Volume 20, Issue 10, Pages 2328-2340 (September 2017) Cbln1 and Cbln4 Are Structurally Similar but Differ in GluD2 Binding Interactions  Chen Zhong, Jinlong Shen, Huibing Zhang, Guangyi Li, Senlin Shen, Fang Wang, Kuan Hu, Longxing Cao, Yongning He, Jianping Ding  Cell Reports  Volume 20, Issue 10, Pages 2328-2340 (September 2017) DOI: 10.1016/j.celrep.2017.08.031 Copyright © 2017 The Authors Terms and Conditions

Cell Reports 2017 20, 2328-2340DOI: (10.1016/j.celrep.2017.08.031) Copyright © 2017 The Authors Terms and Conditions

Figure 1 Homogeneity and Stability of Cbln1 and Cbln4 Hexamers (A) SEC analysis of Cbln1 showing two peaks with some overlap. (B) DLS analysis of the minor peak fraction (upper panel) and the major peak fraction (lower panel) of Cbln1. (C) SEC analysis of Cbln4 showing just one peak. (D) DLS analysis of the peak fraction of Cbln4. (E) Optimization of the buffer system for Cbln1 using the TSA assay. Data are represented as mean ± SD. (F) SEC analysis of Cbln1 in a buffer containing 150 mM K2SO4. Only one peak was detected. (G) DLS analysis of the peak fraction. See also Figure S1. Cell Reports 2017 20, 2328-2340DOI: (10.1016/j.celrep.2017.08.031) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Crystal Structures of the Cbln1C1q and Cbln4C1q Homotrimers (A) Comparison of the crystal structures of the Cbln1C1q and Cbln4C1q homotrimers. The structures in cartoon representations are superimposed and viewed in different orientations. Monomers A, B, and C of the Cbln4C1q homotrimer are colored in green, red, and cyan, respectively. For clarity, the Cbln1C1q homotrimer is colored in gray. The α helix and β strands of monomer A in the structure of the Cbln4C1q homotrimer are labeled in the side view, and some loops are labeled in the bottom view. The NAG molecules in the Cbln1C1q homotrimer are shown with ball-and-stick models. (B) Comparison of the 58GSA60 loop of Cbln1 and the equivalent 64ANS66 loop of Cbln4 in the side view. The residues are shown with ball-and-stick models. (C) Comparison of the loop CD of Cbln4 and loop CD of Cbln1 in the Cbln1C1q-GluD2ATD chimera. Monomer A of the Cbln4C1q structure (green) is superposed onto the structure of the Cbln1C1q-GluD2ATD chimera (gray). The region corresponding to the Cbln1C1q-GluD2ATD interface is shown in the magnified figure. (D) Comparison of the loop CD in the apo Cbln1 structure reported here and the loop CD of Cbln1 in the Cbln1C1q-GluD2ATD chimera. The apo Cbln1 (yellow) is superposed onto the structure of the Cbln1C1q-GluD2ATD chimera (gray). (E) Comparison of the loop CD of Cbln1 in different apo structures. The apo Cbln1 structure reported here and those reported by Elegheert et al. (2016) and Cheng et al. (2016) (PDB: 5KC5, 5KC6, and 5KWR) are superposed and colored in yellow, salmon, slate, and cyan, respectively. See also Figures S2–S4. Cell Reports 2017 20, 2328-2340DOI: (10.1016/j.celrep.2017.08.031) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Negative-Stain Single-Particle EM Reconstruction of the Hexamer of Full-Length Cbln1 (A) A typical negative-stain EM micrograph of Cbln1. Representative particles are marked with white boxes. (B) Different views of the EM density map of the Cbln1 hexamer at 13 Å resolution. (C) Comparison of the projections (top) with reference-based 2D class averages (bottom). (D) Predicted topology of the N-terminal region of Cbln1. De novo folding of the N-terminal region of Cbln1 was performed using Rosetta3.2. (E) Manual fitting of the predicted model of the N-terminal region of Cbln1 and the crystal structure of the Cbln1C1q trimer into the EM density of the Cbln1 hexamer. See also Figure S5. Cell Reports 2017 20, 2328-2340DOI: (10.1016/j.celrep.2017.08.031) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Development of a Semiquantitative Method to Determine the Binding Affinities of Cbln Proteins for Nrxn1β (A) Specific binding of the Cbln1 hexamer to 293T cells transfected with pCDH-Nrxn1β-IRES-RFP. Omission of the primary antibody served as a negative control, and omission of the Cbln1 protein served as a mock control. Data are represented as mean ± SD. (B) Specific binding of the Cbln4 hexamer to 293T cells transfected with pCDH-Nrxn1β-IRES-RFP. Data are represented as mean ± SD. (C) Typical fluorescence images of Nrxn1β-transfected 293T cells bound with Cbln1. Nrxn1β-transfected 293T cells emitted red fluorescence, and the cells bound with Cbln1 were detected with FITC488-conjugated secondary antibody and hence emitted green fluorescence. (D and E) Determination of the binding affinity of Cbln1 (D) or Cbln4 (E) to Nrxn1β with the semiquantitative cell surface binding assay. Data are represented as mean ± SD. Cell Reports 2017 20, 2328-2340DOI: (10.1016/j.celrep.2017.08.031) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Cbln4 Forms a Stable Complex with the LNS Domain of Nrxn1β at 1:1 Stoichiometry (A) SEC analysis of the Cbln4/Nrxn1β complex. (B) SDS-PAGE and western blotting analyses of Cbln1, Cbln1/Nrxn1β, Cbln4, and Cbln4/Nrxn1β. The anti-His antibody was specific for His-Cbln1 or His-Cbln4, and the anti-Strep antibody was specific for Strep-Nrxn1β. (C) SEC analyses of Cbln4 alone, Nrxn1β alone, and the Cbln4 and Nrxn1β mixtures with molar ratios of 1:1, 1:2, and 1:3, respectively. (D) Determination of the molecular masses of Cbln4, Nrxn1β, and the Cbln4/Nrxn1β complex using SEC-MALS. Cell Reports 2017 20, 2328-2340DOI: (10.1016/j.celrep.2017.08.031) Copyright © 2017 The Authors Terms and Conditions

Figure 6 Negative-Stain Single-Particle EM Reconstruction of the Cbln4/Nrxn1β Complex (A) A typical negative-stain EM micrograph of the Cbln4/Nrxn1β complex. Representative particles are marked with white boxes. (B) Different views of the EM density map of the Cbln4/Nrxn1β complex at 19 Å resolution. (C) Comparison of the projections (top) with reference-based 2D class averages (bottom). (D and E) Manual fitting of the crystal structure of murine Nrxn1β (PDB: 3MW2) (D) or rat Nrxn1β (PDB: 2R1B) (E) into the EM density map of the Cbln4/Nrxn1β complex. The EM density map of the Cbln4/Nrxn1β complex is colored in cyan and that of Cbln1 in gray. In the Nrxn1β structure, the part formed by a segment of the S4 insert is colored in red and the rest in blue. See also Figure S6. Cell Reports 2017 20, 2328-2340DOI: (10.1016/j.celrep.2017.08.031) Copyright © 2017 The Authors Terms and Conditions