biostudies-literatureLyu YSichuan University (SCU)National Natural Science Foundation of ChinaSichuan UniversityNational Natural Science Foundation of China (National Science Foundation of China)6511https://www.ebi.ac.uk/biostudies/studies/S-EPMC11297040biostudies-literatureUnknown15(1)Vesicular monoamine transporter 2 (VMAT2) belongs to the major facilitator superfamily (MFS), and mediates cytoplasmic monoamine packaging into presynaptic vesicles. Here, we present two cryo-EM structures of VMAT2, with a frog VMAT2 adopting a canonical MFS fold and an engineered sheep VMAT2 adopting a non-canonical fold. Both VMAT2 proteins mediate uptake of a selective fluorescent VMAT2 substrate into cells. Molecular docking, substrate binding and transport analysis reveal potential substrate binding mechanism in VMAT2. Meanwhile, caution is advised when interpreting engineered membrane protein structures.Nature communicationsEngineering of a mammalian VMAT2 for cryo-EM analysis results in non-canonical protein folding.PMC11297040317707832023SCUH0067Fu CLyu YMa HSu ZZhou XSun ZfalseEngineering of a mammalian VMAT2 for cryo-EM analysis results in non-canonical protein folding.Vesicular monoamine transporter 2 (VMAT2) belongs to the major facilitator superfamily (MFS), and mediates cytoplasmic monoamine packaging into presynaptic vesicles. Here, we present two cryo-EM structures of VMAT2, with a frog VMAT2 adopting a canonical MFS fold and an engineered sheep VMAT2 adopting a non-canonical fold. Both VMAT2 proteins mediate uptake of a selective fluorescent VMAT2 substrate into cells. Molecular docking, substrate binding and transport analysis reveal potential substrate binding mechanism in VMAT2. Meanwhile, caution is advised when interpreting engineered membrane protein structures.2024-01-01T00:00:00Z2024 Aug2026-05-02T12:42:13.886Z2025-02-19T02:27:30.91ZS-EPMC112970403909542810.1038/s41467-024-50934-5