Exploring the transport cycle of DgoT, a bacterial homolog of human vesicular glutamate transporters.

Abstract

The solute carrier 17 (SLC17) family belongs to the major facilitator superfamily (MFS) and encompasses vesicular glutamate transporters (VGLUTs), inorganic phosphate transporters, the lysosomal H+/sialic acid cotransporter sialin, and the vesicular nucleotide transporter (VNUT). SLC17 members mediate symport or exchange of various substrates with H+ at distinct stoichiometries. A bacterial D-galactonate transporter (DgoT) from E. coli shows high sequence similarity with mammalian VGLUTs and sialin. We used a combination of experimental and computational approaches to explore DgoT transport mechanism. In solid-supported membrane (SSM)-based electrophysiology, rapid galactonate application triggers a transient current generated by substrate binding and subsequent conformational changes, followed by a slower electrogenic transport. Transport current at different pH values demonstrates inhibition of H+ release and binding with apparent pK values of 6.7 and 9.2, respectively. We demonstrated that DgoT functions as galactonate/H+ symporter with 1:2 stoichiometry. There are four charged residues present in the membrane spanning region of DgoT, of which three (R47, R126 and E133) are conserved among SLC17 family. Mutations D46N, E133Q, R47Q and R126Q lead to loss of transport activity, while selective binding of galactonate remains intact. Analysis of pre-steady currents indicates that D46 and E133 are involved in proton translocation, while R47 and R126 are necessary for conformational changes. In all-atom molecular dynamics simulations, protonation of D46 and E133 leads to rearrangement of transmembrane (TM) helices TM1 and TM7 that gate access to the substrate-binding site from the extracellular side. Substrate binding then induces the closure of the periplasmic gate and transition to the inward-open conformation, where substrate is released, in agreement with the alternating access mechanism used by other MFS transporters. Based on our experimental and computational results, we propose a detailed transport model for galactonate/H+ cotransport in DgoT.