Uracil/H+ symport by the FurE transporter challenges the rocking-bundle mechanism of transport in APC transporters.

Transporters mediate the uptake or efflux of solutes, metabolites and drugs across the cell membrane. The FurE symporter of uracil-allantoin-uric acid/H+ of Aspergillus nidulans has been used as a model eukaryotic transporter to address structure-function relationships and the mechanism of transport in the NCS1/APC family of transporters. Extensive genetic, functional and cellular studies, as well as homology modeling based on a prokaryotic transporter with a similar fold (Mhp1) have provided important information on specific structural elements of FurE. However, the exact mechanism by which substrates and proton or other cations are translocated by FurE or transporters with a similar fold remains elusive. In this study, we reveal the binding mode, translocation and release pathway of uracil/H+ from the extracellular space to the cytoplasm, using novel metadynamics calculations and rationally designed mutational analysis. In particular, Metadynamics Free Energy Surface maps provide the relative order of internalization of uracil and proton, permitting also selection of intermediate conformational states related to transport cycle. Funnel Metadynamics allow the sampling of specific interactions of both uracil and proton with residues during their internalization, generating a holistic model of the transport events in conjunction with experimental mutation studies. Our work not only complements and extends the existing knowledge on FurE same-fold transporters, but also challenges the so-called rocking bundle mechanism associated with biomedically interesting members of the APC superfamily of transporters.