Investigation of sugar binding kinetics of the E. coli sugar/H+ symporter XylE using solid-supported membrane-based electrophysiology

Andre Bazzone,Laura Tesmer,Derya Kurt,H Ronald Kaback,Klaus Fendler,M Gregor Madej

doi:10.1016/j.jbc.2021.101505

Andre Bazzone, Laura Tesmer + Show 4 more

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https://doi.org/10.1016/j.jbc.2021.101505

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Abstract

Bacterial transporters are difficult to study using conventional electrophysiology because of their low transport rates and the small size of bacterial cells. Here, we applied solid-supported membrane–based electrophysiology to derive kinetic parameters of sugar translocation by the Escherichia coli xylose permease (XylE), including functionally relevant mutants. Many aspects of the fucose permease (FucP) and lactose permease (LacY) have also been investigated, which allow for more comprehensive conclusions regarding the mechanism of sugar translocation by transporters of the major facilitator superfamily. In all three of these symporters, we observed sugar binding and transport in real time to determine KM, Vmax, KD, and kobs values for different sugar substrates. KD and kobs values were attainable because of a conserved sugar-induced electrogenic conformational transition within these transporters. We also analyzed interactions between the residues in the available X-ray sugar/H+ symporter structures obtained with different bound sugars. We found that different sugars induce different conformational states, possibly correlating with different charge displacements in the electrophysiological assay upon sugar binding. Finally, we found that mutations in XylE altered the kinetics of glucose binding and transport, as Q175 and L297 are necessary for uncoupling H+ and d-glucose translocation. Based on the rates for the electrogenic conformational transition upon sugar binding (>300 s−1) and for sugar translocation (2 s−1 − 30 s−1 for different substrates), we propose a multiple-step mechanism and postulate an energy profile for sugar translocation. We also suggest a mechanism by which d-glucose can act as an inhibitor for XylE.

Highlights

The exceptionally diverse major facilitator superfamily (MFS), one of the two largest families of membrane transporters found on earth, includes membrane transport proteins from Archaea to Homo sapiens [1, 2]
The imposition of ΔμeH+ does not affect the rates of equilibrium exchange or counterflow with lactose permease (LacY) [8,9,10], suggesting that the conformational change resulting in alternating access of the sugar-binding and H+-binding sites to either side of the membrane is a consequence of sugar binding and dissociation, and not ΔμeH+ [3]
The X-ray crystallographic studies of single-Cys122 LacY with covalently bound MTSGal [11] and the conformationally trapped mutant cocrystallized with β-D-galactopyranosyl-1-thio-β-D-galactopyranoside (TDG) [12] or an α-substituted galactoside [13] as well as the structures of the human MFS uniporters, GLUT1 with βnonylglucoside trapped in the binding site [14] and GLUT3 with bound D-glucose [15], indicate that only a fully liganded substrate affects the transition into the occluded state of the transporter

Summary

Introduction

The exceptionally diverse major facilitator superfamily (MFS), one of the two largest families of membrane transporters found on earth, includes membrane transport proteins from Archaea to Homo sapiens [1, 2]. The imposition of ΔμeH+ does not affect the rates of equilibrium exchange or counterflow with LacY [8,9,10], suggesting that the conformational change resulting in alternating access of the sugar-binding and H+-binding sites to either side of the membrane is a consequence of sugar binding and dissociation, and not ΔμeH+ [3] In this regard, the X-ray crystallographic studies of single-Cys122 LacY with covalently bound MTSGal [11] and the conformationally trapped mutant cocrystallized with β-D-galactopyranosyl-1-thio-β-D-galactopyranoside (TDG) [12] or an α-substituted galactoside [13] as well as the structures of the human MFS uniporters, GLUT1 with βnonylglucoside trapped in the binding site [14] and GLUT3 with bound D-glucose [15], indicate that only a fully liganded substrate affects the transition into the occluded state of the transporter. Together with the kinetic findings, this analysis indicates that the substrates are identified beyond the level of the binding site of the transporter

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