Fig. 7: Proposed mechanism of spermine transport by hVPAT.

The N- and C-domains of hVPAT are drawn as blue and orange ovals, the spermine- and H⁺-binding protein residues as yellow (deprotonated) or green (protonated) pentagons. R137, spermine, H⁺, membrane bilayer, the inter-domain H-bond and salt bridge are simplified as a red pentagon, red squiggle, green droplet, gray rectangle, and gray zones, respectively. As the spermine-bound hVPAT (state I) becomes protonated, the neutralization of E171 and D255 induce the spermine dissociation, whereas R137 disengages from the protonated D140. Diversion of R137 to the spermine-binding site facilitates the release of spermine from the protonated hVPAT (state II) and discourages substrate rebinding due to charge-charge repulsion. The lumen-facing, protonated hVPAT then switches to the cytosol-facing conformation (state III) and the binding of spermine to E83, E171 and D255 triggers the release of three protons. The binding of spermine to hVPAT (state IV) draws R137 away from the substrate-binding site and induces the formation of salt bridge between R137 and D140, which evokes the release of a fourth proton into the cytosol and completes the transport cycle. Notably, the spermine-bound and low pH structures, which were obtained at pH 8.0 and 4.9, respectively, emulate states I and II, respectively.