Harrison OJ et al. (2020), Family-wide Structural and Biophysical Analysis...

XB-ART-57934

Cell Rep 2020 Feb 25;308:2655-2671.e7. doi: 10.1016/j.celrep.2020.02.003.

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Family-wide Structural and Biophysical Analysis of Binding Interactions among Non-clustered δ-Protocadherins.

Harrison OJ , Brasch J , Katsamba PS , Ahlsen G , Noble AJ , Dan H , Sampogna RV , Potter CS , Carragher B , Honig B , Shapiro L .

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Non-clustered δ1- and δ2-protocadherins, close relatives of clustered protocadherins, function in cell adhesion and motility and play essential roles in neural patterning. To understand the molecular interactions underlying these functions, we used solution biophysics to characterize binding of δ1- and δ2-protocadherins, determined crystal structures of ectodomain complexes from each family, and assessed ectodomain assembly in reconstituted intermembrane junctions by cryoelectron tomography (cryo-ET). Homophilic trans (cell-cell) interactions were preferred for all δ-protocadherins, with additional weaker heterophilic interactions observed exclusively within each subfamily. As expected, δ1- and δ2-protocadherin trans dimers formed through antiparallel EC1-EC4 interfaces, like clustered protocadherins. However, no ectodomain-mediated cis (same-cell) interactions were detectable in solution; consistent with this, cryo-ET of reconstituted junctions revealed dense assemblies lacking the characteristic order observed for clustered protocadherins. Our results define non-clustered protocadherin binding properties and their structural basis, providing a foundation for interpreting their functional roles in neural patterning.

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Genes referenced: pcdh1 pcdh10 pcdh12 pcdh17 pcdh18 pcdh19 pcdh7 pcdh8 pcdh8.2 pcdh9 spr
GO keywords: cell adhesion [+]

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	Graphical Abstract
	Figure 1. SPR Analysis of trans Binding Interactions in the Î´-Protocadherin Family (A) Phylogenetic tree of human Î´-protocadherins from aligned full-length amino acid sequences. Atypical members italicized. Scale indicates protein distance. (B) SPR binding profiles of Î´-protocadherin analytes (columns) over surfaces coated with the same set of proteins (rows). Analyte concentrations of 27, 9, and 3 Î¼M are plotted on each panel. Responses are normalized for molecular weight and scaled for each surface, permitting comparison across rows only. Homophilic combinations are highlighted in green; heterophilic interactions within Î´1- and Î´2-subfamilies are boxed in teal and red. See Figure S1.
	Figure 2. Structures of Adhesive EC1â€“EC4 Fragments of Î´1- and Î´2-Protocadherins (Aâ€“F) Ribbon representations showing two orthogonal views (upper and lower panels) of human EC1â€“EC4 fragment structures of (A) pcdh1; (B) pcdh10 monomer; (C) pcdh10 dimer; (D) pcdh17; (E) pcdh18.; and (F) pcdh19. Single trans dimers, formed between symmetry-related protomers (A) or in the crystallographic asymmetric unit (Câ€“F) are shown. Interdomain calcium ions are shown as green spheres; N-linked and O-linked glycans as wheat and magenta spheres. See Figures S3 and S4 and Tables S1 and S2.
	Figure 3. Conserved and Variable Molecular Interactions in the trans Dimer Interface (A) Residue views of interface regions EC1:EC4 (top) and EC2:EC3 (bottom) in trans dimers of pcdh-1, -10, -18, and -19 (left to right). Side chains of interfacial residues (>5% buried) are shown as sticks. Interactions conserved between multiple structures are highlighted in gold. Conserved hydrophobic residues chosen for mutation are boxed. Green spheres: calcium ions. (B) Molecular surfaces of representative Î´1 (pcdh1, left) and Î´2 (pcdh10, right) trans dimer structures opened to display interfacial residues color-coded according to their conservation within the respective subfamily (yellow: conserved in character; magenta: variable). Non-interface residues are shown in gray. Half of each 2-fold symmetric dimer is shown for clarity. See Figures S3âS5.
	Figure 4. SPR Analysis of Targeted trans Interface Mutations (A) Homophilic binding of wild-type and trans interface mutant Î´1 EC1â€“EC4 fragments of pcdh-1, -7, and -9 over their respective wild-type surfaces. (B) Effects of trans interface mutations in pcdh1 and -7 on heterophilic binding to pcdh-9 (left) and -11 (right). (C) Homophilic binding of wild-type and trans interface mutants of Î´2-pcdh-10, -12, -17, -18, and -19 over wild-type surfaces. Three analyte concentrations (27, 9, and 3 Î¼M) are plotted and responses are scaled independently for each surface.
	Figure 5. Membrane-proximal Regions of Î´2-Protocadherins Lack cis Interface Signatures (A and B) Crystal structures of Xenopus pcdh 8.1 EC1â€“EC6 (A) and human pcdh10 EC1â€“EC6 (B), shown as ribbons. Green spheres: calcium ions; wheat and magenta spheres: N- and O-linked glycans. (C) Superposition of two molecules of pcdh10 EC1â€“EC6 (gold) over the trans dimer structure of pcdh10 EC1â€“EC4 (orange). (D) Crystal structure of human pcdh8 EC5â€“EC6 membrane-proximal fragment, shown as ribbon. (E) Superposition of EC5â€“EC6 membrane-proximal regions of pcdh8 (magenta) and pcdh10 (gold) over EC5â€“EC6 from clustered pcdh Î³B7 (cyan, PDB: 5V5X; Goodman et al., 2017). (F) Sequence logo plots of aligned mouse clustered protocadherins Î², Î³A, and Î³B (top) or human Î´-protocadherins (bottom). Only residue positions with side chains > 20% buried in the Î³B7 cis dimer (PDB: 5V5X) are shown. Positions conserved only in clustered protocadherinpcdhs are highlighted orange. Numbering refers to pcdh Î³B7. (G) Close-up view of the cis interface of clustered pcdh Î³B7 (Goodman et al., 2017) showing differentially conserved interface residues from (E). Protomers colored slate and cyan. See Figure S6 and Table S1.
	Figure 6. Assembly of Î´-Protocadherin Ectodomains in Reconstituted Liposome Junctions (A) Fluorescence microscopy of liposome aggregation mediated by pcdh1 and pcdh10 ectodomains or trans dimer mutants pcdh1 L359D and pcdh10 L362D. Scale bar: 0.5 mm. (B and C) Representative slices of reconstructed tomograms showing aggregated liposomes of pcdh1 (B) or pcdh10 (C). Protocadherin ectodomains enrich at liposome contact sites seen in â€œside viewsâ€ (black arrowheads) where membranes appear parallel, and â€œtop viewsâ€ (arrows) where liposomes are stacked vertically. Unbound ectodomains protrude from non-junctional membranes (white arrowheads). See Videos S1, S2, S3, S4, and S5. (D) Side views of pcdh1, pcdh10, and pcdh gB6 junctions showing ordered assembly only for pcdh gB6. Intensity plots below each image show intermembrane distances and shallow minima where trans dimers overlap (brackets). Lipid bilayers are indicated with dashed lines. (E) Comparison of top views showing formation of a regular lattice by pcdh gB6 ectodomains only. Scale bars in (B)â€“(E): 400 Ã…. See Table S3.

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Adams, PHENIX: a comprehensive Python-based system for macromolecular structure solution. 2010, Pubmed