A Proteomic Screen of Neuronal Cell-Surface Molecules Reveals IgLONs as Structurally Conserved Interaction Modules at the Synapse

Abstract

In the developing brain, cell-surface proteins play crucial roles, but their protein-protein interaction network remains largely unknown. A proteomic screen identified 200 interactions, 89 of which were not previously published. Among these interactions, we find that the IgLONs, a family of five cell-surface neuronal proteins implicated in various human disorders, interact as homo- and heterodimers. We reveal their interaction patterns and report the dimeric crystal structures of Neurotrimin (NTRI), IgLON5, and the neuronal growth regulator 1 (NEGR1)/IgLON5 complex. We show that IgLONs maintain an extended conformation and that their dimerization occurs through the first Ig domain of each monomer and is Ca²⁺ independent. Cell aggregation shows that NTRI and NEGR1 homo- and heterodimerize in trans. Taken together, we report 89 unpublished cell-surface ligand-receptor pairs and describe structural models of trans interactions of IgLONs, showing that their structures are compatible with a model of interaction across the synaptic cleft.

Keywords: ELISA; IgLON; SAXS; ligand-receptor pair; protein crystallography.

Figure 1 –. Flowchart of the ELISA-based ligand receptor assay –

Top panel – Flowchart of the main steps composing the experimental methodology of the ELISA-based assay. Bottom panel: A – Schematic diagram of the vectors used in the assay and their relevant features. B – Schematic of the ELISA setup to show the orientation of the bait and prey. HRP develops a blue color. C – Typical results of a negative 384-well plate. Red boxed wells are false positives (present in every plate of the same batch of one experiment, compare with D). D – 384-well plate containing a potential positive interaction (green box, the interaction appears only in one plate).

Figure 2 –. List of genes used in this study and interaction results –

A – The list contains information about the topology of each protein. Black font indicates genes that have been used as both F_c- and AP fusions, Blue font indicates genes that have been used as F_c-fusions only as positive interactions. In the topology column: S, secreted; MP, multipass membrane; T1, type I protein; GPI, GPI anchor protein. * The RELN gene encodes for Reelin, which is composed by 8 Reelin repeats. Because of its size and intrinsic cleavage, we have cloned this gene into four fragments. B – Pie chart showing the topology distribution of the proteins. C – Graph indicating the number of F_c-fusion detected by western blot expression, shown in Figure S5. D – Pie chart diagram of the number of wells used to identify the 200 interactions and their breakdown as “new” and “known.” E – The matrix of data for 181 × 207 pairwise interactions. Rows contain AP-fusion baits, including the AP-only construct as negative control, whereas columns contain 207 F_c-fusion preys. The green color scale represents values from <2 (White) to 20 (dark green). The largest cluster boxed in green represents all IgLON interactions. Red preys were used as positive control F_c proteins. Source data file attached as supplemental excel file.

Figure 3 –. Solution characterization of NTRI –

A – Coomassie blue staining of purified NEGR1 and NTRI used for SEC experiment. EndoF indicates that the purified protein was treated with Endo F1 under native conditions, causing faster migration in SDS-PAGE. B, C – Four different concentrations of purified NEGR1 and NTRI were subjected to SEC. Higher concentrations of the protein produce a leftward shift in the elution profile. In each graph, the inset contains the concentration of the injected protein and the elution volume. D, E – Sedimentation velocity analysis of two concentrations of purified NTRI, two-dimensional spectrum/Monte Carlo analyses: D shows the presence of two main species (total concentration ~51% and ~40%) of the appropriate MW; E shows that at a higher concentration, one main species of ~59kDa (total concentration ~87%) becomes prevalent; see numerical tables below D and E images. F – Cell aggregation assay of four different conditions as shown in the labels. Three independent experiments were performed, and representative images are shown.

Figure 4 –. Three-dimensional structure of NTRI –

A – “Side” and “top” views of the surface rendering of the crystal structure of the NTRI homodimer. The three Ig domains belonging to each monomer are visible through the semitransparent surface. Dotted double arrows show measurements in Å. B – Top view of the Ig1-Ig1 interface of NTRI showing the hydrophobic residues in the center of the image and salt bridge between the two protomers (D80 and R65). C – Surface conservation map to identify the biologically-relevant dimer – Top, the biological dimeric interface (i) is identified inside the square where most of the residues are conserved (purple). Arrows indicate conserved junctions between Ig domains. Bottom, surface of the artefactual crystal dimer (ii) contains mostly variable residues (white to blue). D – Overlay of two distinct SAXS models obtained by ab initio reconstructions (green and beige bead representation) with the NTRI crystal structure (red and pink, surface representation) to highlight the similarity between model pairs.

Figure 5 –. Superimposed IgLON dimers –

A – Overall superimposition of all the IgLON dimeric crystal structures, in cartoon representation. B – Cα-traces of superimposed Ig1-Ig1 dimeric assemblies as observed in the different crystals. C – Superimposition of Ig1 domains of all the monomers, illustrating an overall flexibility of the IgLONs, to highlight their structural and architectural similarities.

Figure 6 –. Determination of the associating domains between IgLON protomers –

A – BLI curves of purified NEGR1 (100 mM) interacting with NTRI-F_c or its shorter constructs immobilized on a sensor tip, highlighting the associating deletion constructs (Ig1-F_c, Ig1–2-F_c). B – IgLON5 (195 μM) was used to test the binding of four NTRI-F_c mutants. C – The same NTRI mutants were tested by using 100 μM of purified NEGR1. D – Relative expression of the NTRI-F_c mutants in the cell culture medium. As expected, the introduction of an N-linked glycosylation site (T114N) causes a slightly slower migration of the mutant (white arrowhead).

Figure 7 –. ELISA apparent affinity of all IgLONs –

A – Table of apparent affinities among all IgLON combinations and five representative titrations used to calculate these affinities. Numbers on top of the plates (0 to 500) indicate each FLAG purified IgLON-F_c concentration (nM) used in the specific column. B – Titration binding curves used to calculate the affinities reported in A. For clarity, only the first part of the curve is shown, typically to 70 nM to 250 nM concentrations. C – Coomassie blue staining of the five IgLON-F_c purified proteins used as prey. D – Alignment of the five human IgLON sequences at the Ig1-Ig1 interface region (numbering using NTRI sequence). Conserved residues are in red and group similarities are in pink. Using the structures determined in this study (sequence names in blue), interface-buried residues were computed using PDBePISA and boxed. They fall into two categories: a strongly conserved area (green) that include the key interface residues described in Figure 4b (blue font) and a loosely conserved area (cyan). The sequence alignment was done using ESPript 3 (Robert and Gouet, 2014).