Role of the butyrophilin juxtamembrane region in ligand binding and T cell activation

Abstract

Abstract The B7 protein butyrophilin 3A1 (BTN3A1) is an immune receptor for pathogen-derived small isoprenoid diphosphates such as (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP). Binding to these ligands promotes a multifaceted immune response including activation of Vγ2Vδ2 T cells. Here, we use small-angle x-ray scattering (SAXS) to visualize the internal domain of BTN3A1 as it undergoes a conformational change following ligand binding. To better understand the cause of the conformational change, we used nuclear magnetic resonance (NMR) spectroscopy to determine that the juxtamembrane region is important to BTN3A1 activation by HMBPP. The spectral location of the affected amide peaks, partial NMR assignments, and juxtamembrane mutants (ST297AA or T304A) investigated confirm that the backbone amide of at least one threonine residue (T304), adjacent to conserved serine, comes close to HMBPP, while double mutation of non-conserved residues (ST297) perturbs the local fold of the protein. Threonine residue mutation reduces the ability of BTN3A1 in K562 cells to trigger activation of primary human Vγ2Vδ2 T cells in response to HMBPP. Our results show that ligand binding to BTN3A1 induces a conformational change within the intracellular domain that is mediated by the juxtamembrane region and required for activation. These results provide insight into the unique activation mechanism of butyrophilin-type immune receptors.