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Conformational transitions of the sodium-dependent sugar transporter, vSGLT

Aviv Paz,D. P. Claxton,J. P. Kumar,Kelli N Kazmier,P. Bisignano,Shruti Sharma,Shannon A. Nolte,Terrin M. Liwag,V. Nayak,E. M. Wright,M. Grabe,H. Mchaourab,J. Abramson

Published 2018 in Proceedings of the National Academy of Sciences of the United States of America

ABSTRACT

Significance Transporters isomerize between conformations to shuttle cargo across membranes, but the mechanism is not understood. Double electron–electron resonance measurements on the sodium-dependent sugar transporter (vSGLT) were used to explore the conformational state of the transporter under specific ligand conditions. Although sugar transport by vSGLT is driven by sodium gradients, vSGLT adopts an inward-open conformation irrespective of the presence of sodium. In the presence of sodium and galactose, the transporter transitions to an occluded conformation. We propose that the cell’s negative membrane potential aids in driving vSGLT toward the outward-facing state to bind sugar and begin the transport cycle. These findings could be applicable to other transporters whereby the inherent cellular membrane potential is integrated into the transport cycle. Sodium-dependent transporters couple the flow of Na+ ions down their electrochemical potential gradient to the uphill transport of various ligands. Many of these transporters share a common core structure composed of a five-helix inverted repeat and deliver their cargo utilizing an alternating-access mechanism. A detailed characterization of inward-facing conformations of the Na+-dependent sugar transporter from Vibrio parahaemolyticus (vSGLT) has previously been reported, but structural details on additional conformations and on how Na+ and ligand influence the equilibrium between other states remains unknown. Here, double electron–electron resonance spectroscopy, structural modeling, and molecular dynamics are utilized to deduce ligand-dependent equilibria shifts of vSGLT in micelles. In the absence and presence of saturating amounts of Na+, vSGLT favors an inward-facing conformation. Upon binding both Na+ and sugar, the equilibrium shifts toward either an outward-facing or occluded conformation. While Na+ alone does not stabilize the outward-facing state, gating charge calculations together with a kinetic model of transport suggest that the resting negative membrane potential of the cell, absent in detergent-solubilized samples, may stabilize vSGLT in an outward-open conformation where it is poised for binding external sugars. In total, these findings provide insights into ligand-induced conformational selection and delineate the transport cycle of vSGLT.

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