Arginine oscillation explains Na
            <sup>+</sup>
            independence in the substrate/product antiporter CaiT

Kalayil, Sissy; Schulze, Sabrina; Kühlbrandt, Werner

doi:10.1073/pnas.1309071110

Cited by 22 publications

(19 citation statements)

References 39 publications

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“…Rather, we hypothesize these two positively-charged residues in exchangers act as permanent surrogates of the Na + ions in cotransporters. Such Na + ion substitutions have previously been observed in other transporters, which makes their substrate transport independent of sodium 37,38 .…”

Section: Structure Determination Of Multiple Statessupporting

confidence: 61%

Structural basis for the reaction cycle of DASS dicarboxylate transporters

Sauer

Trebesch²,

Marden

et al. 2020

eLife

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Citrate, α-ketoglutarate and succinate are TCA cycle intermediates that also play essential roles in metabolic signaling and cellular regulation. These di- and tricarboxylates are imported into the cell by the divalent anion sodium symporter (DASS) family of plasma membrane transporters, which contains both cotransporters and exchangers. While DASS proteins transport substrates via an elevator mechanism, to date structures are only available for a single DASS cotransporter protein in a substrate-bound, inward-facing state. We report multiple cryo-EM and X-ray structures in four different states, including three hitherto unseen states, along with molecular dynamics simulations, of both a cotransporter and an exchanger. Comparison of these outward- and inward-facing structures reveal how the transport domain translates and rotates within the framework of the scaffold domain through the transport cycle. Additionally, we propose that DASS transporters ensure substrate coupling by a charge-compensation mechanism, and by structural changes upon substrate release.

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Section: Structure Determination Of Multiple Statessupporting

confidence: 61%

Structural basis for the reaction cycle of DASS dicarboxylate transporters

Sauer

Trebesch²,

Marden

et al. 2020

eLife

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“…A mechanism where arginine works as an internal cation replacing Na + has previously been revealed for a carnitine transporter (CaiT), which is closely related to Na + -coupled substrate symporters, but is Na + independent, because an arginine undergoing conformational changes mimics Na + binding and unbinding (29). An equivalent observation is the ability of negatively charged amino acid residues to substitute for Cl − in neurotransmitter, Na + ,Cl − -cotransporters to make these transporters independent of Cl − (30).…”

Section: Discussionmentioning

confidence: 99%

Arginine substitution of a cysteine in transmembrane helix M8 converts Na ⁺ ,K ⁺ -ATPase to an electroneutral pump similar to H ⁺ ,K ⁺ -ATPase

Holm

Khandelwal

Einholm

et al. 2016

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

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Na+,K+-ATPase and H+,K+-ATPase are electrogenic and nonelectrogenic ion pumps, respectively. The underlying structural basis for this difference has not been established, and it has not been revealed how the H+,K+-ATPase avoids binding of Na+ at the site corresponding to the Na+-specific site of the Na+,K+-ATPase (site III). In this study, we addressed these questions by using site-directed mutagenesis in combination with enzymatic, transport, and electrophysiological functional measurements. Replacement of the cysteine C932 in transmembrane helix M8 of Na+,K+-ATPase with arginine, present in the H+,K+-ATPase at the corresponding position, converted the normal 3Na+:2K+:1ATP stoichiometry of the Na+,K+-ATPase to electroneutral 2Na+:2K+:1ATP stoichiometry similar to the electroneutral transport mode of the H+,K+-ATPase. The electroneutral C932R mutant of the Na+,K+-ATPase retained a wild-type–like enzyme turnover rate for ATP hydrolysis and rate of cellular K+ uptake. Only a relatively minor reduction of apparent Na+ affinity for activation of phosphorylation from ATP was observed for C932R, whereas replacement of C932 with leucine or phenylalanine, the latter of a size comparable to arginine, led to spectacular reductions of apparent Na+ affinity without changing the electrogenicity. From these results, in combination with structural considerations, it appears that the guanidine+ group of the M8 arginine replaces Na+ at the third site, thus preventing Na+ binding there, although allowing Na+ to bind at the two other sites and become transported. Hence, in the H+,K+-ATPase, the ability of the M8 arginine to donate an internal cation binding at the third site is decisive for the electroneutral transport mode of this pump.

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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%

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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Arginine oscillation explains Na ⁺ independence in the substrate/product antiporter CaiT

Cited by 22 publications

References 39 publications

Structural basis for the reaction cycle of DASS dicarboxylate transporters

Structural basis for the reaction cycle of DASS dicarboxylate transporters

Arginine substitution of a cysteine in transmembrane helix M8 converts Na ⁺ ,K ⁺ -ATPase to an electroneutral pump similar to H ⁺ ,K ⁺ -ATPase

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

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Arginine oscillation explains Na + independence in the substrate/product antiporter CaiT

Cited by 22 publications

References 39 publications

Structural basis for the reaction cycle of DASS dicarboxylate transporters

Structural basis for the reaction cycle of DASS dicarboxylate transporters

Arginine substitution of a cysteine in transmembrane helix M8 converts Na + ,K + -ATPase to an electroneutral pump similar to H + ,K + -ATPase

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

Contact Info

Product

Resources

About

Arginine oscillation explains Na ⁺ independence in the substrate/product antiporter CaiT

Arginine substitution of a cysteine in transmembrane helix M8 converts Na ⁺ ,K ⁺ -ATPase to an electroneutral pump similar to H ⁺ ,K ⁺ -ATPase