How clustered protocadherin binding specificity is tuned for neuronal self-/nonself-recognition
- Zuckerman Mind, Brain and Behavior Institute, Columbia University, United States
- School of Neurobiology, Biochemistry and Biophysics, Tel Aviv University, Israel
- Sagol School of Neuroscience, Tel Aviv University, Israel
- Department of Medicine, Division of Nephrology, Columbia University, United States
- Department of Biochemistry and Molecular Biophysics, Columbia University, United States
- Department of Systems Biology, Columbia University, United States
Figures
Figure 1
Clustered protocadherin (cPcdh) domain organization and extracellular interactions.
(A) Schematic depicting the domain organization of cPcdhs. EC, extracellular cadherin domain; TM, transmembrane domain; ECD, ectodomain; ICD, intracellular domain. (B) Schematic of two cPcdhs …
Figure 2 with 2 supplements
Clustered protocadherins (cPcdhs) show strict homophilic specificity in their trans interactions.
(A) Surface plasmon resonance (SPR) binding profiles of cPcdh trans fragment analytes from all cPcdh subfamilies (denoted in the top row) flowed over six surfaces coated with alternate cPcdh trans …
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Figure 2—source data 1
Sedimentation equilibrium analytical ultracentrifugation data for trans SPR reagents.
- https://cdn.elifesciences.org/articles/72416/elife-72416-fig2-data1-v1.docx
Figure 2—figure supplement 1
Trans interface mutants demonstrate homophilic interactions observed in surface plasmon resonance (SPR) are mediated by the trans dimer interface.
(A) SPR-binding curves for wild-type and trans mutant alternate clustered protocadherins (cPcdhs) flowed over their respective immobilized wild-type molecule. (B) SPR-binding curves for wild-type …
Figure 2—figure supplement 2
Mutagenesis experiments reveal role in trans specificity for the five interfacial residue differences between close pair β61–4 and β81–4.
(A) Structural superposition of the β61–4 and β81–4 trans dimer crystal structures (PDBs: 5DZX and 5DZY) shown in ribbon depiction above, with close-up views of the trans interfacial regions …
Figure 3 with 1 supplement
C-type clustered protocadherin (cPcdh) γC4 adopts an EC1–4-mediated head-to-tail trans dimer like alternate cPcdhs with a comparatively weak dimer affinity.
(A) Ribbon diagrams of the γC4EC1–4 trans dimer crystal structures obtained from two different crystal forms. Bound calcium ions are shown as green spheres and glycans are shown in pale blue …
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Figure 3—source data 1
X-ray crystallography data collection and refinement statistics.
- https://cdn.elifesciences.org/articles/72416/elife-72416-fig3-data1-v1.docx
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Figure 3—source data 2
Overall structural similarity between cPcdh γC4, alternate cPcdhs, and non-clustered Pcdhs trans dimer structures.
- https://cdn.elifesciences.org/articles/72416/elife-72416-fig3-data2-v1.docx
Figure 3—figure supplement 1
γC4 trans dimer crystal structures and trans interface analysis.
(A) Our crystallization experiments with γC4EC1–4 yielded two distinct crystal forms the first of which showed significant X-ray diffraction anisotropy. (i) UCLA Diffraction Anisotropy Server (Strong…
Figure 4 with 3 supplements
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 …
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Figure 4—source data 1
Sedimentation equilibrium analytical ultracentrifugation data for cis SPR reagents.
- https://cdn.elifesciences.org/articles/72416/elife-72416-fig4-data1-v1.docx
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 γC33–6 analyte binding over a β93–6 covered surface. Data are shown in black, and the red traces represent the fit to an 1:1 binding model. (B) Left, surface plasmon …
Figure 4—figure supplement 2
Range of clustered protocadherin (cPcdh) cis and trans dissociation constants, KDs.
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 …
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 …
Figure 5 with 2 supplements
γA4 preferentially forms the EC6-only side and γC3 the EC5–6 side in cis dimers.
(A) Structural model of γA4/γC3 cis dimer based on γB7EC3–6 cis dimer and γA4EC3–6 crystal structures (PDBs: 5V5X and 5SZQ). γA4 is shown adopting the EC6-only side (blue protomer) and γC3 is shown …
Figure 5—figure supplement 1
Structure-guided sequence analysis of γA4 and γC3 cis interactions.
(A) (i) Schematic of the asymmetric γB7EC3–6 cis dimer crystal structure. (ii) Close-up view of the γB7 cis interface: interfacial residue side chains are shown in pink for the EC6-only protomer and …
Figure 5—figure supplement 2
γA4 and γC3 cis fragments behave as monomers in size exclusion-coupled multiangle light scattering (SEC-MALS) and mutating γA4 to make it more like γC3 prevents γA4/γC3 cis heterodimerization.
(A) SEC-MALS data for wild-type γA43–6, wild-type γC33–6, and γC33–6 V560R showing all three molecules are monomeric in SEC-MALS, consistent with their behavior in sedimentation equilibrium AUC. …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (E. coli) | One shot Top10 Competent Cells | Invitrogen | C4040-06 | Plasmid production |
| Cell line (Homo sapiens) | FreeStyle 293 F cells | Thermo Fisher Scientific | R79007 | Cell line for protein expression |
| Cell line (Homo sapiens) | K-562 bone marrow chronic myelogenous leukemia (CML) cells | ATCC | ATCC CCL-243 | Cell line for cell-aggregation assays |
| Transfected construct (M. musculus) | α41–5 | This paper | Pcdhα4 EC1–5, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | α71–5 | Rubinstein et al., 2015 | ||
| Transfected construct (M. musculus) | α121–5 | This paper | Pcdhα12 EC1–5, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | β61–4 | Goodman et al., 2016c | ||
| Transfected construct (M. musculus) | β81–4 | Goodman et al., 2016c | ||
| Transfected construct (M. musculus) | γA11–4 | Goodman et al., 2016a | ||
| Transfected construct (M. musculus) | γA41–4 | Goodman et al., 2016a | ||
| Transfected construct (M. musculus) | γA81–4 | Rubinstein et al., 2015 | ||
| Transfected construct (M. musculus) | γA91–5 | Goodman et al., 2016a | ||
| Transfected construct (M. musculus) | γB21–5 | Goodman et al., 2016a | ||
| Transfected construct (M. musculus) | γB41–5 | This paper | PcdhγB4 EC1–5, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γB51–4 | Goodman et al., 2016a | ||
| Transfected construct (M. musculus) | αC21–4 | Rubinstein et al., 2015 | ||
| Transfected construct (M. musculus) | γC31–4 | Goodman et al., 2016a | ||
| Transfected construct (M. musculus) | γC41–4 | This paper | PcdhγC4 EC1–4, Honig/Shapiro labs | |
| TRansfected construct (M. musculus) | γC51–5 | Rubinstein et al., 2015 | ||
| Transfected construct (M. musculus) | α71–5-AVI | This paper | Biotinylated Pcdhα7 EC1–5, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | β61–4-AVI | This paper | Biotinylated Pcdhβ6 EC1–4, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | β81–4-AVI | This paper | Biotinylated Pcdhβ8 EC1–4, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γA81–4-AVI | This paper | Biotinylated PcdhγA8 EC1–4, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γA91–5-AVI | This paper | Biotinylated PcdhγA9 EC1–5, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γB21–5-AVI | This paper | Biotinylated PcdhγB2 EC1–5, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | αC21–4-AVI | This paper | Biotinylated PcdhαC2 EC1–4, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γC31–4-AVI | This paper | Biotinylated PcdhγC3 EC1–4, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γC41–4-AVI | This paper | Biotinylated PcdhγC4 EC1–4, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γC51–5-AVI | This paper | Biotinylated PcdhγC5 EC1–5, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | α41–4-AVI | This paper | Biotinylated Pcdhα4 EC1–4, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | α71–5 L301R | This paper | Pcdhα7 EC1–5 mutant, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γA81–4 I116R | Rubinstein et al., 2015 | PcdhγA8 EC1–4 mutant, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | β61–4 R41N | This paper | Pcdhβ6 EC1–4 mutant, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γC51–5 S116R | This paper | PcdhγC5 EC1–5 mutant, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | β61–4 S117I | This paper | Pcdhβ6 EC1–4 mutant, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | β61–4 L125P | This paper | Pcdhβ6 EC1–4 mutant, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | β61–4 E369K | This paper | Pcdhβ6 EC1–4 mutant, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | β61–4 Y371F | This paper | Pcdhβ6 EC1–4 mutant, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | β61–4 R41N/S117I | This paper | Pcdhβ6 EC1–4 mutant, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | β61–4 R41N/E369K | This paper | Pcdhβ6 EC1–4 mutant, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | β61–4 S117I/L125P | This paper | Pcdhβ6 EC1–4 mutant, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | β61–4 R41N/S117I/L125P | This paper | Pcdhβ6 EC1–4 mutant, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | β61–4 R41N/S117I/E369K | This paper | Pcdhβ6 EC1–4 mutant, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | β61–4 R41N/S117I/Y371F | This paper | Pcdhβ6 EC1–4 mutant, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | β61–4 R41N/S117I/L125P/E369K/Y371F | This paper | Pcdhβ6 EC1–4 mutant, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γC41–4 E78A | This paper | PcdhγC4 EC1–4 mutant, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γC41–4 E78Q | This paper | PcdhγC4 EC1–4 mutant, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γC41–4 S344R | This paper | PcdhγC4 EC1–4 mutant, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γC41–4 D290A | This paper | PcdhγC4 EC1–4 mutant, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γC41–4 D290N | This paper | PcdhγC4 EC1–4 mutant, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | β13–6 | This paper | Pcdhβ1 EC3–6, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | β61–6 | This paper | Pcdhβ6 EC1–6, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | β93–6 | This paper | Pcdhβ9 EC3–6, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γA33–6 | This paper | PcdhγA3 EC3–6, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γA43–6 | Goodman et al., 2016a | ||
| Transfected construct (M. musculus) | γA93–6 | This paper | PcdhγA9 EC3–6, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γB23–6 | Goodman et al., 2016a | ||
| Transfected construct (M. musculus) | γB53–6 | Goodman et al., 2016a | ||
| Transfected construct (M. musculus) | γB73–6 | Goodman et al., 2016a | ||
| Transfected construct (M. musculus) | αC22–6 | Goodman et al., 2016a | ||
| Transfected construct (M. musculus) | α71–5/γC36 chimera | Goodman et al., 2016a | ||
| Transfected construct (M. musculus) | γC33–6 | Goodman et al., 2016a | ||
| Transfected construct (M. musculus) | γC52–6 | This paper | PcdhγC5 EC2–6, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | β93–6-AVI | This paper | Biotinylated Pcdh β9 EC3–6, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γA43–6-AVI | This paper | Biotinylated PcdhγA4 EC3–6, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γA93–6-AVI | This paper | Biotinylated PcdhγA9 EC3–6, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γB23–6-AVI | This paper | Biotinylated PcdhγB2 EC3–6, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | αC23–6-AVI | This paper | Biotinylated Pcdh αC2 EC3–6, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γC33–6-AVI | This paper | Biotinylated PcdhγC3 EC3–6, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γC52–6-AVI | This paper | Biotinylated PcdhγC5 EC2–6, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γA43–6 V560R | This paper | PcdhγA4 EC3–6 mutant, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γC33–6 V560R | This paper | PcdhγC3 EC3–6 mutant, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γA43–6 K558R | This paper | PcdhγA4 EC3–6 mutant, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γC33–6 R558K | This paper | PcdhγC3 EC3-6 mutant, Honig/Shapiro labs | |
| Transfected construct (M. musculus) | γB73–6 Y532G | Goodman et al., 2017 | ||
| Transfected construct (M. musculus) | γB73–6 A570R | This paper | PcdhγB7 EC3–6 mutant, Honig/Shapiro labs | |
| Peptide, recombinant protein | NeutrAvidin-HRP | Thermo Fisher Scientific | 31,030 | Biotinylated protein western bot |
| Peptide, recombinant protein | NeutrAvidin protein | Thermo Fisher Scientific | 31,000 | SPR assays |
| Peptide, recombinant protein | BSA | Sigma-Aldrich | A7906 | SPR assays |
| Commercial assay or kit | Spin Miniprep Kit | Qiagen | 27,106 | |
| Commercial assay or kit | Hi-speed Plasmid Maxi Kit | Qiagen | 12,663 | |
| Commercial assay or kit | SF Cell Line 4D-Nucleofector X Kit S | Lonza | V4XC-2032 | |
| Commercial assay or kit | Amine-coupling kit | Cytiva | BR100050 | SPR experiments |
| Commercial assay or kit | Morpheus Amino Acids | Molecular Dimensions | MD2-100-77 | Crystallography |
| Commercial assay or kit | Morpheus Buffer System II | Molecular Dimensions | MD2-100-101 | Crystallography |
| Chemical compound | Polyethylenimine | Polysciences | 24765-2 | Transfection |
| Chemical compound | Biotin | Sigma-Aldrich | B4501 | Protein biotinylation |
| Chemical compound | Tris Base | Fisher Scientific | BP152-5 | |
| Chemical compound | Sodium Chloride | Fisher Scientific | S271-10 | |
| Chemical compound | Calcium Chloride Dihydrate | JT Baker | 1336-01 | |
| Chemical compound | Imidazole | ACROS | 301870025 | |
| Chemical compound | HEPES | Sigma-Aldrich | H3375 | |
| Chemical compound | Tween-20 | Sigma-Aldrich | P7949 | |
| Chemical compound | Sodium Acetate | Sigma-Aldrich | S7545 | |
| Chemical compound | IMAC Sepharose 6 Fast Flow | Cytiva | 17092109 | |
| Chemical compound | Penicillin Streptomycin | Thermo Fisher Scientific | 15070063 | |
| Chemical compound | PEG 6000 | Sigma-Aldrich | 81,260 | |
| Chemical compound | PEG 8000 | Sigma-Aldrich | 89,510 | |
| Chemical compound | Ethylene Glycol | Fluka | 03760 | |
| Chemical compound | Lithium Chloride | Sigma-Aldrich | L8895 | |
| Chemical compound | MES | Sigma-Aldrich | M3671 | |
| Chemical compound | Glycerol | ACROS | 332031000 | |
| Software, algorithm | UCLA Diffraction Anisotropy Server | Strong et al., 2006 | https://srv.mbi.ucla.edu/Anisoscal/ | |
| Software, algorithm | SednTerp | Thomas Laue | http://bitcwiki.sr.unh.edu/index.php/Main_Page | |
| Software, algorithm | HeteroAnalysis | https://core.uconn.edu/auf | ||
| Software, algorithm | Scrubber 2.0 | BioLogic Software | http://www.biologic.com.au | |
| Software, algorithm | Phaser | McCoy et al., 2007 | Implemented in CCP4 or Phenix (see below) | |
| Software, algorithm | CCP4 | Winn et al., 2011 | https://www.ccp4.ac.uk/ | |
| Software, algorithm | Phenix | Liebschner et al., 2019 | http://www.hkl-xray.com/ | |
| Software, algorithm | XDS | Kabsch, 2010 | http://xds.mpimf-heidelberg.mpg.de | |
| Software, algorithm | AIMLESS | Evans and Murshudov, 2013 | http://www.ccp4.ac.uk | |
| Software, algorithm | Coot | Emsley et al., 2010 | https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ | |
| Software, algorithm | PISA | Krissinel and Henrick, 2007 | http://www.ebi.ac.uk/pdbe/protint/pistart.html | |
| Software, algorithm | Pymol | Schrödinger | https://pymol.org | |
| Software, algorithm | UCSF Chimera | Pettersen et al., 2004 | https://www.cgl.ucsf.edu/chimera/ | |
| Software, algorithm | Clustal Omega | Sievers et al., 2011 | https://www.ebi.ac.uk/Tools/msa/clustalo/ | |
| Software, algorithm | WebLogo 3.0 | Crooks et al., 2004 | http://weblogo.threeplusone.com/ | |
| Software, algorithm | SignalP 4.0 | Petersen et al., 2011 | https://services.healthtech.dtu.dk/service.php?SignalP-5.0 | |
| Software, algorithm | ASTRA | Wyatt | https://www.wyatt.com/products/software/astra.html | |
| Other | Freestyle 23 Expression Media | Thermo Fisher Scientific | 12338-018 | Protein expression media |
| Other | Opti-MEM Reduced Serum Media | Thermo Fisher Scientific | 31985-070 | Protein expression media |
| Other | Series S CM4 chip | Cytiva | BR100539 | SPR assays |
| Other | Fetal Bovine Serum | Thermo Fisher Scientific | 16141079 | Cell-aggregation assays media |
| Other | DMEM with GlutaMAX | Thermo Fisher Scientific | 10569010 | Cell-aggregation assays media |
