Author response: Antiparallel protocadherin homodimers use distinct affinity- and specificity-mediating regions in cadherin repeats 1-4
2016-07-26

Article Figures and data Abstract eLife digest Introduction Results and discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Protocadherins (Pcdhs) are cell adhesion and signaling proteins used by neurons to develop and maintain neuronal networks, relying on trans homophilic interactions between their extracellular cadherin (EC) repeat domains. We present the structure of the antiparallel EC1-4 homodimer of human PcdhγB3, a member of the γ subfamily of clustered Pcdhs. Structure and sequence comparisons of α, β, and γ clustered Pcdh isoforms illustrate that subfamilies encode specificity in distinct ways through diversification of loop region structure and composition in EC2 and EC3, which contains isoform-specific conservation of primarily polar residues. In contrast, the EC1/EC4 interface comprises hydrophobic interactions that provide non-selective dimerization affinity. Using sequence coevolution analysis, we found evidence for a similar antiparallel EC1-4 interaction in non-clustered Pcdh families. We thus deduce that the EC1-4 antiparallel homodimer is a general interaction strategy that evolved before the divergence of these distinct protocadherin families. https://doi.org/10.7554/eLife.18449.001 eLife digest As the brain develops, nerve cells or neurons connect with one another to form complex networks. These connections form between branch-like structures, called dendrites, that project from the cell body of each neuron. To prevent unneeded connections from forming, dendrites that belong to the same neuron need a way to recognize and avoid one another. A family of proteins called protocadherins supports this process of self-avoidance. Protocadherins have three main parts or domains: an extracellular domain that faces outwards away from the cell, a transmembrane domain that sits within the cell’s surface membrane and an intracellular domain that faces into the cell’s interior. There are two major groups of protocadherins – clustered and non-clustered – and the former are responsible for the self-avoidance behavior between dendrites. Clustered protocadherins in turn comprise three subfamilies, each of which consists of multiple variants with slightly different structures (known as isoforms). The particular set of protocadherin isoforms that a neuron displays on its surface distinguishes that neuron from all others, a little like a barcode. When two dendrites meet, the protocadherins in their membranes come into contact with one another. If both dendrites come from the same neuron and therefore possess identical sets of protocadherins, then all protocadherins can form two-subunit complexes containing one copy of the same isoform from each dendrite. These complexes are called homodimers and their formation acts as a signal that informs the cell that it has encountered one of its own dendrites and should therefore not establish a connection. By using X-rays to determine the structure of a crystallized protocadherin fragment down to the level of its individual atoms, Nicoludis et al. now reveal exactly how clustered protocadherins form homodimers. The results show that each protocadherin subfamily uses a slightly different type of interaction due to differences in the structure of their extracellular domains. The next challenge is to identify the signaling cascade that is triggered by the formation of clustered protocadherin homodimers, and to work out how activation of this cascade prevents a permanent connection from forming. In addition, the results of Nicoludis et al. predict that some non-clustered protocadherins form dimers with a similar architecture to that of clustered protocadherins. This possibility should also be tested experimentally. https://doi.org/10.7554/eLife.18449.002 Introduction Protocadherins (Pcdhs) encompass about 70% of the cadherin superfamily in mammals, and are involved in cell adhesion in the nervous system of higher animals (Hulpiau and van Roy, 2011; Hulpiau et al., 2013; Keeler et al., 2015; Sotomayor et al., 2014). Pcdhs segregate into two groups: clustered (expressed from a large gene cluster) and non-clustered. Clustered Pcdhs, comprising the α, β, and γ subfamilies, mediate neuronal survival, self-avoidance and self/nonself discrimination, and promote dendritic arborization in neuronal cells (Emond and Jontes, 2008; Garrett et al., 2012; Kostadinov and Sanes, 2015; Ledderose et al., 2013; Lefebvre et al., 2012; Molumby et al., 2016; Suo et al., 2012; Wang et al., 2002; Weiner et al., 2005). Clustered Pcdhs thus function analogously to insect Dscam isoforms to mediate neuronal identity (Zipursky and Sanes, 2010). Clustered Pcdhs are also broadly involved in synapse formation and maintenance, neuronal connectivity and neuronal survival (Hayashi and Takeichi, 2015; Keeler et al., 2015). Non-clustered Pcdhs play key roles in neuronal development (Keeler et al., 2015; Kim et al., 2011). For example, Pcdh7 is involved in germ layer differentiation (Rashid et al., 2006; Yoshida, 2003), and Pcdh1 and Pcdh8 mediate cell sorting and migration during gastrulation (Kim et al., 1998; Kuroda et al., 2002). Both clustered and non-clustered Pcdhs control these phenotypes through homophilic interactions of their extracellular cadherin (EC) repeat domains (Hirano et al., 1999; Hoshina et al., 2013; Kim et al., 1998; Kuroda et al., 2002; Schreiner and Weiner, 2010; Thu et al., 2014; Yamagata et al., 1999; Yoshida, 2003). Clustered Pcdh subfamilies show distinct phenotypes. In zebrafish, α and γ Pcdhs express in overlapping but distinct brain regions (Biswas et al., 2012). In mammals, α Pcdhs regulate sorting of olfactory sensory neuron axons into glomeruli, serotonergic axon maturation, and dendritic patterning in CA1 pyramidal neurons (Hasegawa et al., 2012, 2008; Katori et al., 2009; Suo et al., 2012). The γ subfamily is important for self/non-self discrimination in retinal starburst amacrine cells and Purkinje neurons (Kostadinov and Sanes, 2015; Lefebvre et al., 2012). Thus, available data suggest that the different Pcdh subfamilies may function independently or cooperatively, perhaps depending on the brain region and/or neuronal cell type. Our recent PcdhγA1 and PcdhγC3 EC1-3 structures revealed dimer interactions between EC2 and EC3 (Nicoludis et al., 2015), consistent with previous biochemical and bioinformatics data (Schreiner and Weiner, 2010; Wu, 2005). Using sequence co-evolution analysis, we predicted intersubunit EC1-EC4 interactions, and proposed that clustered Pcdhs form extended antiparallel homodimers engaging EC1-4. A complementary biochemical and structural study arrived at a very similar docking model (Rubinstein et al., 2015), which was recently confirmed for α and β clustered Pcdhs (Goodman et al., 2016). We determined the crystal structure of PcdhγB3 EC1-4, the first full antiparallel dimer for a γ isoform. We analyzed the clustered Pcdhs structures in light of biological, biochemical and evolutionary data to further resolve how clustered Pcdhs encode specificity. We describe how structural differences between the α, β and γ subfamilies generate distinct modes of specificity encoding. We also provide evidence that the EC1/EC4 and EC2/EC3 interfaces are functionally different: EC1/EC4 provides nonselective dimerization affinity while EC2/EC3 is generally responsible for enforcing specificity. Finally, we extend our sequence coevolution analysis to the non-clustered Pcdhs and provide evidence that the EC1-4 interaction is broadly used by Pcdhs. Results and discussion Structure of the PcdhγB3 EC1-4 extended antiparallel dimer The in vitro-refolded recombinantly-expressed PcdhγB3 EC1-4 (47 kDa) yielded two peaks on size exclusion chromatography (SEC; Figure 1—figure supplement 1). Based on multi-angle light scattering (MALS; Figure 1—figure supplement 1), peak 1 was wide and polydisperse (~200–300 kDa). Peak 2 was monodisperse at 80 kDa – consistent with a dimer – and readily yielded a crystal structure (Figure 1—source data 1). As expected, each EC forms a seven-stranded Greek key β-sandwich motif (Figure 1A), similarly to other clustered Pcdh structures (Goodman et al., 2016; Nicoludis et al., 2015; Rubinstein et al., 2015). Notably, EC4 has a unique β-strand arrangement compared to EC1-EC3 (Figure 1B,C) and all known cadherin repeat structures. Strand β1a is extended by 4–5 residues, while β1b is correspondingly shortened. Additionally, while in EC1-3 strand β2 splits into β2a and β2b, interacting with strands β5 and β1a, respectively, in EC4 it forms a continuous strand interacting with both simultaneously. This distinct structural feature contributes to intersubunit EC1/EC4 interactions (see below). Figure 1 with 4 supplements see all Download asset Open asset PcdhγB3 EC1-4 extended antiparallel dimer relies on unusual EC4 β-strand arrangement and is similar to other clustered Pcdh dimers. (A) Structure of the PcdhγB3 EC1-4 antiparallel dimer, with each EC a different shade of blue and the Ca2+ ions in grey. (B) Superposition of PcdhγB3 EC2 and EC4 highlighting the differences in β-strands 1 and 2. (C) Comparison of the canonical cadherin (top) and EC4 (bottom) β-strand arrangement. (D) The structures of Pcdh dimers α4 EC1-4, α7 EC1-5, β6 EC1-4, and β8 EC1-4 (grey) were superimposed using the dimeric EC2-3 region onto γB3 EC1-4 (blue), illustrating variations in twist/corkscrew. (E) The EC4 domains of clustered Pcdh structures (colored as labeled) were superimposed, highlighting EC1 position differences that correlate with subfamily. Point of view (eye symbol) shown in (D). See Figure 1—figure supplements 1–4. https://doi.org/10.7554/eLife.18449.003 Figure 1—source data 1 Statistics for PcdhγB3 EC1-4 structure. https://doi.org/10.7554/eLife.18449.004 Download elife-18449-fig1-data1-v2.docx Although the asymmetric unit contains a single PcdhγB3 molecule, a crystallographic two-fold axis generates an antiparallel dimer with intersubunit EC1/EC4 and EC2/EC3 interactions (Figure 1A). This dimer is consistent with the PcdhγA1 EC1-3 crystal structure, validating the previously predicted interface (Nicoludis et al., 2015; Rubinstein et al., 2015), and with recent α and β Pcdhs structures (Goodman et al., 2016), confirming that this interaction mechanism is conserved among all clustered Pcdh subfamilies (Figure 1D). The structures do differ noticeably in overall twist, including subfamily-specific differences in relative EC1/EC4 orientation (Figure 1E). The linear architecture of clustered Pcdhs enables extended antiparallel dimer interfaces. Overall, the tilt and azimuthal angles between adjacent clustered Pcdh repeats are distinct from those of classical cadherins (Figure 1—figure supplement 2) (Nicoludis et al., 2015). Classical cadherins, which typically dimerize through EC1/EC1 interfaces, exhibit smaller tilt angles and thus an overall curved structure (Boggon et al., 2002). Notably, the clustered Pcdh repeat orientation is such that EC1 and EC3 use the same face for intersubunit contacts, as do EC2 and EC4 (Figure 1—figure supplement 3), suggesting that longer cadherins could readily form even more extended interfaces. Clustered protocadherin subfamilies have distinct specificity mechanisms dictated by structural differences Clustered Pcdh subfamilies control different phenotypes in vivo and have discrete expression patterns (Biswas et al., 2012; Keeler et al., 2015), suggesting that they encode specificity using distinct modes, which may relate to subfamily-specific structural features. To investigate this hypothesis, we calculated the isoform conservation ratio (ICR) within individual subfamilies, which quantifies the extent to which individual residue positions are conserved among orthologs (same isoform in different species) and diversified in paralogs (different isoforms in the same species) (Nicoludis et al., 2015), resulting in three ICR value sets for the α, β and γ subfamilies, respectively (Figure 2—figure supplement 1). To account for subfamily differences in sequence conservation, we normalized the ICR values by dividing by the subfamily average. We then mapped them onto the Pcdhα7 EC1-5, Pcdhβ8 EC1-4 and PcdhγB3 EC1-4 structures (Figure 2A). Comparing the structures and isoform-specific conservation in the different subfamilies allowed us to identify key specificity determinant regions for individual subfamilies. We illustrate three examples of how the subfamilies have encoded specificity using unique structural features. Figure 2 with 1 supplement see all Download asset Open asset Isoform-specific conservation and structural differences reveal subfamily differences in diversity generation. (A) Subfamily-specific ICR values mapped onto the surfaces of Pcdhα7 (top, green), Pcdhβ8 (middle, magenta) and PcdhγB3 (bottom, blue). The black outline marks the dimer interface footprint. (B, C, D) Unique structural features of the α (left), β (center), and γ (right) structures (colored according to Figure 1). ICR values for highlighted residues shown below and normalized amino acid frequencies for these positions shown on the right. See Figure 2—figure supplement 1. https://doi.org/10.7554/eLife.18449.009 In α isoforms, the EC2 β4-β5 loop is enriched in high-ICR and chemically diverse residues, and differs in conformation in the Pcdhα4 and Pcdhα7 structures (Figure 2B): the Pcdhα4 EC2 β4-β5 loop contacts β1b of EC3, while the corresponding loop in Pcdhα7 does not, suggesting variable interactions in other isoforms. In comparison, the EC2 β4-β5 loop residues in both β and γ isoforms have lower ICR values, more similar loop structure, and do not contact β1b of EC3. Thus, this loop may have evolved to generate diversity within α isoforms, but not in other subfamilies. In β isoforms, the Phe-X10-Phe loop between β3 and β4 of EC3 has limited diversity compared to α and γ isoforms and wedges between the EC2 β4-β5 loop and β2b strand (Figure 2C). In contrast, the Phe-X10-Phe loop of α and γ isoforms has a helical conformation, and has residues with higher ICR values and greater chemical diversity. Therefore alterations in secondary structure can affect how specificity is encoded within the subfamilies. The short loop following the extended β1a strand in EC4 contacts the EC1 β6-β7 loop (Figure 2D), and there are large structural differences in the EC1/EC4 interaction between subfamilies (Figure 1E). α Isoforms have low-ICR residues at this interface, whereas β and γ isoforms have higher ICR value residues. This thus suggests that the large structural differences drive inter-subfamily specificity, on which may be layered additional isoform specificity. In all cases, sequence regions with high isoform-specific conservation correlate with interface contacts, revealing the interplay between dimer structure and how subfamilies encode specificity. Diversity in the composition and conformation of loop regions provides distinct specificity mechanisms to subfamilies. Phylogenetic analysis indicates that isoforms are more similar within than across subfamilies (Sotomayor et al., 2014; Wu, 2005), and the available structures show that the interface architecture is more similar within subfamilies as well (Figure 1E) (Goodman et al., 2016; Nicoludis et al., 2015). With this insight, the dimer interface seen in the PcdhγC3 EC1-3 crystal structure may represent a unique dimer architecture for C-type isoforms (Nicoludis et al., 2015), as these isoforms are transcriptionally, functionally and evolutionarily distinct from the subfamilies in which they reside (Chen et al., 2012; Frank et al., 2005; Kaneko et al., 2006). Distinct expression of the clustered Pcdh subfamilies in different tissues and at different developmental stages supports the necessity for intra-subfamily specificity (Biswas et al., 2012; Frank et al., 2005). Differences in subfamily structure and isoform-specific conservation suggest that homophilic specificity mechanisms emerged independently in each subfamily through diversification of subfamily-specific interface contacts. The EC1/EC4 interaction provides affinity of dimerization The EC2/EC3 interaction is integral to clustered Pcdh dimerization specificity, as evidenced by bioinformatics and cell-aggregation assays (Nicoludis et al., 2015; Rubinstein et al., 2015; Schreiner and Weiner, 2010; Thu et al., 2014; Wu, 2005). We sought to understand the functional purpose of the EC1/EC4 interaction, and made three observations. First, for all isoforms with available structures, fewer EC1/EC4 interface residues have high isoform-specific conservation compared to the EC2/EC3 interface residues (Figure 2A, Figure 2—figure supplement 1). Second, interface residues shared by most isoforms are more hydrophobic in EC1/EC4 than in EC2/EC3 (Figure 3A). Third, the PcdhγB3 EC1/EC4 interface is much larger (BSA = 976 Å2 per protomer) than the EC2/EC3 interface (555 Å2 per protomer). The lack of isoform specificity, the hydrophobic nature, and large interface area together suggest that the EC1/EC4 interface promotes binding with little specificity. Figure 3 Download asset Open asset The EC1/EC4 interface is enriched in affinity-driving hydrophobic residues, while the EC2/EC3 interface contains high-ICR residues driving specificity. (A) Amino acid frequencies in clustered Pcdhs of conserved interface residues (see Figure 1—figure supplement 2). (B) Plot of ICR value and ∆∆Gcalc of interface residues of Pcdh α4 EC1-4 (yellow), α7 EC1-5 (green), β6 EC1-4 (salmon), β8 EC1-4 (magenta), γB3 EC1-4 (blue). Two subsets of interface residues segregate from the main cluster: high-∆∆Gcalc and low-ICR residues (‘affinity’; black box) and low-∆∆Gcalc and high-ICR residues (‘specificity’; crimson box). Residue F86 from PcdhγB3 EC1-4 is labeled. (C) High-∆∆Gcalc and low-ICR residues (black) map primarily to EC1 and EC4, while low-∆∆Gcalc and high-ICR residues (crimson) primarily map to EC2 and EC3. N253 (*) is found in the ‘specificity’ region for γB3 and in the ‘affinity’ region for β6 and β8. (D) The EC1/EC4 interface features a hydrophobic cluster, with EC1 F86 near its center. (E) SEC-MALS profiles of WT PcdhγB3 EC1-4 (blue; molecular weight 82 kDa) and F86A (black; molecular weight 52 kDa) run on a Superdex S200 10/300 column, are consistent with dimeric and monomeric proteins, respectively. https://doi.org/10.7554/eLife.18449.011 To probe this hypothesis, we used the Computational Interface Alanine Scanning Server to assess each interface residue’s contribution to the free energy of complex formation (ΔΔGcalc) when computationally mutated to alanine (Kortemme and Baker, 2002; Kortemme et al., 2004). Using the five available EC1-4 interfaces, two residue groups emerged from comparing the ICR values to ΔΔGcalc: one group with low ICR values and high ΔΔGcalc, the other with high ICR values and low ΔΔGcalc (Figure 3B). These residue groups can be regarded as contributing to the affinity and specificity of the complex, respectively. When mapped on PcdhγB3, predicted affinity residues concentrated on EC1 and EC4, and predicted specificity residues on EC2 and EC3, corroborating the distinction between EC1/EC4 and EC2/EC3 interactions (Figure 3C). In the PcdhγB3 EC1/EC4 interface, F86, one of the predicted affinity-driving residues from EC1, wedges into a cavity created by hydrophobic EC4 residues (Figure 3D). A PcdhγB3 EC1-4 F86A mutant indeed disrupted dimerization, resulting in a monomeric protein as measured by MALS (Figure 3E). Thus, the hydrophobic interactions between EC1 and EC4 are crucial to dimerization. Analogously, purified EC1-3 constructs failed to dimerize in vitro whereas EC1-4 constructs did (Nicoludis et al., 2015; Rubinstein et al., 2015), and K562 cells expressing ΔEC1 or ΔEC4-6 constructs did not aggregate while cells expressing chimeras in which EC1 and EC4 derived from different paralogs did (Schreiner and Weiner, 2010; Thu et al., 2014). Together, these results indicate that the EC1/EC4 interaction is not strictly required for the specificity of dimerization but it drives dimerization affinity through non-specific hydrophobic interactions. Antiparallel EC1-4 interaction is predicted in non-clustered Pcdhs The antiparallel EC1-4 interaction architecture can encode diverse specificities within the clustered Pcdh family. Is this architecture unique to clustered Pcdhs or is it ancestral, and thus also found in non-clustered Pcdhs? These include the δ-1 (Pcdh1, Pcdh7, Pcdh9, Pcdh11) and δ-2 (Pcdh8, Pcdh10, Pcdh17, Pcdh18, Pcdh19) families that are integral to the development and maintenance of the nervous system (Keeler et al., 2015; Kim et al., 2011). We used sequence coevolution analysis, which successfully predicted the clustered Pcdh interface (Nicoludis et al., 2015) (Figure 4—figure supplement 1),to look for evidence of an antiparallel interface in non-clustered Pcdhs (Figure 4A). As in clustered Pcdhs, most covarying residue pairs in non-clustered Pcdhs were intra-domain structural contacts of the well-conserved cadherin fold. Additionally, several covarying pairs are found between EC2 and EC3, or EC1 and EC4, similar to those observed for the clustered Pcdhs. When mapped onto the PcdhγB3 dimer, these covarying pairs are somewhat further apart than true interface contacts (Figure 4B), which could be due to differences in dimerization interfaces, as we observe between the clustered Pcdh families, or in the δ-1 or δ-2 Pcdhs secondary structure, for which there are no available structures. This analysis thus predicts an antiparallel EC1-4 interaction in members of the non-clustered Pcdhs. Notably, we cannot determine whether all members or only a subset – and if so, which – likely use this architecture. However maximum parsimony suggests that the ancestral Pcdh used the antiparallel EC1-4 dimer interaction, and Pcdh members which do not show this interaction mechanism either diverged before it evolved or lost it subsequently. Figure 4 with 2 supplements see all Download asset Open asset Evolutionary couplings in non-clustered Pcdhs predict an antiparallel interface engaging EC1-EC4. (A) The top 38 covarying pairs are shown in black, and include a number of EC1-EC4 and EC2-EC3 covarying residue pairs. The intramolecular contact maps of PcdhγB3 EC1-4, Pcdhα4 EC1-4, Pcdhα7 EC1-4, Pcdhβ6 EC1-4, Pcdhβ8 EC1-4 and PcdhγA1 EC1-3 are in gray for reference. The observed interface contact residues are also mapped (α4, yellow; α7, green; β6, salmon; β8, magenta; γB3, blue; γA1, dark blue). (B) Covarying residue pairs across EC1-EC4 or EC2-EC3 are mapped onto the PcdhγB3 EC1-4 structure with a line between coupled residues. Alignments and evolutionary couplings in Figure 4—source data 1 and 2. (C) Amino acid frequencies at non-clustered Pcdh alignment positions corresponding to the conserved interface residue positions identified in clustered Pcdhs (Figure 1—figure supplement 2). See Figure 4—figure supplements 1 and 2. https://doi.org/10.7554/eLife.18449.012 Figure 4—source data 1 Alignment of non-clustered Pcdhs EC1-4. https://doi.org/10.7554/eLife.18449.013 Download Figure 4—source data 2 Evolutionary couplings from the non-clustered Pcdh Download Figure 4—source data 3 Alignment of clustered Pcdhs EC1-4. Download Figure 4—source data 4 Evolutionary couplings from the clustered Pcdh Download Finally, we at the composition of a predicted non-clustered Pcdh interface, by residues to those found at clustered Pcdh interfaces. The predicted EC1/EC4 interface residues are while EC2/EC3 residues have more polar and (Figure Notably, positions and in EC1 and and in EC4 are more hydrophobic in non-clustered than clustered Pcdhs, that these may form contacts in some non-clustered Pcdhs. Thus, like in the clustered Pcdhs, the EC1/EC4 interaction may promote dimer affinity while the EC2/EC3 interaction provides specificity. we and predicted that clustered Pcdhs form homophilic antiparallel EC1-EC4 complexes on crystal structures, and bioinformatics (Nicoludis et al., 2015; Rubinstein et al., 2015). Our structure of PcdhγB3 EC1-4 our sequence coevolution analysis, the of the analysis and revealing the molecular of this we extended this to other non-clustered Pcdhs using sequence coevolution of the PcdhγB3 EC1-4 structure in to α or β subfamily dimers revealed structural differences that with differences in isoform-specific conservation, distinct specificity the isoforms at regions et al., 2016; et al., et al., 2008; et al., the clustered Pcdh subfamilies are and thus can encode specificity in different We identified a hydrophobic interaction between EC1 and EC4 that contributes to dimerization whereas its conservation among clustered Pcdh isoforms suggests that this interaction is not a of specificity. Overall, our data a general for conserved hydrophobic EC1/EC4 interactions in and for diversified polar EC2/EC3 contacts in specificity, and sequence suggest that this is conserved in at some non-clustered Pcdhs. Materials and methods and of PcdhγB3 EC1-4 a PcdhγB3 EC1-4 not the signal was into with a in cells in were at = with at for 4 and by in 2 and were to and onto with and and with protein was at 1 in the from to and in 1 and protein was purified by chromatography on a Superdex in and 2 Two peaks were and each peak was run by before concentrated for light scattering a molecular of PcdhγB3 EC1-4 protein or F86A was determined using a Superdex S200 10/300 with and at 4 was and run at in were normalized and using as a data and structure and analysis a were by at in and in a protein to then with with before in data (Figure 1—source data were in and The PcdhγB3 EC1-4 structure was determined by an molecular with single domains of the PcdhγA1 EC1-3 structure in et al., 2010). was in and and in et al., 2010). We analyzed the of the dimer interface using and In the structure, we found a near the EC2/EC3 interface that forms a with N253 and (Figure 1—figure supplement MALS data were with as the (Figure 1—figure supplement 1), that is not required for dimerization. ICR value a identity of the most residue at each position was used to ICR values, dividing the identity across subfamily orthologs by the identity across subfamily ICR values were then normalized by dividing by the sequence ICR within each subfamily. The alignment and identity data are (Nicoludis et al., 2015). Computational Interface Alanine Scanning Server a Pcdhα4