Identification of glutamine synthetase as a novel contryphan-Bt binding protein by his-tag pull down

(1) BackgroundContryphan-Bt is a D-tryptophan-containing disulfide-constrained decapeptide recently isolated from the venom of Conus betulinus. The molecular targets of contryphans are controversial, and the identification of its interacting proteins may be of great importance. (2) MethodsHis-tag pull down assays were performed to investigate binding proteins of contryphan-Bt from rat brain lysate. Bt-Acp-[His]6, a contryphan-Bt derivative containing hexahistidine tag, was synthesized and used as the bait. As a control, Acp-[His]6 was used to exclude nonspecific bindings. (3) ResultsGlutamine synthetase was identified as a potential contryphan-Bt binding protein by pull down assays and subsequent LC-MS/MS. The binding of contryphan-B to glutamine synthetase was confirmed and determined using microscale thermophoresis, with a Kd of 74.02 {+/-} 2.8 M. The binding did not affect glutamine synthetase activity, suggesting that the interaction site was distinct from the catalytic center. (4) ConclusionsGlutamine synthetase was identified as a novel contryphan-Bt binding protein. This is the first report that the conopeptide binds to an intracellular protein, therefore offering a new concept and methodology for developing peptide toxins. Key ContributionThis is the first report that the conopeptide binds to an intracellular protein, therefore offering a new concept and methodology for developing peptide toxins.