The butyrophilin 3A1 intracellular domain undergoes a conformational change involving the juxtamembrane region

Small isoprenoid diphosphates, such as (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP), are ligands of the internal domain of BTN3A1. Ligand binding in target cells promotes activation of V gamma 9V delta 2 T cells. We demonstrate by small-angle X-ray scattering (SAXS) that HMBPP binding to the internal domain of BTN3A1 induces a conformational change in the position of the B30.2 domain relative to the juxtamembrane (JM) region. To better understand the molecular details of this conformational rearrangement, NMR spectroscopy was used to discover that the JM region interacts with HMBPP, specifically at the diphosphate. The spectral location of the affected amide peaks, partial NMR assignments, and JM mutants (ST(296)AA or T(304)A) investigated, confirm that the backbone amide of at least one Thr (Thr(304)), adjacent to conserved Ser, comes close to the HMBPP diphosphate, whereas double mutation of nonconserved residues (Ser/Thr(296/297)) may perturb the local fold. Cellular mutation of either of the identified Thr residues reduces the activation of V gamma 9V delta 2 T cells by HMBPP, zoledronate, and POM2-C-HMBP, but not by a partial agonist BTN3 antibody. Taken together, our results show that ligand binding to BTN3A1 induces a conformational change within the intracellular domain that involves the JM region and is required for full activation.