No abstract

An increasing number of solute transporters have been shown to function with the so-called sliding-elevator mechanism. Despite structural and functional differences all elevator-type transporters use a common mechanism of substrate translocation via reversible movements of a mobile core domain (the elevator) hosting the substrate binding site along a rigid scaffold domain stably anchored in the plasma membrane via homodimerization. One of the best studied elevator transporters is the UapA uric acid-xanthine/H+ symporter of the filamentous fungus Aspergillus nidulans. Here, we present a genetic analysis for deciphering the role of transmembrane segments (TMS) 5 and 12 in UapA transport function. We show that specific residues in both TMS5 and TMS12 control, negatively or positively, the dynamics of transport, but also substrate binding affinity and specificity. More specifically, mutations in TMS5 can lead to increased rate of transport, but also to an inactive transporter due to high-affinity substrate-trapping, whereas mutations in TMS12 lead to apparently uncontrolled sliding and broadened specificity, leading in specific cases to UapA-mediated purine toxicity. Our findings shed new light on how elevator transporters function and how this knowledge can be applied to genetically modify their transport characteristics.

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.

An increasing number of solute transporters have been shown to function with the so-called sliding-elevator mechanism. Despite structural and functional differences all elevator-type transporters use a common mechanism of substrate translocation via reversible movements of a mobile core domain (the elevator) hosting the substrate binding site along a rigid scaffold domain stably anchored in the plasma membrane via homodimerization. One of the best studied elevator transporters is the UapA uric acid-xanthine/H+ symporter of the filamentous fungus Aspergillus nidulans. Here, we present a novel genetic analysis for deciphering the role of transmembrane segments (TMS) 5 and 12 in UapA transport function. We show that specific residues in both TMS5 and TMS12 control, negatively or positively, the dynamics of transport, but also substrate binding affinity and specificity. More specifically, mutations in TMS5 can lead to increased rate of transport, but also to an inactive transporter due to high-affinity substrate-trapping, whereas mutations in TMS12 lead to apparently uncontrolled sliding, and thus broadened specificity and UapA-mediated purine toxicity. Our findings shed new light on how elevator transporters function or how their transport characteristics might be altered genetically or have been modified in the course of evolution.