Investigation of structural and dynamic properties of the Butyrophilin BTN3A1/BTN2A1 cytoplasmic complex by F solution NMR.

Butyrophilin 3A1 (BTN3A1) is an integral membrane protein capable of detecting phosphoantigens, like (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP), through its internal B30.2 domain. Detection of phosphoantigens leads to interactions with butyrophilin 2A1 and the subsequent activation of γδ-T cells. Though crystallography and functional assays have been crucial for determining vital residues of the BTN3A1/HMBPP/BTN2A1 complex, the mechanism for signal transduction is still unclear. Here, we utilize F solution NMR to observe potential conformational and dynamic changes of specific residues upon complex formation. With point mutants of BTN3A1, we show that W421C, T449C, and T506C are residues that are influenced by HMBPP and BTN2A1 association, while T304C, G323C, C387, and C511 are not impacted. F labeling of W421C reduces the binding affinity of BTN2A1 toward BTN3A1/HMBPP, which indicates that W421 is located at the binding interface. T506 is located away from the phosphoantigen binding site, so its observable chemical shift perturbation suggests that there is a larger conformational change of the BTN3A1 B30.2 domain upon binding HMBPP and BTN2A1. The juxtamembrane residues, T304C, and G323C are not affected, showing that the changes are localized within the B30.2 domain of BTN3A1. Using BTN3A1 T449C, we were able to detect differential binding modes of synthetic HMBPP analogs, showing that it is possible to assess differences in protein conformations that are induced by binding of different ligands. Taken together, these findings illustrate the dynamic processes involved in phosphoantigen detection by the HMBPP receptor.