A vast repertoire of Dscam binding specificities arises from modular interactions of variable Ig domains

Abstract

Dscam encodes a family of cell surface proteins required for establishing neural circuits in Drosophila. Alternative splicing of Drosophila Dscam can generate 19,008 distinct extracellular domains containing different combinations of three variable immunoglobulin domains. To test the binding properties of many Dscam isoforms, we developed a high-throughput ELISA-based binding assay. We provide evidence that 95% (>18,000) of Dscam isoforms exhibit striking isoform-specific homophilic binding. We demonstrate that each of the three variable domains binds to the same variable domain in an opposing isoform and identify the structural elements that mediate this self-binding of each domain. These studies demonstrate that self-binding domains can assemble in different combinations to generate an enormous family of homophilic binding proteins. We propose that this vast repertoire of Dscam recognition molecules is sufficient to provide each neuron with a unique identity and homotypic binding specificity, thereby allowing neuronal processes to distinguish between self and nonself.

Figure 1. An ELISA-Based Assay for Dscam Binding Specificity

(A) The Dscam gene contains four blocks of alternative exons coding for the first halves of Ig2 (red) and Ig3 (blue), all of Ig7 (green), and the transmembrane region (yellow). All isoforms have the same domain structure. Horseshoes represent Ig domains and black rectangles represent fibronectin type III domains. (B) Two models for preferential homophilic binding are shown. Previous studies(Wojtowicz et al., 2004), data presented here (see Figure 3) and the crystal structure by Meijers et al (Meijers et al., 2007) support the modular model for homophilic binding. In the lower part of the panel, we schematically represent how we envision modular binding occurs. (C) Schematic of ELISA-based binding assay to examine binding between ectodomains. Binding between Dscam ectodomain fused to AP (“receptor”) and Dscam ectodomain fused to Fc (“ligand”) is tested. Dscam-AP receptor is captured onto the plate by an anti-AP antibody. The binding of Dscam-Fc ligand to the receptor is detected by an anti-Fc antibody conjugated to horseradish peroxidase (HRP). HRP activity is measured using a colorimetric assay as a direct readout of binding between ligand and receptor (see Experimental Procedures).

Figure 2. Testing All Dscam Variable Ig Domain Binding Specificities

(A) Three sets of Dscam isoforms were generated as both AP and Fc fusions for the ELISA-based binding assay. Ig3.11, believed to be a pseudo exon, was not tested. (B-D) Binding properties of all Ig2, Ig3 and Ig7 variable domains. Each set of isoforms was tested for binding to all other members of the set. Variable Ig domains are arranged on the grid axes according to their sequence relatedness as shown in the dendrograms. Binding is indicated as fold over background by a color scale and the number in each block. The unrelated control isoform 1.30.30 (denoted “C”) was used to provide a value for background binding. The levels of all AP and Fc proteins were normalized (see Experimental Procedures). The average results of duplicate experiments are shown. (E) Summary of variable domain binding experiments showing the number of Ig2, Ig3 and Ig7 variable domains that exhibit preferential self-binding. From the self-binding properties of these domains, the number of preferential homophilic binding proteins encoded by the Dscam gene is estimated.

Figure 3. Modular Variable Ig Domain Interactions Give Rise to Homophilic Binding Specificity

(A) According to the modular model, the binding properties of each variable domain are independent of the identity of the other two variable domains. Therefore, domains should exhibit the same binding properties (i.e. the same pattern of homophilic and heterophilic binding) regardless of the identity of the other two variable domains (i.e. context). Different Ig7 variants are represented by different shapes and different shades of green. Different Ig2 and Ig3 variants are denoted by different shades of gray and different shapes. (B) The binding properties of a subset of Ig2, Ig3 and Ig7 variable domains were tested in two different contexts. The data in the left hand column (i.e. context #1) were taken from the grids in Figure 2. Binding is indicated as fold over background by a color scale and the number in each block. In context #1 experiments, the context #2 isoform was used as a negative control (denoted “C”) for binding and vice versa. The average results of duplicate experiments are shown.

Figure 4. Characterization of the Ig2 and Ig3 Specificity Elements

(A) Experimental design to assess variable Ig domain specificity determinants. (B) Spacefill models of wild type variable Ig2 domains and A′ β-strand swapped Ig2 domains are shown. Different Ig2 variants are represented by different shades of red. The sequence alignment shows the A′ β-strand sequences (residues 105-114) for these Ig2 domains. The binding properties of isoforms containing wild type and strand-swapped Ig2 domains were tested. Binding is indicated as fold over background by a color scale and the number in each block. The unrelated control isoform 1.30.30 (denoted “C”) was used to provide a value for background binding. The average results of duplicate experiments are shown. Many additional strand swaps (>10) show the same results (see Figure S3). (C) Spacefill models of wild type variable Ig3 domains and A-A′ segment swapped Ig3 domains are shown. Different Ig3 variants are represented by different shades of blue. The sequence alignment shows the A-A′ segment sequences (residues 214-224) for these Ig3 domains. Other segment-swapped Ig3 domains also show swapped binding specificity (see Figure S4). Specificity strands predicted according to Ig2.1 and Ig3.34 interfaces in the Ig1-4 structure (Meijers et al., 2007).

Figure 5. The Specificity Element in Ig7 Comprises Multiple β-Strands

(A) Spacefill model of Ig7.25 generated using homology fold recognition servers (i.e. PHRYE, FUGUE, and ESyPred3D) showing two candidate specificity-determining residues (purple and orange) located on adjacent β-strands. A key shows the identity of these two residues in each wild type and residue-swapped Ig7 domain. Different Ig7 variants are represented by different shades of green. Residue 23 was swapped between Ig7.25 and Ig7.26 (note that these Ig7 variants engage in low levels of heterophilic binding with each other). Residue 61 was swapped between Ig7.20 and Ig7.26. Residues 23 and 61 were swapped between Ig7.20 and Ig7.25. The binding properties of isoforms containing wild-type and residue-swapped Ig7 domains were tested. Binding is indicated as fold over background by a color scale and the number in each block. The unrelated control isoform 1.30.30 (denoted “C”) was used to provide a value for background binding. The average results of duplicate experiments are shown. (B) Distribution of binding modes resulting after symmetrical protein-protein docking of homology models of Ig7.25. Ten different models generated using Rosetta (Das et al., 2007) are used as a starting point for symmetrical docking (Andre et al., 2007). The lowest energy models from each docking experiment are collected and the position of Cα atoms of residue 23 (purple) and 61 (orange) (in the center of the proposed binding region) in one monomer are shown. Residue 63 (blue) is included as a reference point. Left panel, Unconstrained docking models. Middle panel, Docking models constrained using a distance constraint of 8 Å between residue 61 in both monomers. Right panel, Docking models additionally filtered to include models in which residue 23 is also within 8.0 Å of itself in the other monomer. (C) Two docking models for Ig7.25 are shown. One Ig7.25 monomer is shown in cartoon (cyan) and the other is shown in spacefill (green). Two images on the right show the corresponding specificity-determining residues 23 and 61 at the center of the interface of the Ig7.25 monomers in both docking models.