Splice form dependence of beta-neurexin/neuroligin binding interactions

Abstract

Alternatively spliced beta-neurexins (beta-NRXs) and neuroligins (NLs) are thought to have distinct extracellular binding affinities, potentially providing a beta-NRX/NL synaptic recognition code. We utilized surface plasmon resonance to measure binding affinities between all combinations of alternatively spliced beta-NRX 1-3 and NL 1-3 ectodomains. Binding was observed for all beta-NRX/NL pairs. The presence of the NL1 B splice insertion lowers beta-NRX binding affinity by approximately 2-fold, while beta-NRX splice insertion 4 has small effects that do not synergize with NL splicing. New structures of glycosylated beta-NRXs 1 and 2 containing splice insertion 4 reveal that the insertion forms a new beta strand that replaces the beta10 strand, leaving the NL binding site intact. This helps to explain the limited effect of splice insert 4 on NRX/NL binding affinities. These results provide new structural insights and quantitative binding information to help determine whether and how splice isoform choice plays a role in beta-NRX/NL-mediated synaptic recognition.

Figure 1. NRX and NL alternative splicing

(A) NRXs are transcribed from two independent promoters for each gene resulting in the longer α-NRXs and shorter β-NRXs. α-NRXs contain three repeats, each of which consists of an EGF domain flanked by two LNS domains. α-NRX and β-NRXs share the LNS 6 domain, a transmembrane region and a short cytoplasmic domain. The arrows indicate the alternative splicing sites in each molecule. The amino acids sequences for the insert at splice site 4 of each β-NRX are also shown. (B) NLs 1, 2, and 3 contain an extracellular domain, which forms homodimers, a stalk region, transmembrane and cytoplasmic domains. All three NLs undergo splicing at site A and NL1 only can also be spliced at site B, resulting in 10 different NL variants. The amino acid sequences for the inserts introduced as a result of splicing are shown. (C) Structure of a β-NRX1Δ4/NL1Δ complex with β-NRX1Δ4 shown in silver and NL1Δ in yellow (PDB ID 3B3Q) (Chen et al., 2008). The SS4 insertion point for β-NRXs is shaded in magenta, the SSA insertion point for NLs is shaded in orange and the SSB insertion point for NL1 in red.

Figure 2. SPR binding analysis of β-NRXs 1–3 splice variants to NLs 1–3 isoforms

(A) In the SPR binding assay, NL was immobilized to the sensor chip surface (ligand) and increasing concentrations of β-NRX were injected in the solution phase (analyte). Two molecules of β-NRX bind to each dimeric NL. (B–M) β-NRXΔ4 isoforms binding over sensor chip surfaces immobilized with NL isoforms 1Δ, 1A, 1B and 1AB at a concentration range of 8.0-0.039 μM with the exception of panels B and C, where the highest concentration tested was 4 μM. Black traces show the experimental data and red traces show the fit to a 1:1 model with a step to account for mass transport. The K_Ds, along with fold change analyses, are listed in Tables 1, S2-S3, and the kinetic rates are shown in Table S1. (N) K_D values determined by SPR binding analysis for the interaction of the two isoforms of each β-NRX 1, 2 and 3, with the ten different NL isoforms, containing or lacking splice inserts A and for NL1 splice insert B. The values for this plot are listed in Table 1. (O) The K_D values of β-NRX+4 interacting with a given NL, normalized against the K_Ds of the same NL binding to β-NRXΔ4. The fold changes for each β-NRX+4 binding to a certain NL compared to the binding of β-NRXΔ4 are shown in Table S2. (P) A plot of the association rates (k_a) and (Q) a plot of the dissociation rates (k_d) for β-NRX/NL interactions. The values in this plot are shown in Table S1. The association and dissociation rates were used to calculate the K_Ds in (N) using the relationship K_D=k_d/k_a.

Figure 3

Structural rearrangements take place to accommodate the splice insert 4. (A) Superposition of the β-NRX1Δ4 structure (PDB ID 3BOD) in silver and the β-NRX1+4 structure in cyan. The SS4 insert is highlighted in magenta. (B) Superposition of the β-NRX2Δ4 structure (PDB ID 3BOP) in silver and the β-NRX2+4 structure in cyan. The SS4 insert is highlighted in magenta. (C) Superposition of the β-NRX1Δ4 structure (PDB ID 3BOD) in silver and the glycosylated β-NRX3Δ4 structure in green. (D) Superposition of the new β-NRX1+4 structure in cyan with the non-glycosylated β-NRX1+4 structure (PDB ID 2R1B) (Shen et al., 2008) in yellow. The SS4 is shown in red in both structures, highlighting identical sequences adopting completely different secondary structures.

Figure 4

Structural effects of the SS4 insertion on the NL binding site. (A) The β-NRX1+4 structure (cyan) is superposed onto the β-NRX1Δ4 (silver) from the β-NRX1Δ4/NL1Δ complex structure (PDB ID 3BIW). The SS4 insert is shown in magenta in the β-NRX1+4 structure. The electrostatic potential surface is shown for the NL1 molecule in the complex with the indicated color scale. (B) Close-up view of the NL binding interface of β-NRX1Δ4 with the β-NRX1+4 structure superposed. The β-NRX1Δ4 interface residues with a buried surface area greater than 5Ų in the complex structure with NL1 and the same residues in β-NRX1+4 are represented. The colorcoding is the same as in (A). (C) Close-up view of the part of the β10–β11 loop that is the most variable upon SS4 insertion.