As the brain develops, its basic building blocks – cells called neurons – need to form the correct connections with one another in order to give rise to neural circuits. A mistake that leads to the formation of incorrect connections can result in a number of disorders or brain abnormalities.

Proteins called cadherins that are present on the surface of neurons enable them to stick to their correct partners like Velcro. One of these proteins is called Protocadherin-19. However, it was not fully understood how this protein forms an adhesive bond with other Protocadherin-19 molecules, or how some of the proteins within the cadherin family are able to distinguish between one another.

Cooper et al. used X-ray crystallography to visualize the molecular structure of Protocadherin-19 taken from zebrafish in order to better understand the adhesive bond that these proteins form with each other. In addition, the new structure showed the sites of the mutations that cause a form of epilepsy in infant females. From this, Cooper et al. could predict how the mutations would disrupt Protocadherin-19’s shape and function.

The structures revealed that Protocadherin-19 molecules from adjacent cells engage in a “forearm handshake” to form the bond that connects neurons. Some of the mutations that cause epilepsy occur in the region responsible for this Protocadherin-19 forearm handshake. Laboratory experiments confirmed that these mutations impair the formation of the adhesive bond, revealing the molecular basis for some of the mutations that underlie Protocadherin-19-female-limited epilepsy.

Other cadherin molecules may interact via a similar forearm handshake; this could be investigated in future experiments. It also remains to be discovered how brain wiring depends on Protocadherin-19 adhesion in animal development, and how altering these proteins can rewire developing brain circuits.

Nervous system function is critically dependent on the underlying neural architecture, including patterns of neuronal connectivity. Cell-cell recognition by cell surface receptors is central to establishing these functional neural circuits during development (Kiecker and Lumsden, 2005; Steinberg, 2007; Zipursky and Sanes, 2010). The cadherin superfamily is a large and diverse family of cell adhesion molecules that are strongly expressed in the developing nervous system (Hirano and Takeichi, 2012; Suzuki, 1996; Frank and Kemler, 2002; Shapiro et al., 2007; Gumbiner, 2005;Chen and Maniatis, 2013). The differential expression of classical cadherins and protocadherins, the largest groups within the cadherin superfamily, suggests that they play important roles in the development of neural circuitry (Weiner and Jontes, 2013; Hirano and Takeichi, 2012), an idea supported by their involvement in a range of neurodevelopmental disorders (Redies et al., 2012; Hirabayashi and Yagi, 2014). In particular, the non-clustered δ-protocadherins have been linked to autism spectrum disorders, intellectual disability, congenital microcephaly and epilepsy.

Protocadherin-19 (PCDH19) is a member of the non-clustered δ2-protocadherin subfamily (Wolverton and Lalande, 2001; Vanhalst et al., 2005; Gaitan and Bouchard, 2006; Emond et al., 2009; Liu et al., 2010) that is located on the X-chromosome. Mutations in PCDH19 cause an X-linked, female-limited form of infant-onset epilepsy (PCDH19 female epilepsy, PCDH19-FE; OMIM 300088) that is associated with intellectual disability, as well as compulsive or aggressive behavior and autistic features (Dibbens et al., 2008; Scheffer et al., 2008; Depienne and LeGuern, 2012; van Harssel et al., 2013; Leonardi et al., 2014; Thiffault et al., 2016; Terracciano et al., 2016; Walters et al., 2014). To date, well over 100 distinct mutations in PCDH19 have been identified in epilepsy patients, making it the second most clinically relevant gene in epilepsy. Approximately half of these mutations are missense mutations distributed throughout the extracellular domain of the PCDH19 protein. Despite the clear importance of PCDH19 and other non-clustered δ-protocadherins to neural development, their specific roles are only beginning to be revealed. For example, Pcdh7, Pcdh17 and Pcdh18b are involved in axon outgrowth or arborization (Piper et al., 2008; Hayashi et al., 2014; Biswas et al., 2014), while several δ-protocadherins, including Pcdh19, regulate cell motility during early development (Yamamoto et al., 1998; Aamar and Dawid, 2008; Biswas et al., 2010; Emond et al., 2009). In zebrafish, pcdh19, regulates the formation of neuronal columns in the optic tectum, and loss of pcdh19 degrades visually-guided behaviors (Cooper et al., 2015). However, it is not known how mutations in PCDH19 lead to PCDH19-FE.

Cadherins typically mediate adhesion using their extracellular domains, which are made of two or more consecutive extracellular cadherin (EC) repeats (Takeichi, 1990; Brasch et al., 2012). The adhesion mechanism used by classical cadherins is well known and involves a tip-to-tip interaction that is stabilized by the reciprocal exchange of tryptophan residues at the N-terminal EC1 repeat most distant from the membrane (Overduin et al., 1995; Shapiro et al., 1995; Nagar et al., 1996; Boggon et al., 2002; Patel et al., 2006; Zhang et al., 2009; Sivasankar et al., 2009; Harrison et al., 2010; Ciatto et al., 2010; Leckband and Sivasankar, 2012). However, PCDH19 along with the rest of the non-classical cadherins lack these critical tryptophan residues and must mediate adhesion by an alternative mechanism (Emond et al., 2011; Sotomayor et al., 2014; Biswas et al., 2010). In the case of the non-classical protocadherin-15 and cadherin-23 proteins, an adhesive interface is formed by overlapping, antiparallel interactions of their EC1 and EC2 tips (Elledge et al., 2010; Sotomayor et al., 2010; 2012; Geng et al., 2013). For clustered protocadherins, recent binding assays and structures suggest that adhesion is mediated by an antiparallel interaction of fully overlapping EC1 to EC4 domains (Rubinstein et al., 2015; Nicoludis et al., 2015; Goodman et al., 2016). Yet how non-clustered δ-protocadherins and PCDH19 form adhesive bonds and how these bonds are altered by disease-causing mutations is unknown.

Here we present crystals structures of the highly homologous zebrafish Protocadherin-19 (Pcdh19) encompassing repeats EC1-4 and EC3-4. The structures allow us to map >70% of the disease-causing missense mutations and provide a structural framework to interpret their functional impact. In addition, the structures suggest two possible homophilic adhesive interfaces, and complementary binding assays validate one of them, which is affected by multiple PCDH19-FE mutations. This interface involves fully overlapping EC1 to EC4 domains and likely represents a general interaction mechanism for the non-clustered δ-protocadherins.

To understand the mechanism of Pcdh19 function and to determine the structural role of PCDH19-FE mutations, the Danio rerio Pcdh19 EC1-4 and the EC3-4 fragments (70% identity, 83% similarity to Homo sapiens EC1-4) were produced in E. coli, refolded from inclusion bodies, and used for crystallization and structural determination (see Materials and methods). The solved structure for Pcdh19 EC3-4 (2.51 Å, Table 1, Figure 1—figure supplement 1A) includes four molecules in the asymmetric unit, each starting from Pro 213 and continuing to Asp 422 (numbering corresponds to the processed Danio rerio protein, see Materials and methods). Root-mean-square-deviation (RMSD) among these four molecules is <2.4 Å. One of the Pcdh19 EC3-4 molecules was used to solve the Pcdh19 EC1-4 structure (3.59 Å, Table 1, Figure 1—figure supplement 1B), which contains two molecules in the asymmetric unit, each starting from Val one to Asp 422 (RMSD of 1.4 Å). The EC3-4 repeats from both structures superpose well (RMSD 2.1 Å), and good quality electron density maps allowed us to unambiguously position side chains for most residues (Materials and methods and Figure 1—figure supplement 1). Given the similarities among our structures and chains, we will describe features as seen in the more complete chain B of Pcdh19 EC1-4, unless otherwise explicitly stated.

The architecture of all Pcdh19 EC repeats matches that observed for other cadherins (Shapiro et al., 1995; Overduin et al., 1995), with the typical Greek-key motif comprised of seven β strands (A-G) forming a β sandwich fold (Figure 1A). The EC1 repeat has a disulfide bond at the E-F loop, typical of clustered protocadherins, as well as one of two α-helices (at the B-C loop) also found in structures of clustered protocadherins (Morishita et al., 2006; Nicoludis et al., 2015; Rubinstein et al., 2015; Goodman et al., 2016) (Figure 1A,B). The three linker regions of Pcdh19 (EC1-2, EC2-3, EC3-4) have canonical cadherin calcium-binding sites (Nagar et al., 1996) (Figure 1E–G). Overall, our structures show canonical features and provide a unique framework to analyze >70% of the PCDH19-FE mutations.

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PCDH19-FE mutations.

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Antiparallel interfaces in crystal contacts of the Pcdh19 EC1-4 structure

Crystal structures have previously revealed the adhesive interfaces for classical cadherins, clustered protocadherins, and the protocadherin-15 and cadherin-23 complex (Nagar et al., 1996; Boggon et al., 2002; Patel et al., 2006; Ciatto et al., 2010; Sotomayor et al., 2012; Nicoludis et al., 2015; Goodman et al., 2016). Although the Pcdh19 EC3-4 structure does not reveal any relevant interface, the Pcdh19 EC1-4 structure does. The purified Pcdh19 EC1-4 fragment elutes in two well-defined peaks in size exclusion chromatography experiments (SEC), with these peaks most likely representing monomeric and dimeric states in solution (Figure 2—figure supplement 1). Pcdh19 EC1-4 crystals were grown from the putative dimer SEC peak elution, and two plausible adhesive interfaces are observed in our Pcdh19 EC1-4 structure. The first one, which we will refer to as Pcdh19-I1, arises from contacts between the two Pcdh19 EC1-4 molecules in the asymmetric unit, and involves a fully-overlapped antiparallel dimer in which EC1 from one molecule interacts with EC4 from the other (EC1:EC4), EC2 with EC3 (EC2:EC3), EC3 with EC2 (EC3:EC2), and EC4 with EC1 (EC4:EC1; Figure 2A,B). Within the same protein crystal structure, the second antiparallel interface (Pcdh19-I2) involves the opposite side of Pcdh19 with observed EC2:EC4, EC3:EC3, and EC4:EC2 interactions, as well as potential (not observed) EC1:EC5 and EC5:EC1 contacts (Figure 2—figure supplement 2A). Several lines of evidence favor the first interface Pcdh19-I1 as the most likely to mediate biological function.

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Analysis of the Pcdh19-I1 antiparallel interface with the Protein Interfaces, Surfaces and Assemblies (PISA) server (Krissinel and Henrick, 2007) and with the NOXclass classifier (Zhu et al., 2006) revealed a large interface (~1650 Ų), that is unlikely to be a crystal packing artifact (89.21% biological, 81% obligate). In contrast, the possible antiparallel Pcdh19-I2 interface is predicted by NOXclass to be non physiological, as its smaller interface area (~930 Ų) and the nature of its contacts matches those of crystal packing interactions (42.97% biological, 20.21% obligate). Yet, both interface areas are larger than 856 Ų, an empirical cut-off that can distinguish biological interfaces from crystal contacts with 85% accuracy (Ponstingl et al., 2000), and our analysis of the Pcdh19-I2 interface lacks contributions from possible EC1-EC5 contacts, which might be significant. Moreover, shape correlation (Lawrence and Colman, 1993) for Pcdh19-I1 is lower than for the Pcdh19-I2 interface (S_c-I1 = 0.44 vs. S_c-I2 = 0.61), as there is a large gap between the main EC2-EC3:EC3-EC2 contacts and the EC1-EC4:EC4-EC1 interactions zones (Figure 2A).

To further differentiate between the possible Pcdh19-I1 and Pcdh19-I2 interfaces, we evaluated whether any of the six PCDH19-FE mutations altering surface residues at crystal contacts, but not necessarily protein structure, could interfere with binding. Five of these mutations (S139L, T146R, P149S, E313K, and T404I; all at conserved sites) change residues involved in the Pcdh19-I1 interface (S116, T123, P126, E290, T381 respectively in Figure 2B–E), where we define residues at a given interface as those with a buried surface area that is at least 20% of their accessible surface area according to PISA. In all cases we predict altered EC1-4 homophilic binding, as the size and nature of the residue is changed by each mutation (hydrophobic vs. hydrophilic; charged vs. non-charged). The remaining mutation (H203P) involves a non-conserved residue at the Pcdh19-I2 interface, which could impair its formation (R180 in Figure 2—figure supplement 2A). However, the patient with the H203P mutation also carries another PCDH19-FE mutation (F206C) at a location mutated in other epilepsy patients (Marini et al., 2012; Depienne et al., 2011); thus it is unclear if H203P is contributing to epilepsy. In contrast, all five PCDH19-FE mutations at the Pcdh19-I1 interface are likely causal, which suggests that Pcdh19-I1 is relevant in vivo.

We also analyzed predicted glycosylation sites that might interfere with binding and thereby reveal non-physiological interfaces, as observed for VE-cadherin (Brasch et al., 2011). There are 14 glycosylation sites within EC1-4, and none of them involve residues at the Pcdh19-I1 interface (Figure 2—figure supplement 3A). An O-linked glycosylation site is predicted to be at the Pcdh19-I2 interface (T232), and an additional O-linked glycosylation site is predicted for the human PCDH19 protein at S204 (the equivalent N202 in Pcdh19 is predicted to be non-glycosylated), also at the Pcdh19-I2 interface (Figure 2—figure supplement 3B). Glycation sites, for which sugar molecules might be added randomly and to long-lived proteins, are predicted at both interfaces (K156 and K308 in Pcdh19-I1 and K204 in Pcdh19-I2), but may not interfere directly with either, since glycation depends on environmental conditions and it has never been reported for cadherins (Salahuddin et al., 2014; Simm et al., 2015). Thus the lack of glycosylation sites at the Pcdh19-I1 interface renders it as the most likely to be functional.

While not conclusive, all the analyses presented above favor the Pcdh19-I1 antiparallel dimer over the Pcdh19-I2 interface in terms of physiological relevance. The larger surface area of the Pcdh19-I1 dimer, the nature of the residues involved, the number of PCDH19-FE mutations at this interface, and the lack of predicted glycosylation sites, all suggest that the Pcdh19-I1 interface may occur and be functional in vivo.

Binding assays probing Pcdh19 interfaces

To conclusively test which binding interface mediates Pcdh19 adhesion, and whether PCDH19-FE mutations at the protein surface can interfere with one of the two possible Pcdh19 interfaces described above, we used modified bead aggregations assays, mutagenesis, and size exclusion chromatography experiments. Previous cell-based assays showed weak homophilic adhesion for the chicken Pcdh19 (Tai et al., 2010). In addition, previous assays in which the full-length Pcdh19 extracellular cadherin domain fused to Fc (Pcdh19ECFc) was incubated with protein A beads showed calcium-dependent aggregation only when it was co-purified with N-cadherin (Biswas et al., 2010; Emond et al., 2011). To study Pcdh19 homophilic interactions, we modified the previous protocol (Emond and Jontes, 2014) and added a final step in which beads were rocked (Sano et al., 1993) in a controlled fashion for up to two minutes (see Materials and methods). The modified protocol allowed us to identify clear bead aggregates mediated by Pcdh19ECFc alone (Figure 3—figure supplement 1).

To identify the minimal adhesive unit of Pcdh19 we used our modified protocol with truncated versions of Pcdh19 containing different numbers of EC repeats: Pcdh19ECFc (EC1-6), Pcdh19EC1-5Fc, Pcdh19EC1-4Fc, Pcdh19EC1-3Fc, Pcdh19EC1-2Fc, and Pcdh19EC2-6Fc (Figure 3). Bead aggregation was observed only when using Pcdh19ECFc, Pcdh19EC1-5Fc, and Pcdh19EC1-4Fc, thus suggesting that Pcdh19EC1-4 is the minimal adhesive unit and highlighting the biological relevance of the antiparallel Pcdh19-I1 interface, which involves EC1-4 only.

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Quantification of aggregation assays.

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Next, we introduced two PCDH19-FE mutations (T146R and E313K located at the Pcdh19-I1 interface; T123R and E290K in Figure 2B) in the full-length Pcdh19 extracellular domain and tested bead aggregation with these protein constructs (Figure 4). Bead aggregates were not detected when the Pcdh19ECFc carried these mutations under the conditions tested (Figure 4B–C,E–H). In contrast, the mutation R364E, predicted to impair the Pcdh19-I2 interface, did not eliminate bead aggregation (Figure 2—figure supplement 2B–D). Moreover, the presence of a N-cadherin (Ncad) fragment known to enhance Pcdh19-mediated adhesion (Emond et al., 2011) did not qualitatively change the effect of the T146R and E313K mutations. Bead aggregates were greatly diminished for T146R and abolished for E313K in the presence of NcadEC W2A/R14E His, a non-adhesive Ncad mutant previously used to study Pcdh19-mediated homophilic adhesion (Harrison et al., 2010; Emond et al., 2011) (Figure 4—figure supplement 1B–C,E–F). In addition, these mutations did not abolish the interaction between Pcdh19ECFc and NcadEC W2A/R14E His (Figure 4—figure supplement 2). It is possible that the T146R and 313K mutations affect interactions with N-cadherin in a subtle way (directly or allosterically), yet our experimental results suggest that these mutations directly impair Pcdh19 homophilic adhesion.

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We also introduced the T146R and E313K mutations at the Pcdh19-I1 interface into the bacterially expressed Pcdh19 EC1-4 protein fragment, and used analytical size exclusion chromatography to determine whether the mutant fragments were eluting as putative dimers or monomers in solution. Both mutations resulted in a shift of the elution peak that indicated a smaller, monomeric state (Figure 4I). Taken together, our crystallographic structural analyses and binding assays including PCDH19-FE mutations strongly support a model in which fully overlapped EC1-4 domains (Pcdh19-I1 interface) form the functional adhesive unit of Pcdh19 (Figure 4J).

Model for PCDH19 adhesive interaction and implications for other protocadherins

The antiparallel Pcdh19-I1 dimer interface validated above reveals a homophilic 'forearm handshake' binding mechanism for PCDH19, involving overlap of 4 ECs from each protomer wrapping around each other. This is different from the mechanism used by classical cadherins, only involving EC1 (Brasch et al., 2012) or the heterophilic 'extended handshake' used by protocadherin-15 and cadherin-23, involving overlap of only EC1-2 of each protein (Sotomayor et al., 2012). The forearm handshake is similar to the binding mechanism recently reported for clustered protocadherins (Goodman et al., 2016; Rubinstein et al., 2015; Nicoludis et al., 2015) and might be used by other non-clustered protocadherins.

The Pcdh19-I1 interface involves extended and mostly symmetric, in-register contacts between repeats EC2:EC3 that account for ~58% of the interfacial area, as well as smaller, separate EC1:EC4 contacts (~350 Ų × 2) that are slightly off-register. The EC1:EC4 contacts arise as both repeats bend to meet after the C-terminal end of EC3 and the N-terminal end of EC2 separate from each other. In this arrangement, the EC2-3 linkers from each protomer are right next to each other, while the EC3-EC4 linker in one protomer is separated from the EC1-2 linker of the binding partner by a large opening. The interface is generally amphiphilic, with ~49% of the interfacial area involving hydrophobic residues, ~28% hydrophilic, and ~23% charged residues (Figure 5—figure supplement 1). Interestingly, the contact formed by EC1:EC4 is more hydrophobic (58%; 22%; 20%) than the one formed by EC2:EC3 (41%; 33%; 26%), yet salt-bridge pairs across protomers are present in both: R40-E328 and E81-R39 enhance the EC1 to EC4 contacts (Figure 2C) and R158-E290 links EC2 to EC3 (Figure 2D). While the R40-E328 pair seems to be zebrafish specific, the other two salt-bridges are highly conserved across sequenced species, along with most of the residues involved in the Pcdh19 EC1-4 interface (Figure 5A and Figure 5—figure supplement 2). The same set of residues is highly variable across different members of the δ1, δ2, and the clustered protocadherins (Figure 5B and Figure 5—figure supplement 3), suggesting that binding mechanisms might differ across subfamilies or that residue variability might confer specificity within a common binding mechanism.

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Protocadherin-19 sequences.

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Sequences for selected clustered and δ-protocadherins.

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A comparison of our Pcdh19-I1 interface to recently reported models and structures of clustered protocadherin interfaces (Nicoludis et al., 2015; Goodman et al., 2016) reveals multiple similarities among them. The most complete models of α and β-protocadherins show similar, fully overlapped antiparallel EC1-4 dimers (Figure 5—figure supplement 4A–D), with the same extended EC2:EC3 antiparallel connection accompanied with smaller EC1:EC4 contacts and salt-bridges across protomers. Structural alignments show that the relative arrangements of protomers within the antiparallel dimers for Pcdh19, Mm Pcdhα4 (5DZW), and Mm Pcdhα7 (5DZV) are the most similar to each other with slight shifting in some EC repeats (Figure 5—figure supplement 4A,B). The Mm Pcdhβ6 (5DZX) and Mm Pcdhβ8 (5DZY) structures show similar dimeric interfaces, but the relative arrangement of protomers within the dimer is slightly shifted for all EC repeats (Figure 5—figure supplement 4C,D). Similarly, the Mm PcdhγA1 EC1-3 interface (4ZI9) matches and aligns well with the Pcdh19 EC1-4 dimer (Figure 5—figure supplement 4E). Mapping of all interaction sites to the Pcdh19 EC1-4 topology diagram (Figure 5C) reveals a pattern for common interacting domains in odd and even EC repeats across these structures, which include the F-G β hairpin and β strand A for repeats EC1 and EC3, as well as the A-B and D-E β hairpins for EC2 and EC4. While there are differences in some of the interacting domains, dimeric arrangements, and contact details, including diversity of interfacial residues, clustered protocadherins seem to use the same binding mechanism that Pcdh19 uses to mediate adhesion.

A conserved RGD sequence motif within Pcdh19 EC2 (residues 158 to 160) at its D-E loop is similar to an integrin-binding RGD site within EC1 (C-D loop) in the α-protocadherins (Ruoslahti, 1996; Mutoh et al., 2004). The EC1 RGD motif is exposed in the Mm Pcdhα4 and Mm Pcdhα7 homo-dimers while the EC2 RGD motif of Pcdh19 (also present in Pcdh17 [Kim et al., 2011]) is buried at the EC2:EC3 contacts in the Pcdh19-I1 interface. This suggests that homophilic binding could regulate the availability of this potential, untested, integrin-binding site.

Pcdh19 belongs to the δ2-protocadherin subfamily, and given the sequence similarity among subfamily members, it is likely that all use the same dimer interface to mediate adhesion. This is less obvious for the δ1 subfamily, with members that have seven EC repeats and that display some critical differences at interaction sites, such as the presence of a positively charged residue (R or K) at position 290, where most δ2 members have a negatively charged glutamate that interacts with an arginine at position 158 (Figure 2D, Figure 5—figure supplements 3 and 5). The PCDH19-FE E313K mutation at this site (E290) prevents binding (Figure 4C,H–I and Figure 4—figure supplement 1), suggesting that δ1-protocadherins, which effectively carry the same mutation, should use a different interface to mediate adhesion. Yet, residues at position 157 and 158 in δ1-protocadherins are also charge swapped, with aspartates and glutamates that would restore this critical salt-bridge interaction at the EC2:EC3 interface, and at the same time prevent heterophilic interactions with δ2-protocadherins (Figure 5—figure supplements 3 and 5). Thus, it is likely that all non-clustered δ-protocadherins use fully overlapped EC1-4 antiparallel interfaces, like the one observed for Pcdh19, to mediate adhesion.

The non-clustered δ-protocadherins are increasingly linked to human neurodevelopmental disorders, emphasizing both their importance to brain development and their relevance to human health (Redies et al., 2005; Redies et al., 2012; Hirabayashi and Yagi, 2014). In particular, mutations in PCDH19 cause a female-limited form of infant-onset epilepsy (Dibbens et al., 2008; Scheffer et al., 2008; Depienne and LeGuern, 2012; van Harssel et al., 2013; Leonardi et al., 2014; Thiffault et al., 2016; Terracciano et al., 2016). Therefore, it is imperative to understand the developmental roles of PCDH19 and other non-clustered δ-protocadherins, the structural basis of homophilic adhesion by these molecules, and the functional impact of pathogenic missense mutations. The structural and biochemical data presented here provide a first view on the molecular mechanism of Pcdh19 adhesion, which is likely used by all non-clustered δ and clustered protocadherins. Moreover, our Pcdh19 EC1-4 structural model shows > 70% of the missense mutations identified in PCDH19-FE patients, and reveals the biochemical basis for the deleterious effects for many of these mutations.

The Pcdh19 EC1-4 structure reveals an antiparallel dimer that is consistent with a trans adhesive interface, a conclusion supported by multiple lines of evidence. Notably, several missense mutations identified in PCDH19-FE patients localize to this interface. Two of these missense mutations (T146R and E313K) impair dimerization, as assessed by analytical gel filtration, and adhesion as assessed in bead aggregation assays, with and without N-cadherin. Sequence analysis suggests that the antiparallel adhesive mechanism presented here is broadly relevant to other, related δ-protocadherins. Recent work with clustered protocadherins, implicated in self-avoidance and self/non-self recognition (Lefebvre et al., 2012; Kostadinov and Sanes, 2015; Yagi, 2012), have revealed a similar antiparallel adhesive interface for these clustered protocadherins (Rubinstein et al., 2015; Nicoludis et al., 2015; Goodman et al., 2016). Thus, the Pcdh19-I1 adhesive interface observed in our Pcdh19 EC1-4 structure likely represents the mechanism used by both non-clustered δ-protocadherins and clustered protocadherins, which, together, represent the largest group within the cadherin superfamily.

Our structural data for Pcdh19, as well as recent work with the clustered protocadherins raises an interesting conundrum. The adhesive interface for protocadherins is extensive and involves interactions extending throughout EC1-4. This contrasts sharply with the adhesive interface of classical cadherins, which is restricted to EC1 and involves the reciprocal swap of Aβ-strands that is stabilized by burying Trp2 in a hydrophobic pocket (Brasch et al., 2012). However, the K_D for dimerization of α- and β-protocadherins is in the micromolar range (similar to classical cadherins), bead aggregation and cell-based assays have consistently shown weak adhesion by both non-clustered and clustered protocadherins, and protocadherins are widely recognized as being only weakly adhesive (Schreiner and Weiner, 2010; Thu et al., 2014; Sano et al., 1993; Rubinstein et al., 2015). This disparity suggests that other mechanisms could modulate protocadherin adhesion in vivo. For instance, cis-oligomerization could compete with trans adhesive interactions, or interactions with other proteins, including N-cadherin, could sequester protocadherins or mask their adhesive interface. Further studies will be required to better understand protocadherin adhesion, how it may be altered in the presence of N-cadherin, and how it is regulated in vivo.

In addition to mutations that disrupt adhesion, our data reveal the potential effects of two other classes of mutations. In the first class, many mutations are predicted to directly impair folding and stability, which could lead to reduced levels of protein on the surface, due to impaired trafficking or enhanced protein degradation. In the second, PCDH19-FE mutations affecting calcium-binding sites are likely to cause shifts in calcium affinity as well as protein instability. Similar mutations in cadherin-23 and protocadherin-15 have been shown to decrease protein affinity for calcium, with K_D shifts that are relevant in the context of the low calcium concentration to which these proteins are exposed (Sotomayor et al., 2010). Yet PCDH19 is expected to be in interstitial space with high calcium concentration, so it is more likely that the relevant effect of PCDH19-FE mutations at calcium-binding sites is compromised stability (even at saturating calcium concentrations), as shown here for the N340S mutation. Finally, analysis of one PCDH19-FE mutation within EC1-4, and three within EC5-6, reveal no obvious predicted consequences at the structural level, as they are exposed residues that should not affect calcium-binding, protein stability or adhesion. These mutations may impact a variety of protein-protein interactions. Although the physiological relevance is unclear, both non-clustered and clustered protocadherins can form cis-homo- or cis-hetero-oligomers (Chen et al., 2007; Schreiner and Weiner, 2010), and mutations affecting the formation of cis-oligomers could adversely impact protocadherin function. Similarly, protocadherins participate in a variety of protein complexes beyond homophilic trans adhesion: Pcdh19 has been shown to associate in cis with N-cadherin (Emond et al., 2011); protocadherins associate with the Wnt co-receptor, RYK (Berndt et al., 2011); PAPC interacts with Frizzled-7 and FLRT3 (Chen et al., 2009; Kraft et al., 2012); and Pcdh17 and Pcdh19 have highly conserved RGD sequences, suggesting that they may interact with integrins (Ruoslahti, 1996; Mutoh et al., 2004; Kim et al., 2011). Thus, further experimental characterization of key mutants in vitro and in vivo will continue to reveal correlations between structural defects, cellular-level defects, and different aspects of PCDH19-FE.

The non-clustered protocadherins are increasingly recognized as a family of molecules that play important roles during neural development. In addition to the role of PCDH19 in epilepsy, mutation of PCDH12 was found to underlie a syndrome of microcephaly that is associated with epilepsy and developmental disability (Aran et al., 2016). Moreover, both PCDH9 and PCDH10 have been associated with autism spectrum disorders (Marshall et al., 2008; Morrow et al., 2008). Ongoing work will likely uncover further links between members of this family and neurodevelopmental disorders. Our Pcdh19 EC1-4 model is the first to show the structural basis of adhesion by the non-clustered δ-protocadherins, and reveals that some of the missense mutations identified in PCDH19-FE occur at the adhesive interface and act by abolishing adhesion. This represents an initial stage in understanding the mechanisms of non-clustered δ-protocadherin homophilic adhesion and provides insight into the biochemical basis of protocadherin-based neurodevelopmental disease.

1. 1. Cooper SR
   2. Jontes JD
   3. Sotomayor M

   (2015) Crystal Structure of Zebrafish Protocadherin-19 EC3-4

   Publicly available at the RCSB Protein Data Bank (accession no: 5CO1).

   http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5CO1
2. 1. Cooper SR
   2. Jontes JD
   3. Sotomayor M

   (2016) Crystal Structure of Zebrafish Protocadherin-19 EC1-4

   Publicly available at the RCSB Protein Data Bank (accession no: 5IU9).

   http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5IU9

1. 1. Aamar E
   2. Dawid IB

   (2008) Protocadherin-18a has a role in cell adhesion, behavior and migration in zebrafish development

   Developmental Biology 318:335–346.

   https://doi.org/10.1016/j.ydbio.2008.03.040

   • PubMed
   • Google Scholar
2. 1. Aran A
   2. Rosenfeld N
   3. Jaron R
   4. Renbaum P
   5. Zuckerman S
   6. Fridman H
   7. Zeligson S
   8. Segel R
   9. Kohn Y
   10. Kamal L
   11. Kanaan M
   12. Segev Y
   13. Mazaki E
   14. Rabinowitz R
   15. Shen O
   16. Lee M
   17. Walsh T
   18. King MC
   19. Gulsuner S
   20. Levy-Lahad E

   (2016) Loss of function of PCDH12 underlies recessive microcephaly mimicking intrauterine infection

   Neurology 86:2016–2024.

   https://doi.org/10.1212/WNL.0000000000002704

   • PubMed
   • Google Scholar
3. 1. Ashkenazy H
   2. Abadi S
   3. Martz E
   4. Chay O
   5. Mayrose I
   6. Pupko T
   7. Ben-Tal N

   (2016) ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules

   Nucleic Acids Research 44:W344–W350.

   https://doi.org/10.1093/nar/gkw408

   • PubMed
   • Google Scholar
4. 1. Barry J
   2. Gu Y
   3. Jukkola P
   4. O'Neill B
   5. Gu H
   6. Mohler PJ
   7. Rajamani KT
   8. Gu C

   (2014) Ankyrin-G directly binds to kinesin-1 to transport voltage-gated Na+ channels into axons

   Developmental Cell 28:117–131.

   https://doi.org/10.1016/j.devcel.2013.11.023

   • PubMed
   • Google Scholar
5. 1. Berndt JD
   2. Aoyagi A
   3. Yang P
   4. Anastas JN
   5. Tang L
   6. Moon RT

   (2011) Mindbomb 1, an E3 ubiquitin ligase, forms a complex with RYK to activate Wnt/β-catenin signaling

   Journal of Cell Biology 194:737–750.

   https://doi.org/10.1083/jcb.201107021

   • PubMed
   • Google Scholar
6. 1. Biswas S
   2. Emond MR
   3. Duy PQ
   4. Hao le T
   5. Beattie CE
   6. Jontes JD

   (2014) Protocadherin-18b interacts with Nap1 to control motor axon growth and arborization in zebrafish

   Molecular Biology of the Cell 25:633–642.

   https://doi.org/10.1091/mbc.E13-08-0475

   • PubMed
   • Google Scholar
7. 1. Biswas S
   2. Emond MR
   3. Jontes JD

   (2010) Protocadherin-19 and N-cadherin interact to control cell movements during anterior neurulation

   Journal of Cell Biology 191:1029–1041.

   https://doi.org/10.1083/jcb.201007008

   • PubMed
   • Google Scholar
8. 1. Boggon TJ
   2. Murray J
   3. Chappuis-Flament S
   4. Wong E
   5. Gumbiner BM
   6. Shapiro L

   (2002) C-cadherin ectodomain structure and implications for cell adhesion mechanisms

   Science 296:1308–1313.

   https://doi.org/10.1126/science.1071559

   • PubMed
   • Google Scholar
9. 1. Brasch J
   2. Harrison OJ
   3. Ahlsen G
   4. Carnally SM
   5. Henderson RM
   6. Honig B
   7. Shapiro L

   (2011) Structure and binding mechanism of vascular endothelial cadherin: a divergent classical cadherin

   Journal of Molecular Biology 408:57–73.

   https://doi.org/10.1016/j.jmb.2011.01.031

   • PubMed
   • Google Scholar
10. 1. Brasch J
    2. Harrison OJ
    3. Honig B
    4. Shapiro L

    (2012) Thinking outside the cell: how cadherins drive adhesion

    Trends in Cell Biology 22:299–310.

    https://doi.org/10.1016/j.tcb.2012.03.004

    • PubMed
    • Google Scholar
11. 1. Chen WV
    2. Maniatis T

    (2013) Clustered protocadherins

    Development 140:3297–3302.

    https://doi.org/10.1242/dev.090621

    • PubMed
    • Google Scholar
12. 1. Chen X
    2. Koh E
    3. Yoder M
    4. Gumbiner BM

    (2009) A protocadherin-cadherin-FLRT3 complex controls cell adhesion and morphogenesis

    PLoS One 4:e8411.

    https://doi.org/10.1371/journal.pone.0008411

    • Google Scholar
13. 1. Chen X
    2. Molino C
    3. Liu L
    4. Gumbiner BM

    (2007) Structural elements necessary for oligomerization, trafficking, and cell sorting function of paraxial protocadherin

    Journal of Biological Chemistry 282:32128–32137.

    https://doi.org/10.1074/jbc.M705337200

    • PubMed
    • Google Scholar
14. 1. Ciatto C
    2. Bahna F
    3. Zampieri N
    4. VanSteenhouse HC
    5. Katsamba PS
    6. Ahlsen G
    7. Harrison OJ
    8. Brasch J
    9. Jin X
    10. Posy S
    11. Vendome J
    12. Ranscht B
    13. Jessell TM
    14. Honig B
    15. Shapiro L

    (2010) T-cadherin structures reveal a novel adhesive binding mechanism

    Nature Structural & Molecular Biology 17:339–347.

    https://doi.org/10.1038/nsmb.1781

    • PubMed
    • Google Scholar
15. 1. Cooper SR
    2. Emond MR
    3. Duy PQ
    4. Liebau BG
    5. Wolman MA
    6. Jontes JD

    (2015) Protocadherins control the modular assembly of neuronal columns in the zebrafish optic tectum

    Journal of Cell Biology 211:807–814.

    https://doi.org/10.1083/jcb.201507108

    • PubMed
    • Google Scholar
16. 1. Dechavanne V
    2. Barrillat N
    3. Borlat F
    4. Hermant A
    5. Magnenat L
    6. Paquet M
    7. Antonsson B
    8. Chevalet L

    (2011) A high-throughput protein refolding screen in 96-well format combined with design of experiments to optimize the refolding conditions

    Protein Expression and Purification 75:192–203.

    https://doi.org/10.1016/j.pep.2010.09.008

    • PubMed
    • Google Scholar
17. 1. Depienne C
    2. LeGuern E

    (2012) PCDH19-related infantile epileptic encephalopathy: an unusual X-linked inheritance disorder

    Human Mutation 33:627–634.

    https://doi.org/10.1002/humu.22029

    • PubMed
    • Google Scholar
18. 1. Depienne C
    2. Trouillard O
    3. Bouteiller D
    4. Gourfinkel-An I
    5. Poirier K
    6. Rivier F
    7. Berquin P
    8. Nabbout R
    9. Chaigne D
    10. Steschenko D
    11. Gautier A
    12. Hoffman-Zacharska D
    13. Lannuzel A
    14. Lackmy-Port-Lis M
    15. Maurey H
    16. Dusser A
    17. Bru M
    18. Gilbert-Dussardier B
    19. Roubertie A
    20. Kaminska A
    21. Whalen S
    22. Mignot C
    23. Baulac S
    24. Lesca G
    25. Arzimanoglou A
    26. LeGuern E

    (2011) Mutations and deletions in PCDH19 account for various familial or isolated epilepsies in females

    Human Mutation 32:E1959–E1975.

    https://doi.org/10.1002/humu.21373

    • PubMed
    • Google Scholar
19. 1. Dibbens LM
    2. Tarpey PS
    3. Hynes K
    4. Bayly MA
    5. Scheffer IE
    6. Smith R
    7. Bomar J
    8. Sutton E
    9. Vandeleur L
    10. Shoubridge C
    11. Edkins S
    12. Turner SJ
    13. Stevens C
    14. O'Meara S
    15. Tofts C
    16. Barthorpe S
    17. Buck G
    18. Cole J
    19. Halliday K
    20. Jones D
    21. Lee R
    22. Madison M
    23. Mironenko T
    24. Varian J
    25. West S
    26. Widaa S
    27. Wray P
    28. Teague J
    29. Dicks E
    30. Butler A
    31. Menzies A
    32. Jenkinson A
    33. Shepherd R
    34. Gusella JF
    35. Afawi Z
    36. Mazarib A
    37. Neufeld MY
    38. Kivity S
    39. Lev D
    40. Lerman-Sagie T
    41. Korczyn AD
    42. Derry CP
    43. Sutherland GR
    44. Friend K
    45. Shaw M
    46. Corbett M
    47. Kim HG
    48. Geschwind DH
    49. Thomas P
    50. Haan E
    51. Ryan S
    52. McKee S
    53. Berkovic SF
    54. Futreal PA
    55. Stratton MR
    56. Mulley JC
    57. Gécz J

    (2008) X-linked protocadherin 19 mutations cause female-limited epilepsy and cognitive impairment

    Nature Genetics 40:776–781.

    https://doi.org/10.1038/ng.149

    • PubMed
    • Google Scholar
20. 1. Elledge HM
    2. Kazmierczak P
    3. Clark P
    4. Joseph JS
    5. Kolatkar A
    6. Kuhn P
    7. Müller U

    (2010) Structure of the N terminus of cadherin 23 reveals a new adhesion mechanism for a subset of cadherin superfamily members

    PNAS 107:10708–10712.

    https://doi.org/10.1073/pnas.1006284107

    • PubMed
    • Google Scholar
21. 1. Emond MR
    2. Biswas S
    3. Blevins CJ
    4. Jontes JD

    (2011) A complex of Protocadherin-19 and N-cadherin mediates a novel mechanism of cell adhesion

    Journal of Cell Biology 195:1115–1121.

    https://doi.org/10.1083/jcb.201108115

    • PubMed
    • Google Scholar
22. 1. Emond MR
    2. Biswas S
    3. Jontes JD

    (2009) Protocadherin-19 is essential for early steps in brain morphogenesis

    Developmental Biology 334:72–83.

    https://doi.org/10.1016/j.ydbio.2009.07.008

    • PubMed
    • Google Scholar
23. 1. Emond MR
    2. Jontes JD

    (2014) Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

    Journal of Visualized Experiments 92:e51762.

    https://doi.org/10.3791/51762

    • Google Scholar
24. 1. Emsley P
    2. Lohkamp B
    3. Scott WG
    4. Cowtan K

    (2010) Features and development of Coot

    Acta Crystallographica Section D Biological Crystallography 66:486–501.

    https://doi.org/10.1107/S0907444910007493

    • PubMed
    • Google Scholar
25. 1. Frank M
    2. Kemler R

    (2002) Protocadherins

    Current Opinion in Cell Biology 14:557–562.

    https://doi.org/10.1016/S0955-0674(02)00365-4

    • PubMed
    • Google Scholar
26. 1. Gaitan Y
    2. Bouchard M

    (2006) Expression of the delta-protocadherin gene Pcdh19 in the developing mouse embryo

    Gene Expression Patterns 6:893–899.

    https://doi.org/10.1016/j.modgep.2006.03.001

    • PubMed
    • Google Scholar
27. 1. Geng R
    2. Sotomayor M
    3. Kinder KJ
    4. Gopal SR
    5. Gerka-Stuyt J
    6. Chen DH
    7. Hardisty-Hughes RE
    8. Ball G
    9. Parker A
    10. Gaudet R
    11. Furness D
    12. Brown SD
    13. Corey DP
    14. Alagramam KN

    (2013) Noddy, a mouse harboring a missense mutation in protocadherin-15, reveals the impact of disrupting a critical interaction site between tip-link cadherins in inner ear hair cells

    Journal of Neuroscience 33:4395–4404.

    https://doi.org/10.1523/JNEUROSCI.4514-12.2013

    • PubMed
    • Google Scholar
28. 1. Goodman KM
    2. Rubinstein R
    3. Thu CA
    4. Bahna F
    5. Mannepalli S
    6. Ahlsén G
    7. Rittenhouse C
    8. Maniatis T
    9. Honig B
    10. Shapiro L

    (2016) Structural basis of diverse homophilic recognition by clustered α- and β-protocadherins

    Neuron 90:709–723.

    https://doi.org/10.1016/j.neuron.2016.04.004

    • PubMed
    • Google Scholar
29. 1. Gumbiner BM

    (2005) Regulation of cadherin-mediated adhesion in morphogenesis

    Nature Reviews Molecular Cell Biology 6:622–634.

    https://doi.org/10.1038/nrm1699

    • PubMed
    • Google Scholar
30. 1. Hansen JE
    2. Lund O
    3. Tolstrup N
    4. Gooley AA
    5. Williams KL
    6. Brunak S

    (1998) NetOglyc: prediction of mucin type O-glycosylation sites based on sequence context and surface accessibility

    Glycoconjugate Journal 15:115–130.

    https://doi.org/10.1023/A:1006960004440

    • PubMed
    • Google Scholar
31. 1. Harrison OJ
    2. Bahna F
    3. Katsamba PS
    4. Jin X
    5. Brasch J
    6. Vendome J
    7. Ahlsen G
    8. Carroll KJ
    9. Price SR
    10. Honig B
    11. Shapiro L

    (2010) Two-step adhesive binding by classical cadherins

    Nature Structural & Molecular Biology 17:348–357.

    https://doi.org/10.1038/nsmb.1784

    • PubMed
    • Google Scholar
32. 1. Hayashi S
    2. Inoue Y
    3. Kiyonari H
    4. Abe T
    5. Misaki K
    6. Moriguchi H
    7. Tanaka Y
    8. Takeichi M

    (2014) Protocadherin-17 mediates collective axon extension by recruiting actin regulator complexes to interaxonal contacts

    Developmental Cell 30:673–687.

    https://doi.org/10.1016/j.devcel.2014.07.015

    • PubMed
    • Google Scholar
33. 1. Higurashi N
    2. Takahashi Y
    3. Kashimada A
    4. Sugawara Y
    5. Sakuma H
    6. Tomonoh Y
    7. Inoue T
    8. Hoshina M
    9. Satomi R
    10. Ohfu M
    11. Itomi K
    12. Takano K
    13. Kirino T
    14. Hirose S

    (2015) Immediate suppression of seizure clusters by corticosteroids in PCDH19 female epilepsy

    Seizure 27:1–5.

    https://doi.org/10.1016/j.seizure.2015.02.006

    • PubMed
    • Google Scholar
34. 1. Hirabayashi T
    2. Yagi T

    (2014) Protocadherins in neurological diseases

    Advances in Neurobiology 8:293–314.

    https://doi.org/10.1007/978-1-4614-8090-7_13

    • PubMed
    • Google Scholar
35. 1. Hirano S
    2. Takeichi M

    (2012) Cadherins in brain morphogenesis and wiring

    Physiological Reviews 92:597–634.

    https://doi.org/10.1152/physrev.00014.2011

    • PubMed
    • Google Scholar
36. 1. Hoshina M
    2. Higurashi N
    3. Abe Y
    4. Mishima H
    5. Hosoya M
    6. Nakayama T
    7. Hirose S

    (2015)

    [A patient with an early diagnosis of PCDH19-related epilepsy]

    No to Hattatsu Brain and Development 47:305–309.

    • PubMed
    • Google Scholar
37. 1. Humphrey W
    2. Dalke A
    3. Schulten K

    (1996) VMD: visual molecular dynamics

    Journal of Molecular Graphics 14:33–38.

    https://doi.org/10.1016/0263-7855(96)00018-5

    • PubMed
    • Google Scholar
38. 1. Jiang M
    2. Chen G

    (2006) High Ca2+-phosphate transfection efficiency in low-density neuronal cultures

    Nature Protocols 1:695–700.

    https://doi.org/10.1038/nprot.2006.86

    • PubMed
    • Google Scholar
39. 1. Johansen MB
    2. Kiemer L
    3. Brunak S

    (2006) Analysis and prediction of mammalian protein glycation

    Glycobiology 16:844–853.

    https://doi.org/10.1093/glycob/cwl009

    • PubMed
    • Google Scholar
40. 1. Julenius K

    (2007) NetCGlyc 1.0: prediction of mammalian C-mannosylation sites

    Glycobiology 17:868–876.

    https://doi.org/10.1093/glycob/cwm050

    • PubMed
    • Google Scholar
41. 1. Keegan RM
    2. Winn MD

    (2007) Automated search-model discovery and preparation for structure solution by molecular replacement

    Acta Crystallographica Section D Biological Crystallography 63:447–457.

    https://doi.org/10.1107/S0907444907002661

    • Google Scholar
42. 1. Kiecker C
    2. Lumsden A

    (2005) Compartments and their boundaries in vertebrate brain development

    Nature Reviews Neuroscience 6:553–564.

    https://doi.org/10.1038/nrn1702

    • PubMed
    • Google Scholar
43. 1. Kim SY
    2. Yasuda S
    3. Tanaka H
    4. Yamagata K
    5. Kim H

    (2011) Non-clustered protocadherin

    Cell Adhesion & Migration 5:97–105.

    https://doi.org/10.4161/cam.5.2.14374

    • PubMed
    • Google Scholar
44. 1. Kostadinov D
    2. Sanes JR

    (2015) Protocadherin-dependent dendritic self-avoidance regulates neural connectivity and circuit function

    eLife 4:e08964.

    https://doi.org/10.7554/eLife.08964

    • PubMed
    • Google Scholar
45. 1. Kraft B
    2. Berger CD
    3. Wallkamm V
    4. Steinbeisser H
    5. Wedlich D

    (2012) Wnt-11 and Fz7 reduce cell adhesion in convergent extension by sequestration of PAPC and C-cadherin

    Journal of Cell Biology 198:695–709.

    https://doi.org/10.1083/jcb.201110076

    • PubMed
    • Google Scholar
46. 1. Krissinel E
    2. Henrick K

    (2007) Inference of macromolecular assemblies from crystalline state

    Journal of Molecular Biology 372:774–797.

    https://doi.org/10.1016/j.jmb.2007.05.022

    • PubMed
    • Google Scholar
47. 1. Kwon M
    2. Firestein BL

    (2013) DNA transfection: calcium phosphate method

    Methods in Molecular Biology 1018:107–110.

    https://doi.org/10.1007/978-1-62703-444-9_10

    • PubMed
    • Google Scholar
48. 1. Lavinder JJ
    2. Hari SB
    3. Sullivan BJ
    4. Magliery TJ

    (2009) High-throughput thermal scanning: a general, rapid dye-binding thermal shift screen for protein engineering

    Journal of the American Chemical Society 131:3794–3795.

    https://doi.org/10.1021/ja8049063

    • PubMed
    • Google Scholar
49. 1. Lawrence MC
    2. Colman PM

    (1993) Shape complementarity at protein/protein interfaces

    Journal of Molecular Biology 234:946–950.

    https://doi.org/10.1006/jmbi.1993.1648

    • PubMed
    • Google Scholar
50. 1. Leckband D
    2. Sivasankar S

    (2012) Cadherin recognition and adhesion

    Current Opinion in Cell Biology 24:620–627.

    https://doi.org/10.1016/j.ceb.2012.05.014

    • PubMed
    • Google Scholar
51. 1. Lefebvre JL
    2. Kostadinov D
    3. Chen WV
    4. Maniatis T
    5. Sanes JR

    (2012) Protocadherins mediate dendritic self-avoidance in the mammalian nervous system

    Nature 488:517–521.

    https://doi.org/10.1038/nature11305

    • PubMed
    • Google Scholar
52. 1. Leonardi E
    2. Sartori S
    3. Vecchi M
    4. Bettella E
    5. Polli R
    6. Palma LD
    7. Boniver C
    8. Murgia A

    (2014) Identification of four novel PCDH19 mutations and prediction of their functional impact

    Annals of Human Genetics 78:389–398.

    https://doi.org/10.1111/ahg.12082

    • PubMed
    • Google Scholar
53. 1. Liu Q
    2. Chen Y
    3. Kubota F
    4. Pan JJ
    5. Murakami T

    (2010) Expression of protocadherin-19 in the nervous system of the embryonic zebrafish

    The International Journal of Developmental Biology 54:905–911.

    https://doi.org/10.1387/ijdb.092882ql

    • PubMed
    • Google Scholar
54. 1. Marini C
    2. Darra F
    3. Specchio N
    4. Mei D
    5. Terracciano A
    6. Parmeggiani L
    7. Ferrari A
    8. Sicca F
    9. Mastrangelo M
    10. Spaccini L
    11. Canopoli ML
    12. Cesaroni E
    13. Zamponi N
    14. Caffi L
    15. Ricciardelli P
    16. Grosso S
    17. Pisano T
    18. Canevini MP
    19. Granata T
    20. Accorsi P
    21. Battaglia D
    22. Cusmai R
    23. Vigevano F
    24. Dalla Bernardina B
    25. Guerrini R

    (2012) Focal seizures with affective symptoms are a major feature of PCDH19 gene-related epilepsy

    Epilepsia 53:2111–2119.

    https://doi.org/10.1111/j.1528-1167.2012.03649.x

    • PubMed
    • Google Scholar
55. 1. Marshall CR
    2. Noor A
    3. Vincent JB
    4. Lionel AC
    5. Feuk L
    6. Skaug J
    7. Shago M
    8. Moessner R
    9. Pinto D
    10. Ren Y
    11. Thiruvahindrapduram B
    12. Fiebig A
    13. Schreiber S
    14. Friedman J
    15. Ketelaars CE
    16. Vos YJ
    17. Ficicioglu C
    18. Kirkpatrick S
    19. Nicolson R
    20. Sloman L
    21. Summers A
    22. Gibbons CA
    23. Teebi A
    24. Chitayat D
    25. Weksberg R
    26. Thompson A
    27. Vardy C
    28. Crosbie V
    29. Luscombe S
    30. Baatjes R
    31. Zwaigenbaum L
    32. Roberts W
    33. Fernandez B
    34. Szatmari P
    35. Scherer SW

    (2008) Structural variation of chromosomes in autism spectrum disorder

    The American Journal of Human Genetics 82:477–488.

    https://doi.org/10.1016/j.ajhg.2007.12.009

    • PubMed
    • Google Scholar
56. 1. McCoy AJ
    2. Grosse-Kunstleve RW
    3. Adams PD
    4. Winn MD
    5. Storoni LC
    6. Read RJ

    (2007) Phaser crystallographic software

    Journal of Applied Crystallography 40:658–674.

    https://doi.org/10.1107/S0021889807021206

    • PubMed
    • Google Scholar
57. 1. Minor W
    2. Cymborowski M
    3. Otwinowski Z
    4. Chruszcz M

    (2006) HKL -3000: the integration of data reduction and structure solution – from diffraction images to an initial model in minutes

    Acta Crystallographica Section D Biological Crystallography 62:859–866.

    https://doi.org/10.1107/S0907444906019949

    • Google Scholar
58. 1. Morishita H
    2. Umitsu M
    3. Murata Y
    4. Shibata N
    5. Udaka K
    6. Higuchi Y
    7. Akutsu H
    8. Yamaguchi T
    9. Yagi T
    10. Ikegami T

    (2006) Structure of the cadherin-related neuronal receptor/protocadherin-alpha first extracellular cadherin domain reveals diversity across cadherin families

    Journal of Biological Chemistry 281:33650–33663.

    https://doi.org/10.1074/jbc.M603298200

    • PubMed
    • Google Scholar
59. 1. Morrow EM
    2. Yoo SY
    3. Flavell SW
    4. Kim TK
    5. Lin Y
    6. Hill RS
    7. Mukaddes NM
    8. Balkhy S
    9. Gascon G
    10. Hashmi A
    11. Al-Saad S
    12. Ware J
    13. Joseph RM
    14. Greenblatt R
    15. Gleason D
    16. Ertelt JA
    17. Apse KA
    18. Bodell A
    19. Partlow JN
    20. Barry B
    21. Yao H
    22. Markianos K
    23. Ferland RJ
    24. Greenberg ME
    25. Walsh CA

    (2008) Identifying autism loci and genes by tracing recent shared ancestry

    Science 321:218–223.

    https://doi.org/10.1126/science.1157657

    • PubMed
    • Google Scholar
60. 1. Murshudov GN
    2. Skubák P
    3. Lebedev AA
    4. Pannu NS
    5. Steiner RA
    6. Nicholls RA
    7. Winn MD
    8. Long F
    9. Vagin AA

    (2011) REFMAC5 for the refinement of macromolecular crystal structures

    Acta Crystallographica Section D Biological Crystallography 67:355–367.

    https://doi.org/10.1107/S0907444911001314

    • PubMed
    • Google Scholar
61. 1. Mutoh T
    2. Hamada S
    3. Senzaki K
    4. Murata Y
    5. Yagi T

    (2004) Cadherin-related neuronal receptor 1 (CNR1) has cell adhesion activity with beta1 integrin mediated through the RGD site of CNR1

    Experimental Cell Research 294:494–508.

    https://doi.org/10.1016/j.yexcr.2003.11.019

    • PubMed
    • Google Scholar
62. 1. Nagar B
    2. Overduin M
    3. Ikura M
    4. Rini JM

    (1996) Structural basis of calcium-induced E-cadherin rigidification and dimerization

    Nature 380:360–364.

    https://doi.org/10.1038/380360a0

    • PubMed
    • Google Scholar
63. 1. Nicoludis JM
    2. Lau SY
    3. Schärfe CP
    4. Marks DS
    5. Weihofen WA
    6. Gaudet R

    (2015) Structure and sequence analyses of clustered protocadherins reveal antiparallel interactions that mediate homophilic specificity

    Structure 23:2087–2098.

    https://doi.org/10.1016/j.str.2015.09.005

    • PubMed
    • Google Scholar
64. 1. Niesen FH
    2. Berglund H
    3. Vedadi M

    (2007) The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability

    Nature Protocols 2:2212–2221.

    https://doi.org/10.1038/nprot.2007.321

    • PubMed
    • Google Scholar
65. 1. Overduin M
    2. Harvey TS
    3. Bagby S
    4. Tong KI
    5. Yau P
    6. Takeichi M
    7. Ikura M

    (1995) Solution structure of the epithelial cadherin domain responsible for selective cell adhesion

    Science 267:386–389.

    https://doi.org/10.1126/science.7824937

    • PubMed
    • Google Scholar
66. 1. Patel SD
    2. Ciatto C
    3. Chen CP
    4. Bahna F
    5. Rajebhosale M
    6. Arkus N
    7. Schieren I
    8. Jessell TM
    9. Honig B
    10. Price SR
    11. Shapiro L

    (2006) Type II cadherin ectodomain structures: implications for classical cadherin specificity

    Cell 124:1255–1268.

    https://doi.org/10.1016/j.cell.2005.12.046

    • PubMed
    • Google Scholar
67. 1. Petersen TN
    2. Brunak S
    3. von Heijne G
    4. Nielsen H

    (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions

    Nature Methods 8:785–786.

    https://doi.org/10.1038/nmeth.1701

    • PubMed
    • Google Scholar
68. 1. Piper M
    2. Dwivedy A
    3. Leung L
    4. Bradley RS
    5. Holt CE

    (2008) NF-protocadherin and TAF1 regulate retinal axon initiation and elongation in vivo

    Journal of Neuroscience 28:100–105.

    https://doi.org/10.1523/JNEUROSCI.4490-07.2008

    • PubMed
    • Google Scholar
69. 1. Ponstingl H
    2. Henrick K
    3. Thornton JM

    (2000) Discriminating between homodimeric and monomeric proteins in the crystalline state

    Proteins 41:47–57.

    https://doi.org/10.1002/1097-0134(20001001)41:1<47::AID-PROT80>3.0.CO;2-8

    • PubMed
    • Google Scholar
70. 1. Redies C
    2. Hertel N
    3. Hübner CA

    (2012) Cadherins and neuropsychiatric disorders

    Brain Research 1470:130–144.

    https://doi.org/10.1016/j.brainres.2012.06.020

    • PubMed
    • Google Scholar
71. 1. Redies C
    2. Vanhalst K
    3. Roy F

    (2005) delta-Protocadherins: unique structures and functions

    Cellular and Molecular Life Sciences 62:2840–2852.

    https://doi.org/10.1007/s00018-005-5320-z

    • PubMed
    • Google Scholar
72. 1. Rubinstein R
    2. Thu CA
    3. Goodman KM
    4. Wolcott HN
    5. Bahna F
    6. Mannepalli S
    7. Ahlsen G
    8. Chevee M
    9. Halim A
    10. Clausen H
    11. Maniatis T
    12. Shapiro L
    13. Honig B

    (2015) Molecular logic of neuronal self-recognition through protocadherin domain interactions

    Cell 163:629–642.

    https://doi.org/10.1016/j.cell.2015.09.026

    • PubMed
    • Google Scholar
73. 1. Ruoslahti E

    (1996) RGD and other recognition sequences for integrins

    Annual Review of Cell and Developmental Biology 12:697–715.

    https://doi.org/10.1146/annurev.cellbio.12.1.697

    • PubMed
    • Google Scholar
74. 1. Salahuddin P
    2. Rabbani G
    3. Khan RH

    (2014) The role of advanced glycation end products in various types of neurodegenerative disease: a therapeutic approach

    Cellular & Molecular Biology Letters 19:407–437.

    https://doi.org/10.2478/s11658-014-0205-5

    • PubMed
    • Google Scholar
75. 1. Sano K
    2. Tanihara H
    3. Heimark RL
    4. Obata S
    5. Davidson M
    6. St John T
    7. Taketani S
    8. Suzuki S

    (1993)

    Protocadherins: a large family of cadherin-related molecules in central nervous system

    The EMBO Journal 12:2249–2256.

    • PubMed
    • Google Scholar
76. 1. Scheffer IE
    2. Turner SJ
    3. Dibbens LM
    4. Bayly MA
    5. Friend K
    6. Hodgson B
    7. Burrows L
    8. Shaw M
    9. Wei C
    10. Ullmann R
    11. Ropers HH
    12. Szepetowski P
    13. Haan E
    14. Mazarib A
    15. Afawi Z
    16. Neufeld MY
    17. Andrews PI
    18. Wallace G
    19. Kivity S
    20. Lev D
    21. Lerman-Sagie T
    22. Derry CP
    23. Korczyn AD
    24. Gecz J
    25. Mulley JC
    26. Berkovic SF

    (2008) Epilepsy and mental retardation limited to females: an under-recognized disorder

    Brain 131:918–927.

    https://doi.org/10.1093/brain/awm338

    • PubMed
    • Google Scholar
77. 1. Schreiner D
    2. Weiner JA

    (2010) Combinatorial homophilic interaction between gamma-protocadherin multimers greatly expands the molecular diversity of cell adhesion

    PNAS 107:14893–14898.

    https://doi.org/10.1073/pnas.1004526107

    • PubMed
    • Google Scholar
78. 1. Shapiro L
    2. Fannon AM
    3. Kwong PD
    4. Thompson A
    5. Lehmann MS
    6. Grübel G
    7. Legrand JF
    8. Als-Nielsen J
    9. Colman DR
    10. Hendrickson WA

    (1995) Structural basis of cell-cell adhesion by cadherins

    Nature 374:327–337.

    https://doi.org/10.1038/374327a0

    • PubMed
    • Google Scholar
79. 1. Shapiro L
    2. Love J
    3. Colman DR

    (2007) Adhesion molecules in the nervous system: structural insights into function and diversity

    Annual Review of Neuroscience 30:451–474.

    https://doi.org/10.1146/annurev.neuro.29.051605.113034

    • PubMed
    • Google Scholar
80. 1. Sievers F
    2. Higgins DG

    (2014) Clustal Omega, accurate alignment of very large numbers of sequences

    Methods in Molecular Biology 1079:105–116.

    https://doi.org/10.1007/978-1-62703-646-7_6

    • PubMed
    • Google Scholar
81. 1. Simm A
    2. Müller B
    3. Nass N
    4. Hofmann B
    5. Bushnaq H
    6. Silber RE
    7. Bartling B

    (2015) Protein glycation - between tissue aging and protection

    Experimental Gerontology 68:71–75.

    https://doi.org/10.1016/j.exger.2014.12.013

    • PubMed
    • Google Scholar
82. 1. Sivasankar S
    2. Zhang Y
    3. Nelson WJ
    4. Chu S
    5. James Nelson W

    (2009) Characterizing the initial encounter complex in cadherin adhesion

    Structure 17:1075–1081.

    https://doi.org/10.1016/j.str.2009.06.012

    • PubMed
    • Google Scholar
83. 1. Sotomayor M
    2. Gaudet R
    3. Corey DP

    (2014) Sorting out a promiscuous superfamily: towards cadherin connectomics

    Trends in Cell Biology 24:524–536.

    https://doi.org/10.1016/j.tcb.2014.03.007

    • PubMed
    • Google Scholar
84. 1. Sotomayor M
    2. Weihofen WA
    3. Gaudet R
    4. Corey DP

    (2010) Structural determinants of cadherin-23 function in hearing and deafness

    Neuron 66:85–100.

    https://doi.org/10.1016/j.neuron.2010.03.028

    • PubMed
    • Google Scholar
85. 1. Sotomayor M
    2. Weihofen WA
    3. Gaudet R
    4. Corey DP

    (2012) Structure of a force-conveying cadherin bond essential for inner-ear mechanotransduction

    Nature 492:128–132.

    https://doi.org/10.1038/nature11590

    • PubMed
    • Google Scholar
86. 1. Steentoft C
    2. Vakhrushev SY
    3. Joshi HJ
    4. Kong Y
    5. Vester-Christensen MB
    6. Schjoldager KT
    7. Lavrsen K
    8. Dabelsteen S
    9. Pedersen NB
    10. Marcos-Silva L
    11. Gupta R
    12. Bennett EP
    13. Mandel U
    14. Brunak S
    15. Wandall HH
    16. Levery SB
    17. Clausen H

    (2013) Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology

    The EMBO Journal 32:1478–1488.

    https://doi.org/10.1038/emboj.2013.79

    • PubMed
    • Google Scholar
87. 1. Steinberg MS

    (2007) Differential adhesion in morphogenesis: a modern view

    Current Opinion in Genetics & Development 17:281–286.

    https://doi.org/10.1016/j.gde.2007.05.002

    • PubMed
    • Google Scholar
88. 1. Suzuki ST

    (1996)

    Protocadherins and diversity of the cadherin superfamily

    Journal of Cell Science 109:2609–2611.

    • PubMed
    • Google Scholar
89. 1. Tai K
    2. Kubota M
    3. Shiono K
    4. Tokutsu H
    5. Suzuki ST

    (2010) Adhesion properties and retinofugal expression of chicken protocadherin-19

    Brain Research 1344:13–24.

    https://doi.org/10.1016/j.brainres.2010.04.065

    • PubMed
    • Google Scholar
90. 1. Takeichi M

    (1990) Cadherins: a molecular family important in selective cell-cell adhesion

    Annual Review of Biochemistry 59:237–252.

    https://doi.org/10.1146/annurev.bi.59.070190.001321

    • PubMed
    • Google Scholar
91. 1. Terracciano A
    2. Trivisano M
    3. Cusmai R
    4. De Palma L
    5. Fusco L
    6. Compagnucci C
    7. Bertini E
    8. Vigevano F
    9. Specchio N

    (2016) PCDH19-related epilepsy in two mosaic male patients

    Epilepsia 57:e51–55.

    https://doi.org/10.1111/epi.13295

    • PubMed
    • Google Scholar
92. 1. Thiffault I
    2. Farrow E
    3. Smith L
    4. Lowry J
    5. Zellmer L
    6. Black B
    7. Abdelmoity A
    8. Miller N
    9. Soden S
    10. Saunders C

    (2016) PCDH19-related epileptic encephalopathy in a male mosaic for a truncating variant

    American Journal of Medical Genetics Part A 170:1585–1589.

    https://doi.org/10.1002/ajmg.a.37617

    • PubMed
    • Google Scholar
93. 1. Thu CA
    2. Chen WV
    3. Rubinstein R
    4. Chevee M
    5. Wolcott HN
    6. Felsovalyi KO
    7. Tapia JC
    8. Shapiro L
    9. Honig B
    10. Maniatis T

    (2014) Single-cell identity generated by combinatorial homophilic interactions between α, β, and γ protocadherins

    Cell 158:1045–1059.

    https://doi.org/10.1016/j.cell.2014.07.012

    • PubMed
    • Google Scholar
94. 1. van Harssel JJ
    2. Weckhuysen S
    3. van Kempen MJ
    4. Hardies K
    5. Verbeek NE
    6. de Kovel CG
    7. Gunning WB
    8. van Daalen E
    9. de Jonge MV
    10. Jansen AC
    11. Vermeulen RJ
    12. Arts WF
    13. Verhelst H
    14. Fogarasi A
    15. de Rijk-van Andel JF
    16. Kelemen A
    17. Lindhout D
    18. De Jonghe P
    19. Koeleman BP
    20. Suls A
    21. Brilstra EH

    (2013) Clinical and genetic aspects of PCDH19-related epilepsy syndromes and the possible role of PCDH19 mutations in males with autism spectrum disorders

    Neurogenetics 14:23–34.

    https://doi.org/10.1007/s10048-013-0353-1

    • PubMed
    • Google Scholar
95. 1. Vanhalst K
    2. Kools P
    3. Staes K
    4. van Roy F
    5. Redies C

    (2005) delta-Protocadherins: a gene family expressed differentially in the mouse brain

    Cellular and Molecular Life Sciences 62:1247–1259.

    https://doi.org/10.1007/s00018-005-5021-7

    • PubMed
    • Google Scholar
96. 1. Vester-Christensen MB
    2. Halim A
    3. Joshi HJ
    4. Steentoft C
    5. Bennett EP
    6. Levery SB
    7. Vakhrushev SY
    8. Clausen H

    (2013) Mining the O-mannose glycoproteome reveals cadherins as major O-mannosylated glycoproteins

    PNAS 110:21018–21023.

    https://doi.org/10.1073/pnas.1313446110

    • PubMed
    • Google Scholar
97. 1. Walters J
    2. Wells-Kilpatrick K
    3. Pandeleos T

    (2014) My epilepsy story--PCDH19 alliance

    Epilepsia 55:968–969.

    https://doi.org/10.1111/epi.12555

    • PubMed
    • Google Scholar
98. 1. Weiner JA
    2. Jontes JD

    (2013) Protocadherins, not prototypical: a complex tale of their interactions, expression, and functions

    Frontiers in Molecular Neuroscience 6:4.

    https://doi.org/10.3389/fnmol.2013.00004

    • PubMed
    • Google Scholar
99. 1. Wolverton T
    2. Lalande M

    (2001) Identification and characterization of three members of a novel subclass of protocadherins

    Genomics 76:66–72.

    https://doi.org/10.1006/geno.2001.6592

    • PubMed
    • Google Scholar
100. 1. Yagi T

     (2012) Molecular codes for neuronal individuality and cell assembly in the brain

     Frontiers in Molecular Neuroscience 5:45.

     https://doi.org/10.3389/fnmol.2012.00045

     • PubMed
     • Google Scholar
101. 1. Yamamoto A
     2. Amacher SL
     3. Kim SH
     4. Geissert D
     5. Kimmel CB
     6. De Robertis EM

     (1998)

     Zebrafish paraxial protocadherin is a downstream target of spadetail involved in morphogenesis of gastrula mesoderm

     Development 125:3389–3397.

     • PubMed
     • Google Scholar
102. 1. Zhang Y
     2. Sivasankar S
     3. Nelson WJ
     4. Chu S

     (2009) Resolving cadherin interactions and binding cooperativity at the single-molecule level

     PNAS 106:109–114.

     https://doi.org/10.1073/pnas.0811350106

     • PubMed
     • Google Scholar
103. 1. Zhu H
     2. Domingues FS
     3. Sommer I
     4. Lengauer T

     (2006) NOXclass: prediction of protein-protein interaction types

     BMC Bioinformatics 7:27.

     https://doi.org/10.1186/1471-2105-7-27

     • Google Scholar
104. 1. Zipursky SL
     2. Sanes JR

     (2010) Chemoaffinity revisited: dscams, protocadherins, and neural circuit assembly

     Cell 143:343–353.

     https://doi.org/10.1016/j.cell.2010.10.009

     • PubMed
     • Google Scholar

Author details

1. Sharon R Cooper

   1. Department of Chemistry and Biochemistry, The Ohio State University, Columbus, United States
   2. Department of Neuroscience, The Ohio State University, Columbus, United States

   Contribution

   SRC, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. James D Jontes

   Department of Neuroscience, The Ohio State University, Columbus, United States

   Contribution

   JDJ, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   jontes.1@osu.edu

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-8954-6127

3. Marcos Sotomayor

   Department of Chemistry and Biochemistry, The Ohio State University, Columbus, United States

   Contribution

   MS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   sotomayor.8@osu.edu

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-3333-1805

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