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丁建平研究组揭示Cbln家族蛋白功能异同的分子机制

来源:Cell

    2017年9月5日,中国科学院上海生命科学研究院生物化学与细胞生物学研究所丁建平研究组在《Cell》子刊《Cell Reports》杂志在线发表了的最新研究成果,研究论文题为Cbln1 and Cbln4 are structurally similar but differ in GluD2 binding interactions,研究揭示了Cbln1和Cbln4之间功能异同的分子机制。

Cbln家族包含4个成员,它们具有很高的序列同源性和相似的结构组成,均在C端包含一个C1q结构域。有意思的是,Cbln1通过在突触间隙中结合Nrxn蛋白和d-谷氨酸受体参与突触形成。然而,尽管Cbln4与Cbln1有高达74%的序列等同性,文献报道Cbln4不能结合Nrxn蛋白和d-谷氨酸受体,而是DCC的配体。

研究人员解析了Cbln1和Cbln4的C1q结构域(Cbln1C1q 和Cbln4C1q)三聚体的晶体结构,分辨率分别为2.2Å和2.3Å。Cbln1C1q和Cbln4C1q总体结构非常相似,但在对应于Cbln1C1q与d-谷氨酸受体2(GluD2)的ATD结构域相互作用界面的区域上有显著不同。利用负染和冷冻电镜单颗粒技术,研究人员获得了分辨率为13Å的全长Cbln1六聚体的三维结构。该结构显示,Cbln1六聚体整体像两个连着的樱桃,Cbln1C1q三聚体占据密度图中的“樱桃”部分,而Cbln1的N端区域形成较平的相互作用界面,介导六聚体的形成。此外,为了解决此前文献中数据有相悖之处的问题,研究人员研发了基于细胞表面结合技术的半定量方法,以测定Cbln1和Cbln4对全长Nrxn1β的亲和力。研究人员发现,Cbln4与全长Nrxn1β的相互作用并不弱,并进一步获得了Cbln4与Nrxn1β的LNS结构域形成的复合物、以及分辨率为19Å的该复合物的三维冷冻电镜结构。结构分析提示,Nrxn1β可能通过其S4片段的β10与Cbln4的N端相结合。这些研究结果揭示了Cbln1和Cbln4之间功能异同的分子机制,为进一步研究Cbln1和Cbln4在神经细胞突触形成中的功能奠定了较好的结构基础。

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.

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 Cbln1C1qhomotrimer 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-GluD2ATDchimera (gray). The region corresponding to the Cbln1C1q-GluD2ATDinterface 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.

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.

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.

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.


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.


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