Fig. 5: Proposed model for the mechanistic roles of Ser 246 and Tyr 333 in Asc1 transport.

a In the exchange mode of transport, the substrate (L-alanine) intercalates between Ser 246 and Tyr 333, thus bridging the scaffold (tinted in orange) with the bundle domain (tinted in cyan) of the transporter. Analogously, in the facilitated diffusion mode of transport, Ser 246 and Tyr 333 form a potential hydrogen bond between the side chains. Our results support a mechanism in which the connection between Ser 246 and Tyr 333 triggers the fully occluded conformation in the exchange mode of transport, with the transporter bound to substrate, but also in the diffusion mode, where the transporter cycles back with an empty binding cavity. b Proposed role of Ser 246 and Tyr 333 in facilitating the transition of the substrate to and from the transport-productive binding pose. In this model, the productive pose of the substrate between TM1 and TM6 (left, corresponding to the PELE pose 1 shown in Fig. 3b) would be destabilized by a rotamer of Tyr 333 towards TM6 (right, corresponding to the second MD replicate at 24 ns), which would have the effect of pulling the substrate away from the GSG motif in TM1, a necessary step for the release of the substrate to the cytosol. The side chain of Ser 246 would stabilize the intermediate step (middle, corresponding to the PELE pose 2 shown in Supplementary Fig. 8a), in which Tyr 333 has rotated but not yet re-bound to the substrate and the substrate itself has experimented a ~ 180° rotation. C atoms in Asc1 residues are colored according with the color of the corresponding TMs as in Fig. 1 whereas substrate C atoms are coloured in pink.