Molecular logic of neuronal self-recognition through protocadherin domain interactions

Abstract

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, and thus providing a size-dependent self-recognition mechanism.

Figure 1. Crystal structures of four Pcdh EC1-EC3 isoforms

A) The Pcdh genomic locus contains three adjacent clusters of variable exons. Each exon encodes an entire ectodomain comprising six EC domains, a transmembrane (TM) domain, and a short cytoplasmic region. Alpha and gamma clusters also contain three constant exons that encode a cluster-specific intracellular domain (ICD) which are joined by pre-mRNA splicing for alpha and gamma clusters. C-type Pcdh exons are shown in pink and light blue for the alpha and gamma clusters, respectively. B) Crystal structures of EC1-EC3 regions from PcdhαC2, Pcdhβ1, PcdhγA8, and PcdhγC5 shown in ribbon representation. Ca²⁺ ions are drawn as green spheres. N-glycans and conserved O-mannose residues are drawn as sticks. The inter-domain calcium binding sites are arranged similarly to those observed in classical cadherins (expanded view). See also Figure S1 and Table S1. C) Comparison of the PcdhγC5 and type I classical C-cadherin structures. The overall architecture of classical cadherin ectodomains have a curved shape with an approximate 90° angle between EC1 and EC5 (Boggon et al., 2002). In contrast, the architecture of Pcdh EC1-EC3 domain regions is characterized by an extended zigzagged conformation. D) EC2-EC3 angles distinct from classical cadherins account for the extended zigzagged conformation of the Pcdh structures. EC1-EC3 domains are drawn as blue (PcdhγC5) and yellow (C-cadherin) ovals. Angles shown are between principal axes of inertia for adjacent domains.

Figure 2. Elements of Pcdh cis and trans binding

A) Correlating multimerization states of truncated Pcdh proteins with their cell-cell recognition properties. Cells transfected with Pcdh deletion series plasmid constructs were tested for aggregation. With the exception of EC2-EC6 Pcdh fragments and PcdhγC5 EC1-EC4, all deletion proteins that formed oligomers in solution also mediated cell aggregation. Full-length Pcdhα4 include the EC6 domain from PcdhγC3 so it could be delivered to cell surface. B) Probing homophilic interaction interface by arginine-scanning mutagenesis. Residues mutated to arginine are drawn in space filling representation. In blue are mutations that did not disrupt recognition, in orange are mutations that weakened recognition and in red are mutations that abolished cell-cell recognition. Excluding residue 142, all the effective arginine mutants are located along one side of the molecule. C) Cell aggregation experiments showing the mutations in part (B) that weakened or abolished interactions. See also Figure S2C. D) In other Pcdh isoforms, residues analogous to the effective PcdhγC5 arginine mutants had similar effects on the cell-cell recognition in the majority of cases.

Figure 3. Pcdh trans binding depends on the four N-terminal domains EC1-EC4

A–C) Domain-shuffled chimeras of closely related isoforms and their wild-type counterparts were assayed for binding specificity. Swapped specificity was noted for chimeras in which either the EC1-EC3 or EC2-EC4 domains were replaced with the corresponding domains of closely related isoforms. See also Figure S3. D) Schematic representation of the domain-shuffled isoforms and their observed binding specificities to their wild-type isoform counterparts.

Figure 4. Candidate specificity determining residues

A) Multiple sequence alignment of the three closely related Pcdh isoform pairs, along with PcdhγC5. Highlighted in gray are positions conserved in all Pcdh sequences. Sequence positions that differ between the closely related isoforms are shown in red; a subset of these residues determines binding specificity. Residues swapped between isoforms and assayed for binding properties are boxed. Secondary structure from PcdhγC5 is shown at the top of the alignment. B) Multiple sequence alignment of the FG-loop region for PcdhγA8 and PcdhγA9 orthologs. Three of the residues that differ between mouse PcdhγA8 and PcdhγA9 are highly conserved in orthologs (highlighted in red), suggesting their functional importance.

Figure 5. Structural elements of the canonical Pcdh trans binding interface

A–C) Assessing specificity-determining residues. Binding properties of wild-type isoforms (left side of each panel) or constructs with shuffled residues (top of each panel) were tested separately for each EC domain. Cases in which shuffled residues swapped specificities are indicated by an orange outline. Residues shuffled between closely related isoforms are shown in magenta on surface representations of the Pcdhα7, Pcdhβ6, and PcdhγA8 structures. Sequence alignments of shuffled regions are shown. See also Figure S4. D) Correspondence between trans interface residues identified by arginine scanning and close-isoform pair analysis. Single arginine mutant residues that abolish or diminish homophilic binding, highlighted in red and orange respectively, are found in the same structural regions as the shuffled residues (see also Figure 2). Residues that swap binding specificity between closely related isoforms are shown in magenta on surface representations of the Pcdh-γC5 crystal structure.

Figure 6. Molecular logic of Pcdh-mediated cell-cell recognition

A) Shown in ribbon representation is the only orientation observed for docking of the four EC1-EC3 domains structures which position the EC2 AB loop in close proximity to the EC3 FG loop. EC2 AB loop residue 116 and FG loop residue 301 are drawn as space filling and colored red and blue respectively. The vast majority of the docked complexes were observed to interact in this mode. See also Figure S5A. B) Cell aggregation assays on chimeric proteins that show EC1 interacts with EC4 and EC2 interacts with EC3. Schematic representation of the head-to-tail interaction between the domain-shuffled chimeras is shown above each panel. Mixed aggregates were formed where all interactions involve “matching” domains (panels 1–3). Separate aggregates were formed when there is a mismatch between EC1/EC4 (panel 4) or between EC2/EC3 (panel 5). C) The EC2 domain AB region recognizes the EC3 domain FG loop. Cells expressing isoforms with single arginine mutants in the EC3 FG loop region, or with double mutations (aspartate at the AB region and arginine at the FG loop), were assayed for aggregation. The double-mutation rescued the non-adhesive phenotype, supporting the head-to-tail binding orientation shown in part (A). D) Two possible models of Pcdh interaction. A discrete tetramer composed of a dimer of dimers is observed in analytical ultracentrifugation, but we suggest that a connected ribbon of molecules can form between cells via the trans and cis interactions. E & F) A model for Pcdh mediated cell-cell recognition based on formation of a superstructure defined by promiscuous cis and specific trans interactions. Growth of the chain of molecules requires matching of all isoforms; a single mismatch can terminate chain extension. Dendrites of the same neuron will have the same isoform repertoire while dendrites of different neurons will differ. In this model, repulsion signaling is triggered, or achieves a sufficient level for response, only through the formation of an extended chain of Pcdhs. G) For the case of 15 distinct Pcdh isoforms expressed per cell, Monte-Carlo simulations were used to estimate the average size of one-dimensional Pcdh assemblies between contacting cells. The average number of cis dimers that comprise such assemblies is shown on a logarithmic scale as a function of the number of mismatched isoforms. Two cases are shown: one for 15000 total Pcdh monomers (1000 per isoform, red), and one for 1500 total copies (100 per isoform). The model assumes that each cell contains a stable set of cis dimers formed from the random association of monomers present in each cell. See also Figure S5B.