A Candidate Ion-Retaining State in the Inward-Facing Conformation of Sodium/Galactose Symporter: Clues from Atomistic Simulations

Bisha, Ina; Laio, Alessandro; Magistrato, Alessandra; Giorgetti, Alejandro; Sgrignani, Jacopo

doi:10.1021/ct3008233

Cited by 26 publications

(34 citation statements)

References 61 publications

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“…Na + binding is predicted to involve H bonds with carbonyl oxygens of A76, I79, and S389 (bond length <4 Å) and the side chain hydroxyl (OH − ) from S392 (bond length 5 Å). The Na + bond with D204 is predicted to occur via a water bridge (35). No Na + bonding is shown to the side chain of S393 in accordance with the experimental data.…”

Section: Resultssupporting

confidence: 74%

Functional identification and characterization of sodium binding sites in Na symporters

Loo

Jiang

Gorraitz

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Sodium cotransporters from several different gene families belong to the leucine transporter (LeuT) structural family. Although the identification of Na + in binding sites is beyond the resolution of the structures, two Na + binding sites (Na1 and Na2) have been proposed in LeuT. Na2 is conserved in the LeuT family but Na1 is not. A biophysical method has been used to measure sodium dissociation constants (K d ) of wild-type and mutant human sodium glucose cotransport (hSGLT1) proteins to identify the Na + binding sites in hSGLT1. The Na1 site is formed by residues in the sugar binding pocket, and their mutation influences sodium binding to Na1 but not to Na2. For the canonical Na2 site formed by two -OH side chains, S392 and S393, and three backbone carbonyls, mutation of S392 to cysteine increased the sodium K d by sixfold. This was accompanied by a dramatic reduction in the apparent sugar and phlorizin affinities. We suggest that mutation of S392 in the Na2 site produces a structural rearrangement of the sugar binding pocket to disrupt both the binding of the second Na + and the binding of sugar. In contrast, the S393 mutations produce no significant changes in sodium, sugar, and phlorizin affinities. We conclude that the Na2 site is conserved in hSGLT1, the side chain of S392 and the backbone carbonyl of S393 are important in the first Na + binding, and that Na + binding to Na2 promotes binding to Na1 and also sugar binding.I on coupled symporters, or cotransporters, such as hSGLT1 use electrochemical potential gradients to drive solutes into cells. A common finding for these transporters is that external Na + binds before the substrate. Na + binding induces a conformational change of the protein, resulting in the substrate vestibule becoming open to the external membrane surface. After substrate binding, the two ligands are transported across the membrane and are released into the cytoplasm. The atomic structures of several sodium dependent transporters have been solved [leucine transporter (LeuT), vibrio parahaemolyticus sodium glucose (vSGLT), sodium hydantoin (Mhp1), and sodium betaine (BetP)] (1-6). They share a common structural fold with a five-helix inverted repeat, the "LeuT fold". The substrate binding sites are located in the middle of the protein, isolated from the external and membrane surfaces by hydrophobic gates, and putative Na + sites have been identified. Testing these binding sites is a problem due to the fact that phenomenological kinetic constants for ligand transport (K 0.5 , half-saturation values) are interdependent on each other (7). Here we have developed a method to estimate the intrinsic Na + dissociation constants (K d ) for human sodium glucose cotransport (hSGLT1) that may be broadly applicable to other symporters. We then use this to investigate the importance of hSGLT1 residues predicted to be at or near the two Na + binding sites, Na1 and Na2.The method is based on the fact that voltage-dependent membrane proteins exhibit transient charge movements (capacitive transients) i...

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Section: Resultssupporting

confidence: 74%

Functional identification and characterization of sodium binding sites in Na symporters

Loo

Jiang

Gorraitz

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…2 and 4; thus, the presence of this transmembrane helix alone is not sufficient to account for increased Na + stability as demonstrated by our results in its absence. Meanwhile, Bisha et al (23) carried out a set of metadynamics simulations to reveal local rearrangements within Na2 that increased Na + coordination resulting in longer dwell time up to 70 ns. Our work suggests that the Na + ion is occasionally well coordinated by multiple protein residues in Na2, but these direct interactions are not required to maintain an occupied binding site.…”

Section: Resultsmentioning

confidence: 99%

Stochastic steps in secondary active sugar transport

Adelman

Ghezzi

Bisignano

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Secondary active transporters, such as those that adopt the leucinetransporter fold, are found in all domains of life, and they have the unique capability of harnessing the energy stored in ion gradients to accumulate small molecules essential for life as well as expel toxic and harmful compounds. How these proteins couple ion binding and transport to the concomitant flow of substrates is a fundamental structural and biophysical question that is beginning to be answered at the atomistic level with the advent of high-resolution structures of transporters in different structural states. Nonetheless, the dynamic character of the transporters, such as ion/substrate binding order and how binding triggers conformational change, is not revealed from static structures, yet it is critical to understanding their function. Here, we report a series of molecular simulations carried out on the sugar transporter vSGLT that lend insight into how substrate and ions are released from the inward-facing state of the transporter. Our simulations reveal that the order of release is stochastic. Functional experiments were designed to test this prediction on the human homolog, hSGLT1, and we also found that cytoplasmic release is not ordered, but we confirmed that substrate and ion binding from the extracellular space is ordered. Our findings unify conflicting published results concerning cytoplasmic release of ions and substrate and hint at the possibility that other transporters in the superfamily may lack coordination between ions and substrate in the inward-facing state.T ransport of sugar molecules across membranes is virtually ubiquitous in biology, and it is of central importance in human health. The uptake of glucose is especially crucial due to its pivotal role in cellular metabolism and energy production. In mammals, glucose is absorbed in the small intestine and kidney via sodium-dependent glucose transporters (SGLTs), which localize to the apical membrane and concentrate glucose in the epithelia. SGLTs fall into the large leucine-transporter (LeuT) structural family of secondary active transporters that have evolved to concentrate a wide array of substrates across membranes using the energy stored in the Na + electrochemical potential gradient. For symporters in this family, transport occurs by an alternating access mechanism (1) in which the transporter first binds ligands in an outward-facing conformation, and then transitions to an inwardfacing conformation that releases the cargo to the cytoplasm. The order of ion and substrate binding and unbinding is likely tied to the function of the transporter, making it possible to convert the energy stored in the ion gradient into a substrate gradient, and vice versa when these proteins operate in reverse.Extensive biochemical uptake assays and electrophysiological studies of SGLTs have led to a view of Na + /glucose cotransport in which Na + binding precedes sugar binding on the external face of the transporter. Kinetic models adhering to this mechanism satisfactorily account fo...

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“…4C–D). Asn309 is at the equivalent position of the key cation-substituting group Arg262 in a sodium-independent antiporter, CaiT 19 , and is one turn above the position equivalent to Asp189 in vSGLT, which is thought to interact with the Na2 ion in that transporter 20, 21 . We tested the importance of this residue for BetP by replacing Asn309 with alanine.…”

Section: Resultsmentioning

confidence: 99%

“…Indeed, solvated, meta-stable sites appear to be a common theme for ion-coupled transporters, and been identified during MD simulations and free energy calculations of vSGLT 20, 21 and Mhp1 24 , as well as LeuT 25 . The important role of individual water molecules binding sodium within transporter proteins is underlined by the ion-water coordination observed in a recent structure of Drosophila melanogaster dopamine transporter (dDAT) 26 , as well as in c-subunits of ATP-synthase rotor rings 27 .…”

Section: Discussionmentioning

confidence: 99%

Substrate-bound outward-open state of the betaine transporter BetP provides insights into Na+ coupling

et al. 2014

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The Na+-coupled betaine symporter BetP shares a highly conserved fold with other sequence unrelated secondary transporters, e.g., with neurotransmitter symporters. Recently, we obtained atomic structures of BetP in distinct conformational states, which elucidated parts of its alternating access mechanism. Here, we report for the first time a structure of BetP in a new outward-open state in complex with an anomalous scattering substrate, adding a fundamental piece to an unprecedented set of structural snapshots for a secondary transporter. In combination with molecular dynamics simulations these structural data highlight important features of the sequential formation of the substrate and sodium-binding sites, in which coordinating water molecules play a crucial role. We observe a strictly interdependent binding of betaine and sodium ions during the coupling process. All three sites undergo progressive reshaping and dehydration during the alternating-access cycle, with the most optimal coordination of all substrates found in the closed state.

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A Candidate Ion-Retaining State in the Inward-Facing Conformation of Sodium/Galactose Symporter: Clues from Atomistic Simulations

Cited by 26 publications

References 61 publications

Functional identification and characterization of sodium binding sites in Na symporters

Functional identification and characterization of sodium binding sites in Na symporters

Stochastic steps in secondary active sugar transport

Substrate-bound outward-open state of the betaine transporter BetP provides insights into Na+ coupling

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