Figure 4. Clustered protocadherin (cPcdh) cis interactions are promiscuous with a preference for interfamily heterodimers.

(A) Surface plasmon resonance (SPR)-binding profiles of cPcdh cis fragment analytes from all cPcdh subfamilies except alphas (shown in columns) flowed over individual surfaces coated with cPcdh cis fragments. Binding profiles for each surface are individually scaled and responses are normalized for molecular weight. (B) Table of dissociation constants calculated from the SPR data for the four monomeric analytes. The number in brackets represents the error of the fit based on analysis of duplicate responses. Binding signals were not detected for interactions labeled NB (no binding) while >50 represents interactions with K_Ds > 50 μM, where an accurate K_D cannot be determined.

Figure 4—figure supplement 1. Calculation of cis interaction dissociation constants and the impact of an α-Pcdh EC5 on family-wide cis interactions.

(A) Kinetic binding analysis of γC3_3–6 analyte binding over a β9_3–6 covered surface. Data are shown in black, and the red traces represent the fit to an 1:1 binding model. (B) Left, surface plasmon resonance (SPR)-binding profiles from Figure 4 for the four monomeric cis fragment analytes over all six cis fragment surfaces. Right, fit of the binding data for these four analytes to 1:1 binding isotherms to calculate K_Ds. γA4_3–6 and γA9_3–6 are monomeric and they are not included in the binding isotherms over their respective surface. (C) SPR-binding profiles for γC3_3–6 (from Figure 4) and an α7_1–5/γC3₆ chimera flowed over the immobilized cis fragment surfaces. Binding profiles for each surface are individually scaled as in Figure 4.

Figure 4—figure supplement 2. Range of clustered protocadherin (cPcdh) cis and trans dissociation constants, K_Ds.

Chart shows the cPcdh trans dimer, homophilic cis dimer, and heterophilic cis dimer interactions for which we have determined binding affinities divided into four subgroups based on their dissociation constant. The trans and homophilic cis dimer affinities were determined using analytical ultracentrifugation (AUC; Figure 2—source data 1 and Figure 4—source data 1) and the heterophilic cis dimer affinities were determined using surface plasmon resonance (SPR; Figure 4B). Of the interactions in the >50 μM group one trans interaction and four homophilic cis interactions are monomeric in solution (>500 μM K_D in AUC). Three of the 11 heterophilic cis interactions in the >50 μM group show no binding in our SPR experiments based on a 40-RU-binding threshold.

Figure 4—figure supplement 3. Amino acid sequence alignment reveals conservation of cis interfacial residues within the alternate clustered protocadherin (cPcdh) subfamilies.

(A) Amino acid sequence alignments of cis interfacial residues from the EC6-only and EC5–6 surfaces for all 58 mouse cPcdhs subdivided by subfamily. Completely conserved residues are highlighted in red with white lettering. Residues 540 and 541 are included in the EC6-only alignments since the crystal structure of γA4 EC3–6 (PDB: 5SZQ) revealed a distinct EC6 A–A′ loop architecture to that observed in the γB2, 4, and 7 (PDBs: 5SZR, 6E6B, and 5V5X) cis fragment crystal structures that would place these residues in the EC6-only interface if maintained in cis interactions. (B) Sequence logos based on the sequence alignment shown in (A) for the EC6-only cis interfacial residues from each of the five cPcdh subfamilies highlighting the similarities and conserved differences between the subfamilies. Residues 540 and 541 are included for all isoforms but grayed out for the non-γA isoforms since their involvement may be γA specific. NB: Previous studies have shown that α-cPcdhs have an impaired EC6-only interface (Thu et al., 2014; Goodman et al., 2017). (C) Sequence logos for the EC5–6 cis interfacial residues from each of the five cPcdh subfamilies.