Probing the Ligand-Binding Pocket of BTN3A1

Small-molecule
phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl
diphosphate stimulate human Vγ9Vδ2 T cells after binding
to the intracellular B30.2 domain of the immune receptor butyrophilin
3 isoform A1 (BTN3A1). To understand the ligand–target interaction
in greater detail, we performed molecular docking. Based on the docking
results, we synthesized the novel ligand (E)-(7-hydroxy-6-methylhept-5-en-1-yl)­phosphonate
and mutated proposed binding site residues. We evaluated the impact
on butyrophilin binding of existing and novel ligands using a newly
developed high-throughput fluorescence polarization assay. We also
evaluated the ability of the compounds to stimulate proliferation
and interferon-γ production of Vγ9Vδ2 T cells. Mutation
of H381 fully blocked ligand binding, whereas mutations to charged
surface residues impacted diphosphate interactions. Monophosphonate
analogs bind similarly to BTN3A1, although they differ in their antigenicity,
demonstrating that binding and efficacy are not linearly correlated.
These results further define the structure–activity relationships
underlying BTN3A1 ligand binding and antigenicity and support further
structure-guided drug design.