Engineering 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. It is common to engineer membrane proteins for cryo-EM analysis. Here, the authors report a cryo-EM structure of an engineered, functional VMAT2 with a non-canonical fold. Caution is advised when interpreting engineered membrane protein structures.