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Molecular Physiology & Biophysics

Conformational transitions of the sodium-dependent sugar transporter, vSGLT.

AUTHORS

Paz A , Claxton DP , Kumar JP , Kazmier K , Bisignano P , Sharma S , Nolte SA , Liwag TM , Nayak V , Wright EM , Grabe M , Mchaourab HS , Abramson J , . Proceedings of the National Academy of Sciences of the United States of America. 2018 3 5; ().

ABSTRACT

Sodium-dependent transporters couple the flow of Naions 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(vSGLT) has previously been reported, but structural details on additional conformations and on how Naand 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 Naand sugar, the equilibrium shifts toward either an outward-facing or occluded conformation. While Naalone 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.

Sodium-dependent transporters couple the flow of Naions 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(vSGLT) has previously been reported, but structural details on additional conformations and on how Naand 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 Naand sugar, the equilibrium shifts toward either an outward-facing or occluded conformation. While Naalone 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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