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. 2016 Dec 6;24(12):2163-2173.
doi: 10.1016/j.str.2016.11.004.

Conformational Plasticity in the Transsynaptic Neurexin-Cerebellin-Glutamate Receptor Adhesion Complex

Shouqiang Cheng  1 , Alpay B Seven  2 , Jing Wang  1 , Georgios Skiniotis  2 , Engin Özkan  3
Affiliations

Conformational Plasticity in the Transsynaptic Neurexin-Cerebellin-Glutamate Receptor Adhesion Complex

Shouqiang Cheng et al. Structure. .

Abstract

Synaptic specificity is a defining property of neural networks. In the cerebellum, synapses between parallel fiber neurons and Purkinje cells are specified by the simultaneous interactions of secreted protein cerebellin with pre-synaptic neurexin and post-synaptic delta-type glutamate receptors (GluD). Here, we determined the crystal structures of the trimeric C1q-like domain of rat cerebellin-1, and the first complete ectodomain of a GluD, rat GluD2. Cerebellin binds to the LNS6 domain of α- and β-neurexin-1 through a high-affinity interaction that involves its highly flexible N-terminal domain. In contrast, we show that the interaction of cerebellin with isolated GluD2 ectodomain is low affinity, which is not simply an outcome of lost avidity when compared with binding with a tetrameric full-length receptor. Rather, high-affinity capture of cerebellin by post-synaptic terminals is likely controlled by long-distance regulation within this transsynaptic complex. Altogether, our results suggest unusual conformational flexibility within all components of the complex.

Keywords: cerebellin; crystal structure; glutamate receptor delta-2; negative-stain single-particle electron microscopy; neurexin; structural flexibility; synapse.

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Figures

Figure 1
Figure 1. Rat Cbln1 hexamers with intact CRN domains bind rat α- or β-Nrxn1 including SS4
A. Domain structure of Cbln1, Nrxn1α and Nrx1β, drawn to scale. The shaded regions, the transmembrane (TM) helix and the unstructured juxtamembrane domains (intracellular and extracellular), are excluded from our constructs. The dotted lines mark the boundaries of the region expressed by exons shared by α and β-Neurexins. SP: Signal peptide. B. Cbln1 (blue curve) runs as hexamers on a Superdex 200 size-exclusion column when its cysteines in the CRN domain are intact. C34,38S (CS) double mutation causes it to run as trimers (green curve). Wild-type (WT) Cbln1 runs as a dimer on non-reducing denaturing gels, while CS runs as a monomer. C. Cbln1 binds Nrxn1α(+SS4) domains LNS2 to LNS6, as observed on a Superose 6 size-exclusion column. Size-exclusion fractions for the Cbln1+Nrxn1α(+SS4) sample (red curve) are run on a non-reducing gel. D. Cbln1 binds Nrxn1β(+SS4) as observed on a Superdex 200 size-exclusion column. Nrxn1β construct contains residues S48 to P292, including the β form-unique N-terminal region and the LNS6 domain. Size-exclusion fractions for the Cbln1+Nrxn1β(+SS4) sample (red curve) are run on a non-reducing gel. For Cbln1 binding to an LNS6-only Nrxn1 construct, see Figure S1A. E. Cbln1 does not bind Nrxn1β-LNS6 without SS4 (−SS4). See also Figure S1.
Figure 2
Figure 2. Rat Cbln1 binds Nrxn1β with high affinity and a stoichiometry of 1 hexamer to 1
A. Isothermal titration calorimetry experiments for Cbln1. Molar ratio represents the ratio of Neurexin monomers to Cerebellin hexamers. B–C. MALS analysis confirms molar mass for Cbln1 and Cbln1-Nrxn1β complex to match one hexamer (B), and one hexamer + one monomer (C), respectively.
Figure 3
Figure 3. Crystal structure of Cbln1
A. Cartoon model of the Cerebellin-1 C1q monomer. N-linked glycan attached to Asn79 side chain is shown in stick representation. The Cα atom of the last residue visible in the electron density is shown as a ball. B. Cbln1 trimer can be formed by applying the crystallographic three-fold symmetry operation. The location of the missing Cysteine-rich N-terminal (CRN) domain is highlighted. C. Looking at the Cbln1 trimer along the symmetry axis from the top, where CRN would be positioned. D. Representative 2mFo-DFc electron density electron density. E. Source of the Cerebellin peptide within the Cbln1 protein. Balls highlight Cα atoms of the first and last residues of the peptide. F. Comparison of Cbln1 C1q domain to the C1q domain of C1QL-1 (PDB: 4D7Y), where most differences are towards the “bottom half” of the domain. For comparisons with other C1q domains, see Figure S2.
Figure 4
Figure 4. Cerebellin binds both α- and β-Neurexin with its flexible CRN domain
A. Domain compositions of Cbln1, Nrxn1α and 1β. The shaded domains were not included in expressed constructs used for electron microscopy. B–D. Representative EM 2D class averages of Cbln1 (B), Cbln1+Nrxn1β (C) and Cbln1+Nrxn1α (D). See also Figure S3. B1–D1. Schematized representations of movements observed in class averages shown in (B) to (D). E. One of the three isothermal titration calorimetry experiments for Cbln1 and Nrxn1α binding. The results are fit to a “one set of sites” binding model.
Figure 5
Figure 5. GluD2-Cbln affinity is weak regardless of oligomeric state
A. Domain structure of GluD2. All domain and structural elements are drawn to scale. B. Cbln1 bind does not bind GluD2 ectodomain (ATD+LBD) with high affinity, as observed by lack of co-elution on size-exclusion columns. Colors of chromatograms match colors of fractions as they are labeled on non-reducing gels. C. GluD2 ATD domain can be tetramerized using a helical coiled coil zipper (tetrameric zipper, or “4Z”). D. Cbln1 bind does not bind tetrameric GluD2 ATD-4Z with high affinity, as observed by lack of co-elution on size-exclusion columns, or by lack of heat release or uptake during ITC experiments in the presence or absence of calcium (Figure S4).
Figure 6
Figure 6. Structure of the symmetric dimers of rat GluD2 ectodomain
A–D. GluD2 ectodomain dimers in four different views. E. Representative 2mFo-DFc electron density from the beta-strands of the C-terminal lobe of the ATD domain at 1.0 σ. F. NCS-averaged 2mFo-DFc electron density for the unmodeled ATD-LTD linker, which packs both on to the ATD and LBD.
Figure 7
Figure 7. GluD2 dimers are in a conformation not observed before in iGluR proteins
A. Schematic representation of iGluR tetramers, with the swing-out motion in the GluD2 ectodomain structure mapped on top. The swinging-out of LBDs in the GluD2 ectodomain dimer structure are shown with arrows. B. GluD2 dimer superposed on GluA2 antagonist bound structure (PDB: 3KG2). C. GluD2 dimer superposed on the desensitized state structure of GluK2 (PDB: 4UQQ). The shorter ATD-LBD linker in GluD2 compared to GluK2 does not allow for GluDs to adopt a similar desensitized state structure. The ATD-LBD linker in the cyan subunit of GluK2 was not resolved in the structure, and is depicted as a thick dashed line. D. GluD2 ectodomain structure is not compatible with tetramerization as observed for GluD2 ATD and the GluD2 ATD + Cbln complex (Elegheert et al.). GluD2 ATD tetramer (PDB: 5KCA) is shown in surface representation, with each monomer as a different hue of pink to purple, while the GluD2 ectodomain dimer is drawn in cartoon representation. The green-colored LBD severely clashes with an ATD (dark purple) from the tetrameric structure. E. GluD2 symmetric dimers can be made to form tetramers if one LBD from each dimer moves out of the way. F. The Nrxn-Cbln-GluD complex (modeled) fits within the synaptic cleft.
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