Conformational Dynamics of hSGLT1 during Na+/Glucose Cotransport

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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...

Section: Resultsmentioning

confidence: 81%

Section: Resultsmentioning

confidence: 93%

Stochastic steps in secondary active sugar transport

Adelman

Ghezzi

Bisignano

et al. 2016

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“…However, it is not generally appreciated that the K 0.5 for Na + and the K 0.5 for substrate are interdependent, and changes in Na + K 0.5 may actually be due to changes in the substrate K 0.5 and vice versa (7,23). New methods are needed to determine the K d of ligand binding to cotransporters in the absence of the cosubstrate, and we have developed a method to determine the Na + K d in electrogenic cotransporters.…”

Section: Resultsmentioning

confidence: 99%

“…The results provide an insight into the voltage dependence of hSGLT1 in that the ligand-free transporter has an apparent valence of 0.7, and the binding of extracellular Na + ions is associated with a total charge of 0.3. Originally, we assumed that the valence of the ligand-free protein was 2, as two Na + bind in the absence of glucose (17,19,23). This led to unaccounted discrepancies between models and the observed shift of V 0.5 with varying [Na + ] o and we had attributed this to a simplifying assumption about simultaneous binding of two external Na + ions (22,23).…”

Section: Resultsmentioning

confidence: 99%

Functional identification and characterization of sodium binding sites in Na symporters

Loo

Jiang

Gorraitz

et al. 2013

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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...

“…Although we speculate, based on our results, that the role of the conserved Na2 site is to stabilize substrate binding without conformational selection, analysis of other Na + -coupled transporters is needed to test this conjecture. Kinetic and conformational investigations of putative LeuT-fold members, hSGLT and PutP, have begun to tease out such details (23,36,37). Such analysis will also test a similarly tantalizing notion that subclasses of ion-coupled LeuT-fold transporters, defined by their transport modes and/or type and number of symported ions, share commonalities in their structural mechanics of alternating access.…”

Section: Discussionmentioning

confidence: 99%

Conformational cycle and ion-coupling mechanism of the Na ⁺ /hydantoin transporter Mhp1

Kazmier

Sharma

Islam

et al. 2014

147

Ion-dependent transporters of the LeuT-fold couple the uptake of physiologically essential molecules to transmembrane ion gradients. Defined by a conserved 5-helix inverted repeat that encodes common principles of ion and substrate binding, the LeuTfold has been captured in outward-facing, occluded, and inwardfacing conformations. However, fundamental questions relating to the structural basis of alternating access and coupling to ion gradients remain unanswered. Here, we used distance measurements between pairs of spin labels to define the conformational cycle of the Na + -coupled hydantoin symporter Mhp1 from Microbacterium liquefaciens. Our results reveal that the inward-facing and outward-facing Mhp1 crystal structures represent sampled intermediate states in solution. Here, we provide a mechanistic context for these structures, mapping them into a model of transport based on ion-and substrate-dependent conformational equilibria. In contrast to the Na + /leucine transporter LeuT, our results suggest that Na + binding at the conserved second Na + binding site does not change the energetics of the inward-and outward-facing conformations of Mhp1. Comparative analysis of ligand-dependent alternating access in LeuT and Mhp1 lead us to propose that different coupling schemes to ion gradients may define distinct conformational mechanisms within the LeuT-fold class.EPR | DEER | Na + -coupled symport | conformational dynamics | transport mechanism