Membrane potential accelerates sugar uptake by stabilizing the outward facing conformation of the Na/glucose symporter vSGLT (original) (raw)
Related papers
Science, 2008
Membrane transporters that use energy stored in sodium gradients to drive nutrients into cells constitute a major class of proteins. We report the crystal structure of a member of the solute sodium symporters (SSS), the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT). Thẽ 3.0Å structure contains 14 transmembrane helices in an inward facing conformation with a core structure of inverted repeats of 5 TM helices (TM2-TM6 and TM7-TM11). Galactose is bound in the center of the core, occluded from the outside solutions by hydrophobic residues. Surprisingly, the architecture of the core is similar to the leucine transporter (LeuT) from a different gene family. Modeling the outward-facing conformation based on the LeuT structure, in conjunction with biophysical data, provides insight into structural rearrangements for active transport.
Conformational transitions of the sodium-dependent sugar transporter, vSGLT
Proceedings of the National Academy of Sciences of the United States of America, 2018
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 do...
Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, 2008
Membrane transporters that use energy stored in sodium gradients to drive nutrients into cells constitute a major class of proteins. We report the crystal structure of a member of the solute sodium symporters (SSS), the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT). Thẽ 3.0Å structure contains 14 transmembrane helices in an inward facing conformation with a core structure of inverted repeats of 5 TM helices (TM2-TM6 and TM7-TM11). Galactose is bound in the center of the core, occluded from the outside solutions by hydrophobic residues. Surprisingly, the architecture of the core is similar to the leucine transporter (LeuT) from a different gene family. Modeling the outward-facing conformation based on the LeuT structure, in conjunction with biophysical data, provides insight into structural rearrangements for active transport. A central question in biology is how energy is harnessed to do work. For the active accumulation of glucose into cells, Crane proposed that energy was obtained from the inward Na + gradient, i.e. Na + /glucose cotransport (symport) (1). This hypothesis has been extensively tested, refined and expanded to include the active transport of solutes and ions in virtually all cell types (2). It is now established that cells maintain a low intracellular [Na + ] through the active pumping of Na + out of the cell. This inward Na + gradient, along with a negative membrane potential, drives the transport of substrates into cells. Despite this paradigm, the structural basis for Na + solute symport is unknown. Solute Sodium Symporters (SSS) (TC# 2.A.21) are a large family of proteins that cotransport Na + with sugars, amino acids, inorganic ions or vitamins (3). Members of this family are important in human physiology and disease where mutations in glucose and iodide symporters (SGLT1 and NIS) result in the congenital metabolic disorders glucosegalactose-malabsoption (GGM) and iodide transport defect (ITD) (4, 5). SGLT1 is the rationale for oral rehydration therapy and SGLTs are currently being targeted in drug trials for type II diabetes. The first member of the SSS family to be cloned was the intestinal Na + /glucose symporter (SGLT1) (6) and since then over 250 other members have been identified across all six kingdoms of life. The functions of the family members have been well characterized, including the mammalian glucose (SGLTs), the iodide (NIS), the Vibrio parahaemolyticus
Cell Biochemistry and Biophysics, 2012
Current advances in structural biology provide valuable insights into structure-function relationship of membrane transporters by solving crystal structures of bacterial homologs of human transporters. Therefore, scientists consider bacterial transporters as useful structural models for designing of drugs targeted in human diseases. The functional homology between Vibrio parahaemolyticus Na ? /galactose transporter (vSGLT) and Na ? /glucose cotransporter SGLT1 has been well established a decade ago. Now the crystal structure of vSGLT is considered quite valuable in explaining not only the cotransport mechanisms, but it also acts as a representative protein in understanding the protein stability and amino acid interactions within the core structure. We investigated the molecular mechanisms of genetic variations in SGLT1 that cause glucose-galactose malabsorption (GGM) defects using the crystal structure of vSGLT as a model sugar transporter. Our in silico mutagenesis and modeling analysis suggest that the GGM genetic variations lead to conformational changes either by structure destabilization or by formation of unnecessary interaction within the core structure of SGLT1 thereby explaining the genetic defects in Na ? dependent sugar translocation across the cell membrane.
Transmembrane IV of the high-affinity sodium-glucose cotransporter participates in sugar binding
AJP: Cell Physiology, 2008
Investigation of the structure/function relationships of the sodium-glucose transporter (SGLT1) is crucial to understanding the cotransporter mechanism. In the present study, we used cysteine-scanning mutagenesis and chemical modification by methanethiosulfonate (MTS) derivatives to test whether predicted transmembrane IV participates in sugar binding. Five charged and polar residues (K139, Q142, T156, K157, and D161) and two glucose/galactose malabsorption missense mutations (I147 and S159) were replaced with cysteine. Mutants I147C, T156C, and K157C exhibited sufficient expression to be studied in detail using the two-electrode voltage-clamp method in Xenopus laevis oocytes and COS-7 cells. I147C was similar in function to wild-type and was not studied further. Mutation of lysine-157 to cysteine (K157C) causes loss of phloridzin and α-methyl-d-glucopyranoside (αMG) binding. These functions are restored by chemical modification with positively charged (2-aminoethyl) methanethiosulf...
Structure, 2016
The phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTS) are found in bacteria, where they play central roles in sugar uptake and regulation of cellular uptake processes. Little is known about how the membrane-embedded components (EIICs) selectively mediate the passage of carbohydrates across the membrane. Here we report the functional characterization and 2.55 Å resolution structure of a maltose transporter, bcMalT, belonging to the Glucose superfamily of EIIC transporters. bcMalT crystallized in an outward-facing occluded conformation, in contrast to the structure of another Glucose superfamily EIIC, bcChbC, which crystallized in an inwardfacing occluded conformation. The structures differ in the position of a structurally conserved substrate-binding domain that is suggested to play a central role in sugar transport. Additionally, molecular dynamics simulations suggest a potential pathway for substrate entry from the periplasm into the bcMalT substrate-binding site. These results provide a mechanistic framework for understanding substrate recognition and translocation for the Glucose superfamily EIIC transporters.
Membrane Topology of the Human Na /Glucose Cotransporter SGLT1
The membrane topology of the human Na /glucose cotransporter SGLT1 has been probed using N-glycosyl-ation scanning mutants and nested truncations. Functional analysis proved essential for establishment of signal anchor topology. The resultant model diverges significantly from previously held suppositions of structure based primarily on hydropathy analysis. SGLT1 incorporates 14 membrane spans. The N terminus resides extracellularly, and two hydrophobic regions form newly recognized membrane spans 4 and 12; the large charged domain near the C terminus is cytoplasmic. This model was evaluated further using two advanced empirically-based algorithms predictive of transmem-brane helices. Helix ends were predicted using thermo-dynamically-based algorithms known to predict x-ray crystallographically determined transmembrane helix ends. Several considerations suggest the hydrophobic C terminus forms a 14th transmembrane helix, differentiating the eukaryotic members of the SGLT1 family from bacterial homologues. Our data inferentially indicate that these bacterial homologues incorporate 13 spans, with an extracellular N terminus. The model of SGLT1 secondary structure and the predicted helix ends signify information prerequisite for the rational design of further experiments on structure/function relationships.
PLoS computational biology, 2014
Sodium-Galactose Transporter (SGLT) is a secondary active symporter which accumulates sugars into cells by using the electrochemical gradient of Na+ across the membrane. Previous computational studies provided insights into the release process of the two ligands (galactose and sodium ion) into the cytoplasm from the inward-facing conformation of Vibrio parahaemolyticus sodium/galactose transporter (vSGLT). Several aspects of the transport mechanism of this symporter remain to be clarified: (i) a detailed kinetic and thermodynamic characterization of the exit path of the two ligands is still lacking; (ii) contradictory conclusions have been drawn concerning the gating role of Y263; (iii) the role of Na+ in modulating the release path of galactose is not clear. In this work, we use bias-exchange metadynamics simulations to characterize the free energy profile of the galactose and Na+ release processes toward the intracellular side. Surprisingly, we find that the exit of Na+ and galact...