synaptic interaction with presynaptic Neurexins, as well as binding to its canonical extracellular ligand Slit. In vivo 2-photon Ca2+ imaging of CA1 PNs during spatial navigation in awake behaving mice shows that preventing Robo2-dependent excitatory synapse formation cell autonomously during development alters place cell properties of adult CA1 PNs. Our results identify a trans-synaptic complex linking the establishment of synaptic specificity to circuit function., In brief Blockus et al. demonstrate that the axon guidance receptors Robo1/2 are synaptogenic cues required postsynaptically for the establishment of synaptic specificity between CA3 axons and dendrites of CA1 pyramidal neurons (PNs). Developmental deletion of Robo2 in CA1 PNs disrupts the emergence of place cell properties in adult hippocampus., Graphical Abstract

The stochastic expression of fewer than 60 clustered protocadherin (cPcdh) isoforms provides diverse identities to individual vertebrate neurons and a molecular basis for self/non-self- discrimination. cPcdhs form chains mediated by alternating cis and trans interactions between apposed membranes, which has been suggested to signal self-recognition. Such a mechanism requires that cPcdh cis dimers form promiscuously to generate diverse recognition units, and that trans interactions have precise specificity so that isoform mismatches terminate chain growth. However, the extent to which cPcdh interactions fulfill these requirements has not been definitively demonstrated. Here we report biophysical experiments showing that cPcdh cis interactions are promiscuous, but with preferences favoring formation of heterologous cis dimers. Trans-homophilic interactions are remarkably precise, with no evidence for heterophilic interactions between different isoforms. A new C-type cPcdh crystal structure and mutagenesis data help to explain these observations. Overall, the interaction characteristics we report for cPcdhs help explain their function in neuronal self/non-self-discrimination.

Antibodies that potently neutralize SARS-CoV-2 target mainly the receptor-binding domain or the N-terminal domain (NTD). Over a dozen potently neutralizing NTD-directed antibodies have been studied structurally, and all target a single antigenic supersite in NTD (site 1). Here, we report the cryo-EM structure of a potent NTD-directed neutralizing antibody 5-7, which recognizes a site distinct from other potently neutralizing antibodies, inserting a binding loop into an exposed hydrophobic pocket between the two sheets of the NTD β sandwich. Interestingly, this pocket was previously identified as the binding site for hydrophobic molecules, including heme metabolites, but we observe that their presence does not substantially impede 5-7 recognition. Mirroring its distinctive binding, antibody 5-7 retains neutralization potency with many variants of concern (VOCs). Overall, we reveal that a hydrophobic pocket in NTD proposed for immune evasion can be used by the immune system for recognition., Graphical abstract, Cerutti et al. report the cryo-EM structure of potent neutralizing antibody 5-7 bound to SARS-CoV-2 spike. While most NTD-directed neutralizing antibodies target the NTD supersite, 5-7 binds to a conserved hydrophobic pocket on NTD and neutralizes many variants of concern.

Antibodies with heavy chains that derive from the VH1-2 gene constitute some of the most potent SARS-CoV-2-neutralizing antibodies yet identified. To provide insight into whether these genetic similarities inform common modes of recognition, we determined structures of the SARS-CoV-2 spike in complex with three VH1-2-derived antibodies: 2-15, 2-43, and H4. All three utilize VH1-2-encoded motifs to recognize the receptor-binding domain (RBD), with heavy chain N53I enhancing binding and light chain tyrosines recognizing F486RBD. Despite these similarities, class members bind both RBD-up and -down conformations of the spike, with a subset of antibodies utilizing elongated CDRH3s to recognize glycan N343 on a neighboring RBD – a quaternary interaction accommodated by an increase in RBD separation of up to 12 Å. The VH1-2-antibody class thus utilizes modular recognition encoded by modular genetic elements to effect potent neutralization, with VH-gene component specifying recognition of RBD and CDRH3 component specifying quaternary interactions., Graphical Abstract, Rapp et al. determine structures of three human VH1-2-derived SARS-CoV-2 neutralizing antibodies and define a prevalent multi-donor antibody class which recognizes the receptor-binding domain (RBD) with convergent heavy and light chain modules. They further show the structural basis for RBD-up and RBD-down recognition and quaternary recognition-mediated spike conformation change.

Numerous antibodies that neutralize SARS-CoV-2 have been identified, and these generally target either the receptor-binding domain (RBD) or the N-terminal domain (NTD) of the viral spike. While RBD-directed antibodies have been extensively studied, far less is known about NTD-directed antibodies. Here we report cryo-EM and crystal structures for seven potent NTD-directed neutralizing antibodies in complex with spike or isolated NTD. These structures defined several antibody classes, with at least one observed in multiple convalescent donors. The structures revealed all seven antibodies target a common surface, bordered by glycans N17, N74, N122, and N149. This site – formed primarily by a mobile β-hairpin and several flexible loops – was highly electropositive, located at the periphery of the spike, and the largest glycan-free surface of NTD facing away from the viral membrane. Thus, in contrast to neutralizing RBD-directed antibodies that recognize multiple non-overlapping epitopes, potent NTD-directed neutralizing antibodies appear to target a single supersite., Graphical Abstract, Cerutti et al. report structural analysis of seven potent neutralizing antibodies targeting the N-Terminal domain of SARS-CoV-2 spike. All antibodies recognize a common glycan-free, electropositive surface comprised of a mobile β-hairpin and flexible loops. While RBD-directed antibodies recognize non-overlapping epitopes, these findings indicate NTD-directed antibodies predominantly target a single supersite.

SummaryNumerous antibodies that neutralize SARS-CoV-2 have been identified, and these generally target either the receptor-binding domain (RBD) or the N-terminal domain (NTD) of the viral spike. While RBD-directed antibodies have been extensively studied, far less is known about NTD-directed antibodies. Here we report cryo-EM and crystal structures for seven potent NTD-directed neutralizing antibodies in complex with spike or isolated NTD. These structures defined several antibody classes, with at least one observed in multiple convalescent donors. The structures revealed all seven antibodies to target a common surface, bordered by glycansN17,N74,N122, andN149. This site – formed primarily by a mobile β-hairpin and several flexible loops – was highly electropositive, located at the periphery of the spike, and the largest glycan-free surface of NTD facing away from the viral membrane. Thus, in contrast to neutralizing RBD-directed antibodies that recognize multiple non-overlapping epitopes, potent NTD-directed neutralizing antibodies target a single 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 affect spike conformation, we determined cryo-electron microscopy 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, single-RBD-up conformations dominated 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 structures provide a foundation for understanding prefusion-spike mechanics governing endosomal entry; we suggest that the low pH all-down conformation potentially facilitates immune evasion from RBD-up binding antibody., Graphical Abstract, Highlights • Determine cryo-EM structures of SARS-CoV-2 spike along its endosomal entry pathway • Reveal structural basis by which a pH-dependent switch mediates RBD positioning • Show spike to exclusively adopt an all-RBD-down conformation at low pH • Suggest low-pH all-RBD-down conformation to provide a basis for immune evasion, Zhou et al. determine 12 structures of the SARS-CoV-2 spike, bound by ACE2 receptor and ligand free, that reveal a pH-dependent switch to mediate positioning of spike receptor-binding domains (RBDs). At low pH, the spike adopts an all-RBD-down conformation, which provides a potential means of immune evasion from RBD-up-recognizing antibody.

Sex-specific synaptic connectivity is beginning to emerge as a remarkable, but little explored feature of animal brains. We describe here a novel mechanism that promotes sexually dimorphic neuronal function and synaptic connectivity in the nervous system of the nematodeCaenorhabditis elegans. We demonstrate that a phylogenetically conserved, but previously uncharacterized Doublesex/Mab-3 related transcription factor (DMRT),dmd-4, is expressed in two classes of sex-shared phasmid neurons specifically in hermaphrodites but not in males. We finddmd-4to promote hermaphrodite-specific synaptic connectivity and neuronal function of phasmid sensory neurons. Sex-specificity of DMD-4 function is conferred by a novel mode of posttranslational regulation that involves sex-specific protein stabilization through ubiquitin binding to a phylogenetically conserved but previously unstudied protein domain, the DMA domain. A human DMRT homolog of DMD-4 is controlled in a similar manner, indicating that our findings may have implications for the control of sexual differentiation in other animals as well.

The SARS-CoV-2 spike employs mobile receptor-binding domains (RBDs) to engage the ACE2 receptor and to facilitate virus entry. But what controls RBD positioning? As SARS-CoV-2 utilizes endosomal pathways as a major mode of entry, we investigated the impact of both low pH and ACE2 binding. Cryo-EM structures –at serological and endosomal pH– delineated spike recognition of up to three ACE2 molecules, revealing RBD to freely adopt the ‘up’ conformation required for ACE2 interaction, primarily through RBD movement combined with subtle alterations in position and orientation of neighboring domains. In the absence of ACE2, single-RBD-up conformations dominated at pH 5.5, resolving into a single all-down conformation at lower pH, which biochemical studies suggested might provide immune evasion from RBD-up-recognizing antibody. Notably, a pH-dependent refolding region (residues 824-858) at the spike-interdomain interface displayed dramatic structural rearrangements and appeared to mediate RBD positioning in concert with coordinated movements through the entire trimer apex. Funding: Support for this work was provided by the Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID), Federal funds from the Frederick National Laboratory for Cancer Research under Contract HHSN261200800001E (A.S., T.S., Y.T.). Cryo-EM data for the spike-ACE2 complexes were collected at Columbia University Cryo-EM Center at the Zuckerman Institute, and at the National Center for CryoEM Access and Training (NCCAT) and the Simons Electron Microscopy Center located at the New York Structural Biology Center, supported by the NIH Common Fund Transformative High Resolution Cryo-Electron Microscopy program (U24 GM129539,) and by grants from the Simons Foundation (SF349247) and NY State Assembly. Cryo-EM datasets for individual spike proteins were collected at the National CryoEM Facility (NCEF) of the National Cancer Institute. This research was, in part, supported by the National Cancer Institute’s National Cryo-EM Facility at the Frederick National Laboratory for Cancer Research under contract HSSN261200800001E. We are especially grateful to U. Baxa, A. Wier, M. Hutchison, and T. Edwards of NCEF for collecting cryo-EM data and for technical assistance with cryo-EM data processing. Frederick Research Computing Environment (FRCE) high-performance computing cluster was used for processing cryo-EM datasets of individual spike proteins. Conflict of Interest: The authors declare no competing interest.

SummaryUnderstanding protective mechanisms of antibody recognition can inform vaccine and therapeutic strategies against SARS-CoV-2. We discovered a new antibody, 910-30, that targets the SARS-CoV-2 ACE2 receptor binding site as a member of a public antibody response encoded by IGHV3-53/IGHV3-66 genes. We performed sequence and structural analyses to explore how antibody features correlate with SARS-CoV-2 neutralization. Cryo-EM structures of 910-30 bound to the SARS-CoV-2 spike trimer revealed its binding interactions and ability to disassemble spike. Despite heavy chain sequence similarity, biophysical analyses of IGHV3-53/3-66 antibodies highlighted the importance of native heavy:light pairings for ACE2 binding competition and for SARS-CoV-2 neutralization. We defined paired heavy:light sequence signatures and determined antibody precursor prevalence to be ~1 in 44,000 human B cells, consistent with public antibody identification in several convalescent COVID-19 patients. These data reveal key structural and functional neutralization features in the IGHV3-53/3-66 public antibody class to accelerate antibody-based medical interventions against SARS-CoV-2.HighlightsA molecular study of IGHV3-53/3-66 public antibody responses reveals critical heavy and light chain features for potent neutralizationCryo-EM analyses detail the structure of a novel public antibody class member, antibody 910-30, in complex with SARS-CoV-2 spike trimerCryo-EM data reveal that 910-30 can both bind assembled trimer and can disassemble the SARS-CoV-2 spikeSequence-structure-function signatures defined for IGHV3-53/3-66 class antibodies including both heavy and light chainsIGHV3-53/3-66 class precursors have a prevalence of 1:44,000 B cells in healthy human antibody repertoires

The developmental transition between axon guidance and synapse formation is critical for circuit assembly but still poorly understood at the molecular level. We hypothesized that this key transition could be regulated by axon guidance cues switching their function to regulate synaptogenesis with subcellular specificity. Here, we report evidence for such a functional switch, describing a novel role for the axon guidance molecule Robo2 in excitatory synapse formation onto dendrites of CA1 pyramidal neurons (PNs) in the mouse hippocampus. Cell-autonomous deletion of Robo2 from CA1 PNs leads to a drastic reduction of the number of excitatory synapses specifically in proximal dendritic compartments. At the molecular level, we show that this novel postsynaptic function of Robo2 depends on both its canonical ligand Slit and a novel interaction with presynaptic Neurexins. Biophysical analysis reveals that Robo2 binds directly to Neurexins via its Ig4-5 domains. In vivo 2-photon Ca2+ imaging of CA1 PNs during spatial navigation in mice shows that sparse deletion of Robo2 during development drastically reduces the likelihood of place cell emergence and alters spatial coding properties of the remaining place cells. Our results identify Robo2 as a novel molecular effector linking synaptic specificity to the acquisition of spatial coding properties characterizing hippocampal circuits.

SUMMARY Type II cadherins are cell-cell adhesion proteins critical for tissue patterning and neuronal targeting but whose molecular binding code remains poorly understood. Here, we delineate binding preferences for type II cadherin cell-adhesive regions, revealing extensive heterophilic interactions between specific pairs, in addition to homophilic interactions. Three distinct specificity groups emerge from our analysis with members that share highly similar heterophilic binding patterns and favor binding to one another. Structures of adhesive fragments from each specificity group confirm near-identical dimer topology conserved throughout the family, allowing interface residues whose conservation corresponds to specificity preferences to be identified. We show that targeted mutation of these residues converts binding preferences between specificity groups in biophysical and co-culture assays. Our results provide a detailed understanding of the type II cadherin interaction map and a basis for defining their role in tissue patterning and for the emerging importance of their heterophilic interactions in neural connectivity., In Brief Type II cadherins are a family of vertebrate cell adhesion proteins expressed primarily in the CNS. Brasch et al. measure binding between adhesive fragments, revealing homophilic and extensive selective heterophilic binding with specificities that define groups of similar cadherins. Structures reveal common adhesive dimers, with residues governing cell-adhesive specificity.

Understanding mechanisms of protective antibody recognition can inform vaccine and therapeutic strategies against SARS-CoV-2. We report a monoclonal antibody, 910-30, targeting the SARS-CoV-2 receptor-binding site for ACE2 as a member of a public antibody response encoded by IGHV3-53/IGHV3-66 genes. Sequence and structural analyses of 910-30 and related antibodies explore how class recognition features correlate with SARS-CoV-2 neutralization. Cryo-EM structures of 910-30 bound to the SARS-CoV-2 spike trimer reveal binding interactions and its ability to disassemble spike. Despite heavy-chain sequence similarity, biophysical analyses of IGHV3-53/3-66-encoded antibodies highlight the importance of native heavy:light pairings for ACE2-binding competition and SARS-CoV-2 neutralization. We develop paired heavy:light class sequence signatures and determine antibody precursor prevalence to be ∼1 in 44,000 human B cells, consistent with public antibody identification in several convalescent COVID-19 patients. These class signatures reveal genetic, structural, and functional immune features that are helpful in accelerating antibody-based medical interventions for SARS-CoV-2., Graphical abstract, Banach et al. report a SARS-CoV-2 neutralizing antibody along with genetic, structural, and functional features of public antibody responses targeting SARS-CoV-2. These data reveal how structural interactions with the SARS-CoV-2 receptor-binding domain correlate with viral neutralization and demonstrate the importance of native antibody heavy:light pairings in convergent antibody responses.

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.

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 opposite-charge attraction. These findings show that Dsg:Dsc heterodimers represent the fundamental adhesive unit of desmosomes and provide a structural framework for understanding desmosome assembly.

SUMMARY Non-clustered δ1- and δ2-protocadherins, close relatives of clustered protocadherins, function in cell adhesion and motility and play essential roles in neural patterning. To understand the molecular interactions underlying these functions, we used solution biophysics to characterize binding of δ1- and δ2-protocadherins, determined crystal structures of ectodomain complexes from each family, and assessed ectodomain assembly in reconstituted intermembrane junctions by cryoelectron tomography (cryo-ET). Homophilic trans (cell–cell) interactions were preferred for all δ-protocadherins, with additional weaker heterophilic interactions observed exclusively within each subfamily. As expected, δ1- and δ2-protocadherin trans dimers formed through antiparallel EC1–EC4 interfaces, like clustered protocadherins. However, no ectodomain-mediated cis (same-cell) interactions were detectable in solution; consistent with this, cryo-ET of reconstituted junctions revealed dense assemblies lacking the characteristic order observed for clustered protocadherins. Our results define non-clustered protocadherin binding properties and their structural basis, providing a foundation for interpreting their functional roles in neural patterning., Graphical Abstract, In Brief Non-clustered δ-protocadherins are adhesion molecules linked to a number of neurological disorders. Harrison et al. apply biophysical and structural methods across the family to show preferential self-binding through a canonical interface and disordered assemblies of adhesive dimers between membranes that diverge from ordered assemblies of close relatives clustered protocadherins.

Summary Drosophila Dpr (21 paralogs) and DIP proteins (11 paralogs) are cell recognition molecules of the immunoglobulin superfamily (IgSF) that form a complex protein interaction network. DIP and Dpr proteins are expressed in a synaptic layer-specific fashion in the visual system. How interactions between these proteins regulate layer-specific synaptic circuitry is not known. Here we establish that DIP-α and its interacting partners Dpr6 and Dpr10 regulate multiple processes, including arborization within layers, synapse number, layer specificity, and cell survival. We demonstrate that heterophilic binding between Dpr6/10 and DIP-α and homophilic binding between DIP-α proteins promote interactions between processes in vivo. Knockin mutants disrupting the DIP/Dpr binding interface reveal a role for these proteins during normal development, while ectopic expression studies support an instructive role for interactions between DIPs and Dprs in circuit development. These studies support an important role for the DIP/Dpr protein interaction network in regulating cell-type-specific connectivity patterns.

Sidekick (Sdk) 1 and 2 are related immunoglobulin superfamily cell adhesion proteins required for appropriate synaptic connections between specific subtypes of retinal neurons. Sdks mediate cell-cell adhesion with homophilic specificity that underlies their neuronal targeting function. Here we report crystal structures of Sdk1 and Sdk2 ectodomain regions, revealing similar homodimers mediated by the four N-terminal immunoglobulin domains (Ig1–4), arranged in a horseshoe conformation. These Ig1–4 horseshoes interact in a novel back-to-back orientation in both homodimers through Ig1:Ig2, Ig1:Ig1 and Ig3:Ig4 interactions. Structure-guided mutagenesis results show that this canonical dimer is required for both Sdk-mediated cell aggregation (via trans interactions) and Sdk clustering in isolated cells (via cis interactions). Sdk1/Sdk2 recognition specificity is encoded across Ig1–4, with Ig1–2 conferring the majority of binding affinity and differential specificity. We suggest that competition between cis and trans interactions provides a novel mechanism to sharpen the specificity of cell-cell interactions. DOI: http://dx.doi.org/10.7554/eLife.19058.001

The tip link, a filament formed by protocadherin 15 (PCDH15) and cadherin 23, conveys mechanical force from sound waves and head movement to open hair-cell mechanotransduction channels. Tip-link cadherins are thought to have acquired structural features critical for their role in mechanotransduction. Here, we biophysically and structurally characterize the unusual cis-homodimeric architecture of PCDH15. We show that PCDH15 molecules form double-helical assemblies through cis-dimerization interfaces in the extracellular cadherin EC2-EC3 domain region and in a unique membrane-proximal domain. Electron microscopy studies visualize the cis-dimeric PCDH15 assembly and reveal the PCDH15 extracellular domain as a parallel double helix with cis cross-bridges at the two locations we defined. The helical configuration suggests the potential for elasticity through helix winding and unwinding. Functional studies in hair cells show that mutations that perturb PCDH15 dimerization contacts affect mechanotransduction. Together, these data reveal the cis-dimeric architecture of PCDH15 and show that dimerization is critical for sensing mechanical stimuli.

Alternatively spliced beta-neurexins (beta-NRXs) and neuroligins (NLs) are thought to have distinct extracellular binding affinities, potentially providing a beta-NRX/NL synaptic recognition code. We utilized surface plasmon resonance to measure binding affinities between all combinations of alternatively spliced beta-NRX 1-3 and NL 1-3 ectodomains. Binding was observed for all beta-NRX/NL pairs. The presence of the NL1 B splice insertion lowers beta-NRX binding affinity by approximately 2-fold, while beta-NRX splice insertion 4 has small effects that do not synergize with NL splicing. New structures of glycosylated beta-NRXs 1 and 2 containing splice insertion 4 reveal that the insertion forms a new beta strand that replaces the beta10 strand, leaving the NL binding site intact. This helps to explain the limited effect of splice insert 4 on NRX/NL binding affinities. These results provide new structural insights and quantitative binding information to help determine whether and how splice isoform choice plays a role in beta-NRX/NL-mediated synaptic recognition.

Previous kinetic investigations of the N-terminal RNA recognition motif (RRM) domain of spliceosomal protein U1A, interacting with its RNA target U1 hairpin II, provided experimental evidence for a 'lure and lock' model of binding in which electrostatic interactions first guide the RNA to the protein, and close range interactions then lock the two molecules together. To further investigate the 'lure' step, here we examined the electrostatic roles of two sets of positively charged amino acids in U1A that do not make hydrogen bonds to the RNA: Lys20, Lys22 and Lys23 close to the RNA-binding site, and Arg7, Lys60 and Arg70, located on 'top' of the RRM domain, away from the RNA. Surface plasmon resonance-based kinetic studies, supplemented with salt dependence experiments and molecular dynamics simulation, indicate that Lys20 predominantly plays a role in association, while nearby residues Lys22 and Lys23 appear to be at least as important for complex stability. In contrast, kinetic analyses of residues away from the RNA indicate that they have a minimal effect on association and stability. Thus, well-positioned positively charged residues can be important for both initial complex formation and complex maintenance, illustrating the multiple roles of electrostatic interactions in protein-RNA complexes.

Stable phosphoinositide (PIP(n))-containing liposomes were prepared using polydiacetylene photochemistry. Tethered pentacosadiynyl inositol polyphosphate (InsP(n)) analogues of Ins(1,3,4)P(3), Ins(1,4,5)P(3), and Ins(1,3,4,5)P(4) were synthesized, incorporated into vesicles made up of diyne-phosphatidylcholine and -phosphatidylethanolamine, and polymerized by UV irradiation. The polymerized liposome nanoparticles showed markedly increased stability over conventional PIP(n)-containing vesicles as a result of the covalent conjugated ene-yne network in the acyl chains. The polymerized liposomes were specifically recognized by PIP(n) binding PH domains in liposome overlay assays and amplified luminescent proximity homogeneous assays. Moreover, the biotin moiety allowed attachment of the nanoparticles to a streptavidin-coated sensor chips in surface plasmon resonance (SPR) sensor. The PIP(n) headgroups displayed on SPR sensors showed higher affinities for PH domains and PIP(n) monoclonal antibodies than did monomeric PIP(n)-analogues with biotinylated acyl chains.

The A protein of the U1 small nuclear ribonucleoprotein particle, interacting with its stem-loop RNA target (U1hpII), is frequently used as a paradigm for RNA binding by recognition motif domains (RRMs). U1A/U1hpII complex formation has been proposed to consist of at least two steps: electrostatically mediated alignment of both molecules followed by locking into place, based on the establishment of close-range interactions. The sequence of events between alignment and locking remains obscure. Here we examine the roles of three critical residues, Tyr13, Phe56 and Gln54, in complex formation and stability using Biacore. Our mutational and kinetic data suggest that Tyr13 plays a more important role than Phe56 in complex formation. Mutational analysis of Gln54, combined with molecular dynamics studies, points to Arg52 as another key residue in association. Based on our data and previous structural and modeling studies, we propose that electrostatic alignment of the molecules is followed by hydrogen bond formation between the RNA and Arg52, and the sequential establishment of interactions with loop bases (including Tyr13). A quadruple stack, sandwiching two bases between Phe56 and Asp92, would occur last and coincide with the rearrangement of a C-terminal helix that partially occludes the RRM surface in the free protein.

RNA recognition motifs (RRMs) are characterized by highly conserved regions located centrally on a beta-sheet, which forms the RNA binding surface. Variable flanking regions, such as the loop connecting beta-strands 2 and 3, are thought to be important in determining the RNA-binding specificities of individual RRMs. The N-terminal RRM of the spliceosomal U1A protein mediates binding to an RNA hairpin (U1hpII) in the U1 small nuclear RNA. In this complex, the beta(2)-beta(3) loop protrudes through the 10-nucleotide RNA loop. Shortening of the RNA loop strongly perturbs binding, suggesting that an optimal "fit" of the beta(2)-beta(3) loop into the RNA loop is an important factor in complexation. To understand this interaction further, we mutated or deleted loop residues Lys(50) and Met(51), which protrude centrally into the RNA loop but do not make any direct contacts to the bases. Using BIACORE, we analyzed the ability of these U1A mutants to bind to wild type RNAs, or RNAs with shortened loops. Alanine replacement mutations only modestly affected binding to wild type U1hpII. Interestingly, simultaneous replacement of Lys(50) and Met(51) with alanine appeared to alleviate the loss of binding caused by shortening of the RNA loop. Deletion of Lys(50) or Met(51) caused a dramatic loss in stability of the U1A.U1hpII complex. However, deletion of both residues simultaneously was much less deleterious. Simulated annealing molecular dynamics analyses suggest this is due to the ability of this mutant to rearrange flanking amino acids to substitute for the two deleted residues. The double deletion mutant also exhibited substantially reduced negative effects of RNA loop shortening, suggesting the rearranged loop is better able to accommodate a short RNA loop. Our results indicate that one of the roles of the beta(2)-beta(3) loop is to provide a steric fit into the RNA loop, thereby stabilizing the RNA.protein complex.

Although structural, biochemical, and genetic studies have provided much insight into the determinants of specificity and affinity of proteins for RNA, little is currently known about the kinetics that underlie RNA-protein interactions. Protein-RNA complexes are dynamic, and the kinetics of binding and release could influence many processes, such as the ability of RNA-binding proteins to compete for binding sites, the sequential assembly of ribonucleoprotein complexes, and the ability of bound RNA to move between cellular compartments. Therefore, to attain a complete and biologically relevant understanding of RNA-protein interactions, complex formation must be studied not only in equilibrated reactions, but also as a dynamic process. BIACORE, a surface plasmon resonance-based biosensor technology, allows intermolecular interactions to be measured in real time, and can provide both equilibrium and kinetic information about complex formation. This technology is a powerful tool with which to study the dynamics of RNA-protein interactions. We have used BIACORE extensively to obtain detailed insight into the interaction between RNA and proteins carrying RNA recognition motif domains. Here we discuss the physical principles on which BIACORE is based, and the required instrumentation. We describe how to design well-controlled RNA-protein interaction experiments aimed at yielding high-quality data, and outline the steps required for data analysis. In addition, we present examples to illustrate how kinetic studies have provided us with unique insights into the interaction of the spliceosomal U1A protein and the neuronal HuD protein with their respective RNA targets.

Type I cadherin cell-adhesion proteins are similar in sequence and structure and yet are different enough to mediate highly specific cell–cell recognition phenomena. It has previously been shown that small differences in the homophilic and heterophilic binding affinities of different type I family members can account for the differential cell-sorting behavior. Here we use a combination of X-ray crystallography, analytical ultracentrifugation, surface plasmon resonance and double electron-electron resonance (DEER) electron paramagnetic resonance spectroscopy to identify the molecular determinants of type I cadherin dimerization affinities. Small changes in sequence are found to produce subtle structural and dynamical changes that impact relative affinities, in part via electrostatic and hydrophobic interactions, and in part through entropic effects because of increased conformational heterogeneity in the bound states as revealed by DEER distance mapping in the dimers. These findings highlight the remarkable ability of evolution to exploit a wide range of molecular properties to produce closely related members of the same protein family that have affinity differences finely tuned to mediate their biological roles.

Binding of the U1A protein to its RNA target U1 hairpin II has been extensively studied as a model for a high affinity RNA/protein interaction. However, the mechanism and kinetics by which this complex is formed remain largely unknown. Here we use real-time biomolecular interaction analysis to dissect the roles various protein and RNA structural elements play in the formation of the U1A.U1 hairpin II complex. We show that neutralization of positive charges on the protein or increasing the salt concentration slows the association rate, suggesting that electrostatic interactions play an important role in bringing RNA and protein together. In contrast, removal of hydrogen bonding or stacking interactions within the RNA/protein interface, or reducing the size of the RNA loop, dramatically destabilizes the complex, as seen by a strong increase in the dissociation rate. Our data support a binding mechanism consisting of a rapid initial association based on electrostatic interactions and a subsequent locking step based on close-range interactions that occur during the induced fit of RNA and protein. Remarkably, these two steps can be clearly distinguished using U1A mutants containing single amino acid substitutions. Our observations explain the extraordinary affinity of U1A for its target and may suggest a general mechanism for high affinity RNA/protein interactions.

Nectins are immunoglobulin superfamily glycoproteins that mediate intercellular adhesion in many vertebrate tissues. Homophilic and heterophilic interactions between nectin family members help mediate tissue patterning. We determined the homophilic binding affinities and heterophilic specificities of all four nectins and the related protein nectin-like 5 (Necl-5) from human and mouse, revealing a range of homophilic interaction strengths and a defined heterophilic specificity pattern. To understand the molecular basis of their adhesion and specificity, we determined the crystal structures of natively glycosylated full ectodomains or adhesive fragments of all four nectins and Necl-5. All of the crystal structures revealed dimeric nectins bound through a stereotyped interface that was previously proposed to represent a cis dimer. However, conservation of this interface and the results of targeted cross-linking experiments showed that this dimer probably represents the adhesive trans interaction. The structure of the dimer provides a simple molecular explanation for the adhesive binding specificity of nectins.

Many cell–cell adhesive events are mediated by the dimerization of cadherin proteins presented on apposing cell surfaces. Cadherin-mediated 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/E-cadherin 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.