2017 - Variations on the Coupling Theme in Secondary Active Transport

Icon N1   SURFE²R N1 Oral Presentation

Gregor Madej, University of Regensburg, Germany

Nanion User Meeting
October 26.-26., 2018


University of Regensburg, Institute of Biophysics and Physical Biochemistry, Regensburg, Germany Numerous physiological processes, i.e. cellular uptake of glucose or hyperosmotic stress response, require specialized proteins that utilize electrochemical membrane potential as an energy source for substrate transport across the cellular membranes. One fundamentally important problem for understanding the mechanism of such secondary active transport is the relationship between binding of the cargo and the coupling ion. Typical of many secondary active transporters is
their selectivity for the coupling ion. In the symporter BetP from Corynebacterium glutamicum, which is a highly effective osmoregulated uptake system for glycine betaine, transport is coupled to the symport of two Na+ ions. Mutations, which affect residues in the Na1 site, were designed to alter its ion specificity. Here a variant BetP has been generated that couples the charge translocation also with K+. For the unrelated sugar/H+ symporter of the major facilitator superfamily (MFS), it has been shown that the affinity for the coupling ion changes during the transport cycle. In lactose  ermease from E. coli (LacY), the poster child of the MFS, sidechains have been identified modulating the pKa of the sugar-binding. However, these positions are not in direct contact to the sugar- or the H+-binding site, and are not conserved in distant MFS sugar transporters; therefore, mechanistic conclusions are difficult to draw. Here we designed mutations near the H+-
binding site in a glucose/H+ symporter GlcP of Staphylococcus epidermidis to alter the affinity for H+. We show experimental results verifying that polarity modification in the vicinity of the H+-binding site results in pKa modulation of the sugar-induced charge transfer.

M.G. Madej, K. Kossmann and C. Ziegler


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