Dimerization of LTβR by LTα1β2 is necessary and sufficient for signal transduction

Abstract

Homotrimeric TNF superfamily ligands signal by inducing trimers of their cognate receptors. As a biologically active heterotrimer, Lymphotoxin(LT)α1β2 is unique in the TNF superfamily. How the three unique potential receptor-binding interfaces in LTα1β2 trigger signaling via LTβ Receptor (LTβR) resulting in lymphoid organogenesis and propagation of inflammatory signals is poorly understood. Here we show that LTα1β2 possesses two binding sites for LTβR with distinct affinities and that dimerization of LTβR by LTα1β2 is necessary and sufficient for signal transduction. The crystal structure of a complex formed by LTα1β2, LTβR, and the fab fragment of an antibody that blocks LTβR activation reveals the lower affinity receptor-binding site. Mutations targeting each potential receptor-binding site in an engineered single-chain variant of LTα1β2 reveal the high-affinity site. NF-κB reporter assays further validate that disruption of receptor interactions at either site is sufficient to prevent signaling via LTβR.

Keywords: biophysics; crystallography; cytokines; mechanism.

Fig. 1.

Anti-LTα binds to a single protomer in LTα₃, and LTα₁β₂ blocks signaling through TNFR2 and LTβR. (A) Anti-LTα mAb and LTβR-Ig block NF-κB activation through LTα₁β₂. HeLa/NF-κB-luc cells endogenously expressing LTβR were stimulated with LTα₁β₂. (B) Anti-LTα mAb and human TNFR2-Ig block NF-κB activation through LTα₃, but LTβR-Ig does not. HeLa/NF-κB-luc reporter cells endogenously expressing TNFR2 were stimulated with WT–LTα₃. In both A and B, NF-κB activity was measured in relative luciferase units; baseline activity in unstimulated cells (no stim, + symbol) and activity in stimulated cells in absence of blockade (x symbol) are indicated. Data are shown as mean ± SD of duplicate wells from duplicate plates. Data are representative of at least two experiments. (C) Anti-LTα mAb and LTβR-Ig cobind to LTα− and LTβ-expressing 293 cells. Surface LTα and LTβ expression on 293-hLTαβ cells is shown using anti-LTα mAb (blue) or LTβR-Ig (red). Light-shaded histogram represents staining with isotype control antibody. Cobinding of both LTα-specific mAb and LTβR-Ig was determined by preincubating 293-hLTαβ cells with LTα-specific mAb or LTβR-Ig followed by staining for surface LT. (D) Crystal structure of the LTα₃–(anti-LTα Fab)₃ complex shows each anti-LTα Fab molecule (gray) recognizing a single protomer within the LTα₃ homotrimer (shades of yellow). (E) Anti-LTα Fab (gray) binding to LTα (blue) induces a conformational change in the DE- and AA’-loops altering positions of residues Y142 and D84 relative to LTα (yellow) in complex with TNFR1 (green) (PDB ID code 1TNR).

Fig. 2.

Representative ITC curves suggest two LTβR binding sites in LTα₁β₂ with distinct affinities. (A) LTβR (200 μM) was titrated into LTα₁β₂ (10 μM). Upper, baseline-corrected power-versus-time plot for the titration. Lower, integrated heats and molar ratios of LTβR binding to LTα₁β₂. The data were corrected for heats of dilution and fit to a two-site binding model. (B) LTβR (150 μM) was titrated into LTα₁β₂–anti-LTα Fab complex (7 μM). Upper, baseline-corrected power-versus-time plot for the titration. Lower, integrated heats and molar ratios of LTβR binding to LTα₁β₂. The data were corrected for heats of dilution and fit to a one-site binding model. The higher affinity site is blocked as a result of anti–LTα-Fab bound to LTα₁β₂. Illustrations next to curves depict the reactions with LTβR being added to either LTα₁β₂ (A) or LTα₁β₂–anti-LTα Fab complex (B) to measure heats of reaction against moles of LTβR added. See SI Materials and Methods for details of experimental setup, curve fitting, and data analysis.

Fig. 3.

Structure of LTα₁β₂–LTβR bound to anti-LTα reveals low-affinity LTβR binding site. (A) Crystal structure of the LTα₁β₂–anti-LTα-LTβR complex. Anti-LTα (gray) is bound to LTα as expected from the structure of LTα₃ bound to anti-LTα Fab as shown in Fig. 1. LTβR (red) is bound at the β–β’ interface (light and dark blue). Side view (Upper), top-down view (Lower). (B) Structural alignment (secondary structure) of LTα (yellow, PDB ID code 1TNR) and LTβ (purple, current work) revealing overall similarities. Tyrosine residues in the DE-loops of either molecule important for receptor binding are shown. (C) Structural alignment (Cα trace) of TNFR1 (green, PDB ID code 1TNR) and LTβR (orange, current work) reveals significant overlap between CRD1 and CRD2 regions.

Fig. 4.

The α–β and β–β’ binding sites are essential for signaling. (A) A representation of the various single-chain variants of LTα₁β₂ generated to identify the high-affinity LTβR binding site in LTα₁β₂. Receptor-binding sites affected in each variant are shown on the right. (B) Size exclusion chromatography on the complexes of LTβR with the single-chain LTα₁β₂ variants suggests that charge reversal substitutions in the α–β and β–β sites disrupt receptor binding as predicted. The data also suggest that the α–β site, not the β’ –α site, is the higher affinity binding site for LTβR. (C) Binding of LTβR to both the α–β and β–β’ sites are required for LTβR signaling. 293T/NF-κB-luc cells (Upper Left), 293T/NF-κB-luc transfected with LTβR (Upper Right), or HeLa/NF-κB-luc cells (Lower) were stimulated with increasing concentrations of WT LTα₁β₂ protein or single-chain variants of LTα₁β₂. Luciferase activity induced by stimulation is shown relative to activity in unstimulated cells. Data are shown as mean ± SD of two independent experiments.