Structure and Sequence Analyses of Clustered Protocadherins Reveal Antiparallel Interactions that Mediate Homophilic Specificity

Abstract

SUMMARY Clustered protocadherin (Pcdh) proteins mediate dendritic self-avoidance in neurons via specific homophilic interactions in their extracellular cadherin (EC) domains. We determined crystal structures of EC1-EC3, containing the homophilic specificity-determining region, of two mouse clustered Pcdh isoforms (PcdhγA1 and PcdhγC3) to investigate the nature of the homophilic interaction. Within the crystal lattices, we observe antiparallel interfaces consistent with a role in trans cell-cell contact. Antiparallel dimerization is supported by evolutionary correlations. Two interfaces, located primarily on EC2-EC3, involve distinctive clustered Pcdh structure and sequence motifs, lack predicted glycosylation sites, and contain residues highly conserved in orthologs but not paralogs, pointing towards their biological significance as homophilic interaction interfaces. These two interfaces are similar yet distinct, reflecting a possible difference in interaction architecture between clustered Pcdh subfamilies. These structures initiate a molecular understanding of clustered Pcdh assemblies that are required to produce functional neuronal networks.

γ-Protocadherin structural diversity and functional implications

“…The γA1 EC1–4 crystal structure contained four molecules in the asymmetric unit: Two of which are arranged in an EC1–4-mediated antiparallel dimer, with all four EC domains involved in the dimer interaction (chains A and B); and two are arranged in an EC2–3-mediated antiparallel dimer, in which EC1 and EC4 are not involved in the dimer interaction (chains C and D) (Figure 1A). The EC2–3 portion of the dimer interaction is very similar between the two dimers in the structure (RMSD = 0.98 Å over 415 Cα’s) and closely resembles the partial interaction observed in the previously published γA1 EC1–3 structure (Nicoludis et al, 2015; Figure 1—figure supplement 1B). The main difference between the two dimers in the γA1 crystal is therefore simply the presence or absence of the EC1:EC4 interaction.…”

“…These constructs encompassed the entire Pcdh EC1–4-mediated trans interface (Rubinstein et al, 2015; Nicoludis et al, 2015; Goodman et al, 2016), but lacked EC6, which mediates a distinct cis interface (Thu et al, 2014; Rubinstein et al, 2015). We used sedimentation equilibrium analytical ultracentrifugation (AUC) to characterize the homophilic binding properties of these proteins.…”

“…The specificity-determining cell-cell recognition interface of Pcdhs involves domains EC1–4, as shown experimentally through mutagenesis analysis (Rubinstein et al, 2015) and suggested by mutation correlation analysis (Nicoludis et al, 2015). Structures of the trans dimer formed through this interface have been reported for two α-Pcdhs and two β-Pcdhs which revealed overall-similar recognition-dimer structures, mediated by interfaces populated with diverse residue compositions that determine homophilic specificity (Goodman et al, 2016).…”

Structural determinants of adhesion by Protocadherin-19 and implications for its role in epilepsy

“…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).…”

“…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.…”