The evolution of VMAT2 structural models. Homology modeling proved instrumental in advancing our understanding of VMAT2’s structure, providing a crucial framework that guided many of our biochemical investigations. An initial model was constructed using LacY as a structural template. Approximately a decade later, an updated and more sophisticated model emerged (Yaffe et al., 2013). This second-generation model, developed through aligning VMAT2 with a broader array of homologous MFS transporter structures, offered a significantly more accurate representation of the critical pore-lining helices and the substrate-binding cavity. (A) TM topology of antiporters from the DHA1 family. The structure of the bacterial multidrug transporter YajR and the homology model of BbMAT were aligned to locate the conserved membrane embedded charged residues, which are indicated as ellipses, based on the color-coding shown below. Transparent triangles indicate the location of three-helix structural repeats in the N-terminal (blue and green) and C-terminal halves (pink and yellow) of the MFS fold. (B) Model of rVMAT2 in the lumen-facing conformation indicating the location of membrane embedded charged residues in rVMAT2, and colored by TM helix, with the N-terminal half in shades of blue and green and the C-terminal half in shades of red and yellow (rVMAT2 numbering). (C) Gating residues. Magnification of the cytoplasmic domain of TM5 and TM11. Residues contributing to the cytoplasmic gate are shown as sticks (rVMAT2 numbering). Adapted from Yaffe et al. (2018).