Cryo‐ EM structures of the full‐length human contactin‐2

Figures

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  Fig. 1. Cryo‐EM (Cryogenic electron microscopy) analysis of human CNTN2 (Contactin‐2) Ig1‐6. (A) Schematic of CNTN2 full‐length domain organization. The yellow color represents the Ig domain, which is the structure we studied. The gray represents the FNIII domain. (B, C) Overall cryo‐EM map and structure of CNTN2 Ig1‐6 homodimer shown in two views. One monomer is colored yellow, and the other is colored magenta. (D) Distribution diagram of average sedimentation coefficient of samples at three concentrations (ls‐g (s) model). The experiment was conducted once. (E) CNTN2 mature full‐length gel filtration analysis shows a concentration‐dependent peak shift. The gel filtration analysis was conducted in duplicate. The structural figures were generated utilizing UCSF Chimera software.

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  Fig. 2. CNTN2 (contactin‐2) dimerization is mediated through two small interfaces between the Ig3‐6 interface. (A) A broad view of the CNTN2 Ig 3, 4, 5, and 6 interface interactions underlying the characteristic architecture. One monomer is colored yellow, and the other is colored magenta. (B) CNTN2 Ig1‐6 monomer (domain rainbow colored) and monomer of the contactin 2 Ig1‐6 in surface representation colored according to various properties and with dimerization surfaces outlined (homodimer surfaces purple colored). (C, D) The molecular details of CNTN2 Ig 4 with lg 5 and lg 6 interface interactions. The key residues of dimerization interface are shown as sticks. The black dotted lines represent hydrogen bonding. (E) Close‐up and overlay of cryo‐EM density (gray) of the N498‐glycan on Ig5, which is part of the extensive interface. The N498‐glycan contacts with E40, R62, R64 of Ig1, shown as sticks. The black dotted lines represent hydrogen bonding. (F) Pull‐down assay of Flag‐tagged CNTN2 wild‐type protein and GFP‐tagged CNTN2 point mutations from transgenic cell lysates, utilizing Coomassie Blue staining and western blot analysis. The western blot experiments were conducted in duplicate.…

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  Fig. 3. Comparison of the ectodomain structures of CNTN (Contactin) 2 with CNTN1. (A) Structural alignment of the single protomer CNTN2 Ig1‐6 and IgSFs Ig1‐4 crystal structures (PDB ID: 1CS6, 3JXA, 3P3Y, 4X9H, 5K6U) revealing that their overall architecture is highly similar, as shown by a horseshoe arrangement of Ig1‐4. (B) Superimposition of the three Ig domains of CNTN2 (this study, colored in magenta) and CNTN1 (PDB ID: 7OL4, 7OL2, colored in blue and gray). The superimposition is based on the N‐terminal Ig1‐4 domain. The conformational difference between the two structures is evident from the large deviation between the CNTN2–Ig 5, 6 and CNTN1‐Ig 5, 6 in this superimposition. (C, D) the CNTN2 Ig1‐6 (Left) compared with CNTN1 Ig 1–6 (Right PDB ID: 7OL2) in electrostatic and hydrophobic surface representation colored according to various properties. For hydrophobic potential, the hydrophilic residues are labeled green and the hydrophobic residues are labeled yellow. For electrostatic potential, the negative charged residues are labeled red and the positive charged residues are labeled blue. The Unit of electrostatic potential is kT e−1. (E) Gel filtration studies of CNTN2 (2.0…

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  Fig. 4. The FNIII (fibronectin type III) domain of CNTN2 (contactin‐2) reveals a flexible conformation and does not contribute to the dimerization of CNTN2. (A, B) Three views of the CNTN2 Ig‐FNIII1‐2 cryo‐EM structure. (C) Superimposition of the two protomers of the asymmetric CNTN2 dimer reveals a highly similar Ig domain structure. The black lines and circles represent the rotation direction and angle. (D, E) Superimposition of CNTN2 Ig5‐FNIII1‐2 (colored in magnate) with CNTN5 (colored in orange, PDB ID:5I99) and CNTN2 FNIII1‐2 (colored in yellow) with mouse CNTN2 FNIII1‐3 (colored in blue, PDB ID:5E7L). (F) Representative 2D class averages of the CNTN2 full‐length conformation showing the flexibility of FNIII domain in solution (Scale bar: 2 nm). The structural figures were generated utilizing UCSF Chimera software.

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  Fig. 5. Cell adhesion is mediated by CNTN2 (contactin‐2) in HEK293F cells. (A) A diagram of the CNTN2 constructs with GFP (green) generated for cell adhesion assays. (B) Confocal images of subcellular localization of CNTN2 full length, CNTN2 lg1‐4, CNTN2 lg1‐6, and CNTN2 FNIII domains. The confocal experiments were conducted in duplicate (Scale bar: 10 μm). (C) Cell–cell adhesion was monitored by microscopy. HEK293F cells were transfected with CNTN2 full length, CNTN2 lg1‐4, CNTN2 lg1‐6, CNTN2 FNIII domains to form cell clusters (Scale bar: 100 μm). (D) Aggregation percent; the relative percentage of the 4 cells, 10 cells were classified compared to control as a cluster (Average ± SEM, n = 10). The experiments were conducted in duplicate, with a minimum of 500 cells counted from each data point. One‐way ANOVA was employed to compare the wild‐type (WT) group and the mutant groups for the counts of 4 cells and 10 cells, respectively. The structural figures were generated utilizing UCSF Chimera software.

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  Fig. 6. Cell adhesion mediated by CNTN2 (contactin‐2) Ig4‐6 domains. (A) A diagram of the CNTN2 mutant constructs with GFP (green) generated for cell adhesion assays. (B) A diagram of the CNTN2 point mutation constructs with GFP (green) generated for cell adhesion assays (Scale bar: 10 μm). The red channel was modified to magenta to enhance accessibility for individuals with color vision deficiencies. The confocal experiments were conducted in duplicate. (C) Cell–cell adhesion monitored by fluorescence microscopy. HEK293F cell transfected with L330E, R355A, R432A, R480A, R506A, or R355/432/480/506A mutations (Scale bar: 100 μm). (D) Clustering index; the proportion of the total segmented cell area classified as a cluster (Average ± SEM, n = 10). The experiments were conducted in duplicate, with a minimum of 500 cells counted from each data point. One‐way ANOVA was employed to compare the wild‐type (WT) group and the mutant groups for the counts of 4 cells and 10 cells, respectively.

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  Fig. 7. Schematic diagram of how CNTN2 (contactin‐2) regulates the myelin sheath distance based on concentration. The dimerization of CNTN2 exhibits a concentration‐dependent behavior. At relatively low concentrations of CNTN2, the protein predominantly exists in a monomeric form. However, as the concentration increases, there is a tendency for CNTN2 to form homodimers.