International Journal of Molecular Sciences

Review Structure and Mechanism of the Divalent Anion/Na+

Min Lu

Department of Biochemistry and Molecular , Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064, USA; [email protected]; Tel.: +1-847-578-8357

 Received: 21 December 2018; Accepted: 18 January 2019; Published: 21 January 2019 

Abstract: Integral membrane proteins of the divalent anion/Na+ symporter (DASS) family are conserved from bacteria to humans. DASS proteins typically mediate the coupled uptake of Na+ ions and dicarboxylate, tricarboxylate, or sulfate. Since the substrates for DASS include key intermediates and regulators of energy metabolism, alterations of DASS function profoundly affect fat storage, energy expenditure and life span. Furthermore, loss-of-function mutations in a human DASS have been associated with neonatal epileptic encephalopathy. More recently, human DASS has also been implicated in the development of liver cancers. Therefore, human DASS proteins are potentially promising pharmacological targets for battling obesity, diabetes, kidney stone, fatty liver, as well as other metabolic and neurological disorders. Despite its clinical relevance, the mechanism by which DASS proteins recognize and transport anionic substrates remains unclear. Recently, the crystal structures of a bacterial DASS and its humanized variant have been published. This article reviews the mechanistic implications of these structures and suggests future work to better understand how the function of DASS can be modulated for potential therapeutic benefit.

Keywords: ; anion transporter; sodium symporter; dicarboxylate transporter; substrate recognition; sodium coordination

1. Introduction Integral membrane proteins from the divalent anion/Na+ symporter (DASS) family are found in all domains of life [1–3]. They typically move Krebs cycle intermediates or sulfate across cell membranes by dissipating the electrochemical Na+ gradient. Specifically, mammalian DASS proteins + NaDC1, NaDC3, and NaCT co-transport three or more Na ions and C4-dicarboxylate (such as + succinate) or C6-tricarboxylate (such as citrate), whereas NaS1 and NaS2 co-transport two or three Na ions and sulfate [4–8]. Mammalian DASS proteins carry out their function at the plasma membrane of epithelial cells or cells of the central nervous system. The location of and functional difference among the human DASS proteins has been previously reviewed [9] and therefore not discussed here. Additionally, previous phylogenetic analysis has also suggested that the five human DASS transporters can be divided into three sub-groups [9]. The bacterial DASS proteins, by contrast, are located in the cytoplasmic membrane and catalyze the coupled uptake of two or more Na+ ions and C4-dicarboxylate [10–14]. Although most of the DASS proteins are co-transporters or (Figure1), some members from the non-vertebrates, including INDY from the fruit fly, function as exchangers and are Na+-independent [15,16].

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Figure 1. Two modes of transport identified in the divalent anion/Na+ symporter (DASS) Figure 1. Two modes of transport identified in the divalent anion/Na+ symporter (DASS) proteins. proteins. Many DASS proteins, including VcINDY and five human DASS transporters, function Many DASS proteins, including VcINDY and five human DASS transporters, function as as Na+-dependent co-transporters or symporters (left). Whereas, the fruit fly INDY, which is the Na+-dependent co-transporters or symporters (left). Whereas, the fruit fly INDY, which is the founding member of the DASS family, appears to be a Na+-independent exchanger or (right). founding member of the DASS family, appears to be a Na+-independent exchanger or antiporter For simplicity, the DASS proteins are drawn as cylinders (blue and yellow) and the membrane as a (right). For simplicity, the DASS proteins are drawn as cylinders (blue and yellow) and the grey rectangle. membrane as a grey rectangle. Since the DASS substrates include key intermediates and regulators of energy metabolism, Since the DASS substrates include key intermediates and regulators of energy metabolism, the the modulation of DASS activity can profoundly impact fatty acid synthesis, energy expenditure, modulation of DASS activity can profoundly impact fatty acid synthesis, energy expenditure, and and life span. For example, a number of mutations in a DASS-encoding gene had been found to nearly life span. For example, a number of mutations in a DASS-encoding gene had been found to nearly double the average adult life-span in fruit flies, likely by promoting a metabolic state that mimics double the average adult life-span in fruit flies, likely by promoting a metabolic state that mimics caloric and dietary restriction [17,18]. In addition, the knockdown of the genes encoding NaDC2 and caloric and dietary restriction [17,18]. In addition, the knockdown of the genes encoding NaDC2 and NaCT in worms could decrease their body size and fat content, and/or increase their life span [19,20]. NaCT in worms could decrease their body size and fat content, and/or increase their life span [19,20]. Moreover, the deletion of the gene encoding NaCT protected mice from the adiposity and insulin Moreover, the deletion of the gene encoding NaCT protected mice from the adiposity and insulin resistance induced by high-fat feeding and aging [21]. In addition, several loss-of-function mutations resistance induced by high-fat feeding and aging [21]. In addition, several loss-of-function mutations in human NaCT have been associated with severe epilepsy and encephalopathy early in life, as well in human NaCT have been associated with severe epilepsy and encephalopathy early in life, as well as developmental delay and tooth dysplasia in children [22]. Furthermore, a recent study reported as developmental delay and tooth dysplasia in children [22]. Furthermore, a recent study reported that the loss of NaCT could halt the growth of liver cancer cells, probably by changing both the energy that the loss of NaCT could halt the growth of liver cancer cells, probably by changing both the production and cell signaling in these cells [23]. Apart from the di/tricarboxylate substrates, sulfate is energy production and cell signaling in these cells [23]. Apart from the di/tricarboxylate substrates, one of the most abundant anions in mammalian plasma. As such, mammalian NaS1 and NaS2 have sulfate is one of the most abundant anions in mammalian plasma. As such, mammalian NaS1 and been implicated in regulating sulfate conjugation and the detoxification of xenobiotics [2]. NaS2 have been implicated in regulating sulfate conjugation and the detoxification of xenobiotics Altogether, these studies support human DASS proteins as potentially novel therapeutic targets [2]. for tackling diet-induced obesity, type 2 diabetes, kidney stone, and fatty liver, in addition to other Altogether, these studies support human DASS proteins as potentially novel therapeutic targets metabolic and neurological disorders [1–3]. Despite such importance, the molecular mechanism of for tackling diet-induced obesity, type 2 diabetes, kidney stone, and fatty liver, in addition to other DASS remained unclear, largely owing to the paucity of structural information on any substrate-bound metabolic and neurological disorders [1–3]. Despite such importance, the molecular mechanism of DASS. Recently, the X-ray structures of a bacterial DASS have been reported, elucidating the DASS remained unclear, largely owing to the paucity of structural information on any transporter architecture as well as the Na+- and substrate-binding sites [24,25]. This review discusses substrate-bound DASS. Recently, the X-ray structures of a bacterial DASS have been reported, these structures in the context of relevant biochemical data and suggests future directions towards elucidating the transporter architecture as well as the Na+- and substrate-binding sites [24,25]. This illuminating the general principles underlying DASS-mediated transport. review discusses these structures in the context of relevant biochemical data and suggests future 2.directions Structure towards Determination illuminating of DASS the general principles underlying DASS-mediated transport.

2. StructureThe molecular Determination structure ofof anyDASS DASS remained unknown until 2012, when the 3.2 Å resolution crystal structure of citrate-bound VcINDY (Figure2), a DASS from Vibrio cholerae, was reported [24]. This structureThe molecular (PDB 4F35)structure revealed of any the DASS transporter remained architecture unknown until and implicated2012, when the the amino3.2 Å resolution acids in Nacrystal+- and structure citrate-binding. of citrate-boun However,d VcINDY like other (Figure well-characterized 2), a DASS from bacterial Vibrio DASS cholerae proteins,, was reported VcINDY [24]. is This structure (PDB 4F35) revealed the transporter architecture and implicated the amino acids in known to transport succinate and other C4-dicarboxylates, rather than citrate, a C6-tricarboxylate [14,24]. Furthermore,Na+- and citrate-binding. although VcINDY However, was like suggested other well-cha to catalyzeracterized the bacterial co-transport DASS ofproteins, three NaVcINDY+ ions is known to transport succinate and other+ C4-dicarboxylates, rather than citrate, a C6-tricarboxylate and C4-dicarboxylate [14], only one Na -binding site was observed in the 3.2 Å resolution X-ray [14,24]. Furthermore, although VcINDY was suggested to catalyze the co-transport of three Na+ ions and C4-dicarboxylate [14], only one Na+-binding site was observed in the 3.2 Å resolution X-ray

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Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 3 of 12 structure [24]. Although a second Na+-binding site was predicted, no direct structural evidence was foundstructure and [24]. the assignmentAlthough a ofsecond this site Na was+-binding uncertain. site was Thus, predicted, the 3.2 Å no structure direct structural sheds little evidence light on howwas VcINDYfound and recognizes the assignment substrate of andthis multiplesite was Nauncertai+ ions.n. Thus, the 3.2 Å structure sheds little light on how VcINDY recognizes substrate and multiple Na+ ions.

FigureFigure 2.2.Amino-acid Amino-acid sequence sequence alignment alignment of representativeof representative DASS DASS proteins. proteins. Residues Residues conserved conserved among VcINDYamong VcINDY and three and human three orthologueshuman orth areologues colored are magenta, colored magenta, regions of regions secondary of secondary structural structural elements inelements VcINDY in areVcINDY outlined, are withoutlined transmembrane, with transmembrane helices shown helices as greenshown rectangles. as green rectangles. Red and blue Red dotsand + highlightblue dots aminohighlight acids amino that bindacids succinate/citratethat bind succinate/citrate and Na+, and respectively. Na , respectively. Positions Positions for the relevant for the humanizingrelevant humanizing mutations, mutation which includes, which S200T, include P201G, S200T, V322I, P201G, T379V, V322I, A376T, T379V, S381T, A376T, A382T, S381T, and A382T, A383T, areand marked A383T, by are green marked asterisks. by green For clarity,asterisks. some For residues clarity, in some the human residues DASS in the proteins human were DASS omitted proteins and indicatedwere omitted by “ ...and”. indicated Notably, theby amino-acid“…”. Notably, sequence the amino-acid identity between sequence VcINDY identity and between NaCT is 23%,VcINDY but theand degree NaCT of is sequence 23%, but conservation the degree in of and sequence around theconservation citrate- and in Na +and-binding around sites the is substantiallycitrate- and higher,Na+-binding suggesting sites thatis substantially the VcINDY higher, structure suggestin providesg that a useful the modelVcINDY for structure studying provides the mechanism a useful of NaCTmodel orfor other studying human the DASS. mechanism Sequence of alignment NaCT or was other performed human byDASS. using Sequence the program alignment ClustalW. was performed by using the program ClustalW. To address such critical questions, the structure of succinate-bound VcINDY, determined at a resolutionTo address of 2.8 such Å, was critical published questions, in 2017 the [ 25struct]. Thisure structureof succinate-bound (PDB 5UL7) VcINDY, elucidates determined a previously at a + undiscoveredresolution of Na2.8 Å,-binding was published site in VcINDY in 2017 as [25]. well asThis how structure this protein (PDB selects 5UL7) for elucidates trans-C4-dicarboxylate. a previously undiscovered Na+-binding site in VcINDY as well as how this protein selects for trans-C4-dicarboxylate. In the same study, the structure of a citrate-bound VcINDY (PDB 5UL9) as well as those of the succinate- (PDB 5ULD) and citrate-bound MT5 (PDB 5ULE), a humanized

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Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 4 of 12 In the same study, the structure of a citrate-bound VcINDY (PDB 5UL9) as well as those of the succinate- (PDB 5ULD) and citrate-bound MT5 (PDB 5ULE), a humanized variant of VcINDY, were established variant of VcINDY, were established at 2.8 Å resolution [25]. These crystal structures cast new light at 2.8 Å resolution [25]. These crystal structures cast new light on how citrate competitively inhibits on how citrate competitively inhibits VcINDY-mediated succinate transport as well as how a DASS VcINDY-mediated succinate transport as well as how a DASS distinguishes between C4-dicarboxylate distinguishes between C4-dicarboxylate and C6-tricarboxylate. In combination with mutagenesis and and C -tricarboxylate. In combination with mutagenesis and functional studies, these structures offer functional6 studies, these structures offer a solid framework for understanding how DASS proteins a solid framework for understanding how DASS proteins select and transport anionic substrates. select and transport anionic substrates. 3. Overall Structure of VcINDY 3. Overall Structure of VcINDY The structure of succinate-bound VcINDY [25] reveals a homodimeric arrangement (Figure3), The structure of succinate-bound VcINDY [25] reveals a homodimeric arrangement (Figure 3), with each protomer consisting of eleven membrane-spanning helices (named TM1-TM11), two with each protomer consisting of eleven membrane-spanning helices (named TM1-TM11), two re-entrant helix-turn-helix hairpins (HPin and HPout), and two interfacial helices (H4c and H9c). re-entrant helix-turn-helix hairpins (HPin and HPout), and two interfacial helices (H4c and H9c). As As viewed from the membrane, the VcINDY dimer looks like an inverted bowl with its concave side viewed from the membrane, the VcINDY dimer looks like an inverted bowl with its concave side facing the cytoplasm, thereby allowing for the aqueous solution to reach the midpoint of the membrane. facing the cytoplasm, thereby allowing for the aqueous solution to reach the midpoint of the This protein architecture facilitates substrate diffusion to the binding site and it partially solves the membrane. This protein architecture facilitates substrate diffusion to the binding site and it partially problem of translocating anionic substrate across the hydrophobic , an energetically solves the problem of translocating anionic substrate across the hydrophobic lipid bilayer, an unfavorable process. The N- and C-domains of VcINDY are similarly folded, despite opposite energetically unfavorable process. The N- and C-domains of VcINDY are similarly folded, despite membrane topology and modest amino-acid sequence similarity [25]. opposite membrane topology and modest amino-acid sequence similarity [25].

Figure 3. Structure of the succinate-bound VcINDY. Structure of dimeric VcINDY, as viewed from the membrane bilayer (left panel). VcINDY is shown in ribbon rendition, the N (amino acids 18-231) andFigure C (amino3. Structure acids of 232-462) the succinate-bound domains in one VcINDY. protomer Structure are colored of dimeric cyan VcINDY, and yellow, as viewed respectively, from + whereasthe membrane the other bilayer protomer (left panel is colored). VcINDY magenta. is shown Na ions in ribbon (green) rendition, and succinate the N are(amino drawn acids as spheres.18-231) Theand cytoplasmicC (amino acids view 232-462) of the domains VcINDY in structure one protom (righter panelare colored), highlighting cyan and the yellow, solvent-accessible respectively, + succinatewhereas the (black other arrows) protomer and is buried colored Na magenta.ions. As Na such,+ ions the (green) crystal and structure succinate captures are drawn the transporter as spheres. in theThe inward-opencytoplasmic state.view Unlessof the VcINDY noted otherwise, structure structural (right panel analysis), highlighting in this review the wassolvent-accessible performed by usingsuccinate the program(black arrows) O and and figure buried was Na prepared+ ions. byAs usingsuch, the softwarecrystal stru PyMOL.cture captures the transporter in the inward-open state. Unless noted otherwise, structural analysis in this review was performed Moreover, VcINDY bears structural resemblance to the dimeric AbgT transporters, MtrF and by using the program O and figure was prepared by using the software PyMOL. YdaH [26,27] despite a lack of significant amino-acid sequence similarity. Specifically, the structure of succinate-boundMoreover, VcINDY VcINDY bears can be structural superimposed resemblance onto those to the of MtrF dimeric (PDB AbgT 4R1I) transporters, and YdaH (PDB MtrF 4R0C) and toYdaH yield [26,27] rms deviations despite a lack of 3.1 of and significant 3.5 Å for amino-acid 294 and 305 sequence Cα atoms, similarity. respectively. Specifically, Moreover, the structure VcINDY bearsof succinate-bound 18 and 13% amino-acid VcINDY sequencecan be superimposed identity to MtrF onto and those YdaH, of MtrF respectively. (PDB 4R1I) Although and YdaH most of(PDB the DASS4R0C) proteins,to yield rms including deviations VcINDY, of 3.1 are and co-transporters, 3.5 Å for 294 theand AbgT 305 C transportersα atoms, respectively. function as Moreover, antibiotic effluxVcINDY pumps bears and 18 and they 13% are exchangersamino-acid [sequence26,27]. Apparently, identity to the MtrF AbgT and and YdaH, DASS respectively. proteins constitute Although a newmost groupof the ofDASS secondary proteins, membrane including transporters VcINDY, withare co shared-transporters, dimeric organizationthe AbgT transporters and structural function fold, evenas antibiotic though efflux they seem pumps to haveand they distinct are physiologicalexchangers [26,27]. functions Apparently, and transport the AbgT mechanisms. and DASS It proteins remains unclear,constitute however, a new group whether of thesecond AbgTary and membrane DASS proteins transporters arose fromwith convergentshared dimeric or divergent organization evolution. and structural fold, even though they seem to have distinct physiological functions and transport mechanisms. It remains unclear, however, whether the AbgT and DASS proteins arose from convergent or divergent evolution.

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Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 5 of 12 4. Na+-binding Sites in VcINDY 4. Na+-binding Sites in VcINDY The binding sites of two Na+ ions, designated as Na1 and Na2 (Figure4), were identified in VcINDYThe [ 25binding]. Despite sites compelling of two Na structural+ ions, designated evidence, as it Na1 is generally and Na2 a challenge(Figure 4), to were unambiguously identified in establishVcINDY the [25]. Na Despite+-binding compelling sites in protein structural structures. evidence Therefore,, it is generally to validate a challenge the observed to unambiguously Na+-binding sites,establish one putative the Na+-binding cation-coordinating sites in protein amino structures. acid in Na1Therefore, or Na2 to was validate replaced the byobserved Ala. Both Na+ of-binding these twosites, single one mutantsputative exhibitedcation-coordinating impaired transporter amino acid activity in Na1 and or Na2 substantially was replaced altered by NaAla.+-dependence Both of these oftwo succinate single transport, mutants thereby exhibited confirming impaired the assignedtransporter Na+ -bindingactivity sitesand [25 ].substantially Moreover, mostaltered of theNa Na+-dependence+-binding amino of succinate acids are transport, conserved thereby (Figure 2confirming), suggesting the that assigned both cation Na+-binding binding sites are[25]. sharedMoreover, by the most DASS of the members Na+-binding [25]. Notably, amino acids the bindingare conserved site Na2 (Figure can also 2), suggesting be found in that YdaH both [25 cation,27], furtherbinding supporting sites are shared the notion by the that DASS the AbgTmembers and [25]. DASS Notably, transporters the binding represent site aNa2 group can of also membrane be found transportersin YdaH [25,27], with similar further structure. supporting the notion that the AbgT and DASS transporters represent a group of membrane transporters with similar structure.

+ + FigureFigure 4. 4.Close-up Close-up views views of of the the Na Na-binding+-binding sites sites in in VcINDY. VcINDY. Structure Structure of of the the Na Na-binding+-binding site site in in the the + NN domain domain (left (left panel panel). The). The previously previously unobserved unobserved Na -binding Na+-binding site within site within the C domain the C domain (right panel (right). + Napanelions). Na are+ drawnions are as drawn green as spheres green andspheres relevant and relevant amino acids amino as acids stick models.as stick models. Dashed Dashed lines (blue) lines indicate(blue) indicate the coordination the coordination interactions. interactions.

In VcINDY, each Na+ ion is penta-coordinated to two amino-acid side-chain and three backbone In VcINDY, each Na+ ion is penta-coordinated to two amino-acid side-chain and three backbone carbonyl oxygen atoms (Figure4). The two Na + ions are bound to the pseudo-symmetry-related carbonyl oxygen atoms (Figure 4). The two Na+ ions are bound to the pseudo-symmetry-related HPin HPin and HPout, and thus Na1 and Na2 are structurally similar [25]. The structures of Na1 and and HPout, and thus Na1 and Na2 are structurally similar [25]. The structures of Na1 and Na2 also Na2 also resemble those of the Na+-binding sites found in other transporters, including AbgT, resemble those of the Na+-binding sites found in other transporters, including AbgT, VcCNT (a + + VcCNT (a Na -dependent concentrative ), and GltPh (a Na -coupled aspartate Na+-dependent concentrative nucleoside transporter), and GltPh (a Na+-coupled aspartate symporter). All of these binding sites comprise a helix-turn-helix hairpin and a discontinuous symporter). All of these binding sites comprise a helix-turn-helix hairpin and a discontinuous helix helix [27–29], thereby defining a class of widespread Na+-binding motifs in membrane proteins. [27–29], thereby defining a class of widespread Na+-binding motifs in membrane proteins. By By contrast, yet another common Na+-binding motif can be found in the transporters with the LeuT-like contrast, yet another common Na+-binding motif can be found in the transporters with the LeuT-like structural fold [30], which consists of a substantially bent and discontinuous helix in addition to a long structural fold [30], which consists of a substantially bent and discontinuous helix in addition to a but usually continuous helix. long but usually continuous helix. 5. Di- and Tri-carboxylate Binding Sites in VcINDY 5. Di- and Tri-carboxylate Binding Sites in VcINDY Within the Na+-binding cleft in VcINDY, the binding sites for succinate and citrate were also Within the Na+-binding cleft in VcINDY, the binding sites for succinate and citrate were also observed [25]. Specifically, the bound succinate interacts with VcINDY through H-bonding interactions observed [25]. Specifically, the bound succinate interacts with VcINDY through H-bonding (Figure5) and it is partly exposed to the cytoplasm, indicating that the transporter adopts an interactions (Figure 5) and it is partly exposed to the cytoplasm, indicating that the transporter inward-open conformation (Figure3). Moreover, the alanine substitutions of several succinate-binding adopts an inward-open conformation (Figure 3). Moreover, the alanine substitutions of several amino acids reduced the binding of succinate to VcINDY and impaired transport function, thereby succinate-binding amino acids reduced the binding of succinate to VcINDY and impaired transport confirming the biological relevance of the substrate-binding site [24,25]. Notably, the bound succinate function, thereby confirming the biological relevance of the substrate-binding site [24,25]. Notably, adopts an extended conformation, arguing that VcINDY is specific for C -dicarboxylate in a stretched the bound succinate adopts an extended conformation, arguing 4that VcINDY is specific for conformation. Since most of the succinate-interacting amino acids are evolutionarily conserved C4-dicarboxylate in a stretched conformation. Since most of the succinate-interacting amino acids are (Figure2), this preference for trans-dicarboxylate is likely to be shared by the DASS proteins. evolutionarily conserved (Figure 2), this preference for trans-dicarboxylate is likely to be shared by the DASS proteins.

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FigureFigure 5. 5. Close-upClose-up viewsviews ofof thethe succinate-succinate- andand citrate-bindingcitrate-binding sitessites inin VcINDY.VcINDY. Structure Structure of of the the succinate-bindingsuccinate-binding site site ( left(left panel panel).). Detailed Detailed view view of of the the citrate-binding citrate-binding site site ( right(right panel panel).). Succinate Succinate (grey),(grey), citrate citrate (green) (green) and and relevant relevant amino amino acids acids are are drawn drawn as as stick stick models, models, whereas whereas the the Na Na+ ions+ ions are are shownshown as as green green spheres. spheres. Dashed Dashed lines (blue)lines (blue) highlight highlight the interactions the interactions between VcINDYbetween andVcINDY succinate and orsuccinate citrate. or citrate.

TheThe citrate-bound citrate-bound VcINDY VcINDY is structurallyis structurally similar simi tolar the to succinate-bound the succinate-bound protein, protein, with the with citrate- the andcitrate- succinate-binding and succinate-binding sites overlapping sites overlappin substantially,g substantially, which is consistent which withis consistent the contention with that the citratecontention inhibits that the citrate transport inhibits of succinate the transport by preoccupying of succinate by the preoccupying substrate-binding the substrate-binding site in VcINDY, i.e., site asin a VcINDY, competitive i.e., inhibitor. as a competitive Furthermore, inhibitor. the twoFurthe co-crystalrmore, the structures two co-crystal suggest structures that HPin, suggest HPout, and that theHP unwoundin, HPout, and region the unwound in TM10 constituteregion in TM10 a “trans-dicarboxylate-recognition” constitute a “trans-dicarboxylate-recognition” module in DASS module [25]. Thisin DASS module [25]. lacks This any module protonatable lacks any or positivelyprotonatable charged or positively amino acids,charged starkly amino contrasting acids, starkly the succinate-bindingcontrasting the succinate-binding water-soluble proteins, water-soluble in which proteins, Arg and in which Lys form Arg and charge-charge Lys form charge-charge interactions withinteractions the bound with dicarboxylate the bound dicarboxylate [31–35]. [31–35]. InIn vivo vivo,, atat least least two two carboxylates carboxylates in in citrate citrate are are deprotonated deprotonated and and negatively negatively charged charged [ 14[14].]. In In the the citrate-boundcitrate-bound VcINDY, VcINDY, one one carboxylate carboxylate group group makes makes no no contact contact with with the the transporter. transporter. By By contrast, contrast, bothboth carboxylates carboxylates inin succinatesuccinate areare stabilizedstabilized byby thethe H-bondingH-bonding interactionsinteractions mademade withwith VcINDY.VcINDY. Therefore,Therefore, the the negative negative charges charges in in citrate citrate appears appears not not fully fully “neutralized” “neutralized” by by its its interactions interactions with with VcINDY,VcINDY, which which may may explain explain why why citrate citrate is is less less effective effective in in inhibiting inhibiting VcINDY–mediated VcINDY–mediated transport transport thanthan C C4-dicarboxylates4-dicarboxylates and and why why citrate citrate preferably preferably binds binds to to the the inward-facing inward-facing VcINDY VcINDY [ 14[14,25].,25]. PreviousPrevious studiesstudies also implied implied that that the the coordination coordination of ofNa Na+ promotes+ promotes substrate substrate binding binding to DASS to DASS[10–13]. [10 –In13 VcINDY,]. In VcINDY, the Na the+ ions Na+ inions Na1 in and Na1 Na2 and coordinate Na2 coordinate several several succinate-binding succinate-binding amino amino acids acidsand andthus thus stabilize stabilize the theconformation conformation of ofthese these amino-acid amino-acid side side chains. chains. This arrangementarrangement helpshelps toto explainexplain why why the the transport transport of of succinate succinate and and Na Na+ +is is strictly strictly coupled, coupled, as as they they bind bind to to a a common common subset subset ofof amino amino acids, acids, and and the the binding binding or or unbinding unbinding of of one one likely likely affects affects that that of of the the other other [ 25[25].]. Moreover,Moreover, thethe Na Na+ +ions ions may may attract attract negatively negatively charged charged succinate succinate through through long-range long-range electrostatic electrostatic interactions interactions withinwithin the the low-dielectric low-dielectric intramembrane intramembrane environment. environment.Furthermore, Furthermore, thethe amino amino ends ends of of four four short short heliceshelices from from HP HPinin,, HP HPoutout,, TM5,TM5, andand TM10,TM10, whichwhich possess possess localized localized positive positive dipoles, dipoles, all all point point toward toward thethe bound bound succinate. succinate. The The stabilization stabilization of of negative negative charges charges by by the the opposing, opposing, positive positive helix helix dipoles dipoles withinwithin inverted inverted structural structural repeats repeats seems seems to to be be a a common common strategy strategy in in achieving achieving anion anion selectivity selectivity by by membranemembrane proteins proteins [36 [36–39].–39].

6.6. Structures Structures of of A A Humanized Humanized Variant Variant of of VcINDY VcINDY ToTo gain gain new new insights insights into theinto transport the transport mechanism mechanism of human of DASS,human the DASS, structures the ofstructures a humanized of a varianthumanized of VcINDY variant in of complexes VcINDY within complexes citrate and with succinate citrate were and determinedsuccinate were to 2.8-Å determined resolutions to 2.8-Å [25]. Inresolutions order to generate [25]. In this order mutant, to generate MT5, eight this amino mutant, acids MT5, surrounding eight amino the citrate-binding acids surrounding cleft were the replacedcitrate-binding by their cleft counterparts were replaced in human by their NaCT counte (Figurerparts 2in), human which primarilyNaCT (Figure transports 2), which citrate primarily [ 6]. Althoughtransports the citrate structure [6]. Although of citrate-bound the structure MT5 remains of citrate-bound similar to thatMT5 ofremains VcINDY, similar one importantto that of VcINDY, one important difference centers on one carboxylate of the bound citrate (Figure 6). In contrast to that in VcINDY, this carboxylate group from citrate latches onto the amino ends of TM5b

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differenceInt. J. Mol. Sci. centers 2019, 20 on, x FOR one PEER carboxylate REVIEW of the bound citrate (Figure6). In contrast to that in VcINDY, 7 ofthis 12 carboxylate group from citrate latches onto the amino ends of TM5b and the second helix in HPout inand MT5, the withsecond its putativehelix in negativeHPout in chargeMT5, with being its stabilized putative bynegative the positive charge helix being dipoles. stabilized Since by NaCT the transportspositive helix trianionic dipoles. citrate Since [6 NaCT] and MT5transports was co-crystallized trianionic citrate with [6] citrate and atMT5 pH~7 was [25 co-crystallized], the citrate-bound with MT5citrate structure at pH~7 may [25], predict the citrate-bound the interactions MT5 between structure NaCT may and predict its bound the interactions substrate in between vivo. NaCT and its bound substrate in vivo.

Figure 6. The citrate- and succinate-binding sites in hu humanizedmanized variant of VcINDY. Structure of the citrate-binding sitesite ( left(left panel panel).). Close-up Close-up view view of theof the succinate-binding succinate-binding site (siteright (right panel panel). Citrate). Citrate (light blue),(light blue), succinate succinate (grey) (grey) and relevant and relevant amino amino acids acids are drawn are drawn as stick as stick models, models, while while the Nathe+ Naions+ ions are shownare shown as green as green spheres. spheres. Humanizing Humanizing amino-acid amino-acid substitutions substitutions are highlighted are highlighted in red. in Dashedred. Dashed lines (blue)lines (blue) indicate indicate the interactions the interactions between between MT5 andMT5 citrate and citrate or succinate. or succinate.

Interestingly, thethe succinate-boundsuccinate-bound structure of MT5 revealed that the humanized variant binds succinate virtually in the samesame wayway asas VcINDYVcINDY (Figure(Figure6 ).6). Moreover,Moreover, thethe transporttransport kineticskinetics forfor MT5-mediated succinatesuccinate uptakeuptake is is also also similar similar to to those those of of VcINDY VcINDY [25 [25].]. However, However, citrate citrate inhibited inhibited the MT5-meditatedthe MT5-meditated uptake uptake of succinate of succinate much moremuch effectively more effectively than it did than on VcINDYit did on [ 25VcINDY]. In accordance [25]. In withaccordance this observation, with this observation, MT5 seemed MT5 to interact seemed with to interact citrate morewith stronglycitrate more within strongly the crystals within than the VcINDY.crystals than Although VcINDY. no appreciableAlthough no citrate-transporting appreciable citrate-transporting activity in MT5 activity was detected, in MT5 the was structure detected, of citrate-boundthe structure of MT5 citrate-bound likely recapitulates MT5 likely the recapitula substrate-bindingtes the substrate-binding properties of NaCT properties to a significant of NaCT extent, to a insignificant light of theextent, amino-acid in light of sequence the amino-acid similarity sequence between similarity VcINDY between and NaCT, VcINDY as well and as NaCT, the homolog as well swapas the mutationshomolog swap carried mutations by MT5 (Figurecarried2 ).by It MT5 remains (Fig unclear,ure 2). It on remains the other unclear, hand, whyon the MT5 other failed hand, to transportwhy MT5 citratefailed into vitrotransportdespite citrate its higherin vitro affinity despite for itscitrate higher than affinity that for of VcINDYcitrate than [25 ].that One of plausibleVcINDY explanation[25]. One plausible would explanation be that MT5 would still lacks be that key MT5 structural still lacks elements key structural that somehow elements enable that somehow NaCT to transportenable NaCT citrate to transport more effectively citrate thanmore MT5, effectively which than may MT5, be found which through may be inspection found through of the amino-acid inspection sequenceof the amino-acid alignment sequence (Figure 2alignment) and further (Figure mutagenesis 2) and further study. mutagenesis study.

7. Substrate Recognition By DASS and Other anion Transporters The structures of VcINDY and its humanized variant suggest that the amino ends of TM5b and the secondsecond helixhelix inin HPHPoutout formform a second substrate-recognition module in DASS for differentiatingdifferentiating C6-tricarboxylate-tricarboxylate from from C 4-dicarboxylate-dicarboxylate [25]. [25]. In In a a C 4-dicarboxylate-specific-dicarboxylate-specific VcINDY, VcINDY, this modulemodule includes a Pro and aa ThrThr (Figure(Figure7 ),7), whichwhich selectsselects againstagainst citratecitrate byby pushingpushing awayaway oneone ofof itsits carboxylate groupsgroups and and likely likely gives gives rise torise negative to negative charge surpluscharge withinsurplus the within hydrophobic the hydrophobic membrane environment.membrane environment. In a C6-tricarboxylate-transporting In a C6-tricarboxylate-transporting NaCT, however, NaCT, the however, Pro and the Thr Pro are and superseded Thr are bysuperseded Gly and by Val, Gly respectively, and Val, respectively, which enables which direct enables interaction direct interaction with the same with carboxylatethe same carboxylate group in citrategroup in (Figure citrate7). (Figure Thus, DASS7). Thus appears, DASS to appears be equipped to be withequipped two substrate-recognitionwith two substrate-recognition modules: + onemodules: selective one for selective trans-C4 -dicarboxylatefor trans-C4-dicarboxylate and the other forand C 6the-tricarboxylate. other for C Na6-tricarboxylate.also contributes Na+ to also the bindingcontributes of C to4-dicarboxylate the binding of to C DASS4-dicarboxylate by stabilizing to DASS the first by structural stabilizing module. the first Taken structural together, module. these Taken together, these structures shed new light on how a DASS recognizes its substrate and offer a new angle to understand protein-mediated anion transport in general [25].

Int. J. Mol. Sci. 2019, 20, 440 8 of 12 structures shed new light on how a DASS recognizes its substrate and offer a new angle to understand protein-mediatedInt. J. Mol. Sci. 2019, 20 anion, x FOR transportPEER REVIEW ingeneral [25]. 8 of 12

Figure 7. Structural basis for substrate recognition by DASS. The N and C domains in VcINDY (Figureleft panel 7. Structural) and its humanized basis for substrate variant (right recognition panel) areby DASS. colored The cyan N and and yellow, C domains respectively. in VcINDY Relevant (left aminopanel) acidsand its are humanized drawn as stick variant models, (right whereas panel the) are bound colored succinate cyan and (left yellow, panel) orrespectively. citrate (right Relevant panel), asamino well acids as the are Na drawn+ ions (green)as stick aremode shownls, whereas as spheres. the bo Positiveund succinate helix dipoles (left panel for short) or helicescitrate TM5b(right + andpanel TM10b), as well are as highlighted the Na ions by plus(green) signs, are whereasshown as the spheres. negatively Positive charged helix carboxylates dipoles for short in succinate helices orTM5b citrate and are TM10b indicated are byhighlighted minus signs. by plus The chargedsigns, whereas state of succinatethe negatively or citrate charged is deduced carboxylates based onin thesuccinate crystallization or citrate pH are (~7). indicated Both theby minus helix dipoles signs. Th ande charged Na+ appear state to of contribute succinate toor thecitrate anion is deduced binding. Furthermore,based on the crystallization P201 and T379 pH may (~7). enable Both VcINDYthe helix todipoles select and for succinateNa+ appear but to againstcontribute citrate. to the In anion MT5, P201,binding. and Furthermore, T379 are replaced P201 byand Gly T379 and may Val, enable respectively, VcINDY which to select may for allow succinate the membrane-embedded but against citrate. transporterIn MT5, P201, to bind and citrate T379 more are stronglyreplaced than by VcINDY.Gly and Val, respectively, which may allow the membrane-embedded transporter to bind citrate more strongly than VcINDY. A striking feature of the substrate-binding site in VcINDY is the absence of any positively charged aminoA acid,striking i.e., Lysfeature or Arg. of the Apparently, substrate-binding DASS has evolvedsite in VcINDY such a scheme is the probablyabsence becauseof any positively positively charged amino amino acid, acids i.e., would Lys or discourage Arg. Apparently, the binding DASS ofhas Na evolved+ in their such vicinity a scheme due probably to electrostatic because repulsionpositively and/orcharged cause amino the transporteracids would to binddiscourage anionic substratethe binding much of too Na tightly,+ in their thereby vicinity discouraging due to theelectrostatic dissociation repulsion of substrate and/or fromcause the the transporter transporte [r25 to]. bind Besides anionic DASS, substrate at least much three too families tightly, of transportersthereby discouraging with available the dissociation structures of selectively substrate transportfrom the transporter anionic substrates: [25]. Besides SeCitS DASS, and KpCitSat least fromthree thefamilies citrate-sodium of transporters symporter with familyavailable [40 ,41structures], GltPh and selectively GltTk from transport the excitatory anionic aminosubstrates: acid transporterSeCitS and familyKpCitS [29 from,42], andthe NarKcitrate-sodium and NarU symporter from the nitrate/nitrite family [40,41], porter GltPh family and [Glt43–Tk45 from]. the excitatoryIn contrast amino to acid VcINDY, transporter all the family other [29,42], three anion and NarK transporters and NarU utilize from positively the nitrate/nitrite charged aminoporter acidsfamily to [43–45]. bind the negatively charged groups in the substrate. This difference may reflect the distinct substrate-bindingIn contrast to sitesVcINDY, and/or all thethe couplingother three mechanisms anion transporters [25]. For utilize example, positively an Arg charged residue amino in the substrate-bindingacids to bind the negatively site of GltPh chargedappears groups critical in in the determining substrate. acidic This versusdifference neutral may substrate reflect the selectivity, distinct sincesubstrate-binding the neutralization sites and/or of this the Arg coupling residue increasedmechanisms the [25]. tendency For example, of the transporter an Arg residue to select in the for neutralsubstrate-binding rather than acidicsite of substrate GltPh appears [46]. In GltcriticalPh, however, in determining an Asp residue acidic is versus located neutral in close proximitysubstrate toselectivity, this Arg, since the former the neutralization of which may of neutralize this Arg theresidue positive increased charge the on thetendency Arg side-chain of the transporter and weaken to theselect electrostatic for neutral attraction rather than between acidic the substrate Arg and anionic[46]. In substrate,GltPh, however, thereby an facilitating Asp residue the releaseis located of the in negativelyclose proximity charged to substratethis Arg, duringthe former transport. of which may neutralize the positive charge on the Arg side-chain and weaken the electrostatic attraction between the Arg and anionic substrate, thereby facilitating the release of the negatively charged substrate during transport.

8. Elevator-like Mechanism and Future Perspectives Despite recent progress in the structural studies of VcINDY, great uncertainties remain in the mechanistic understanding of the DASS-mediated anion transport. Particularly, in all the known structures of VcINDY, the substrate-binding site opens into the cytoplasm, i.e., adopting the

Int. J. Mol. Sci. 2019, 20, 440 9 of 12

8. Elevator-like Mechanism and Future Perspectives Despite recent progress in the structural studies of VcINDY, great uncertainties remain in the mechanisticInt. J. Mol. Sci. 2019 understanding, 20, x FOR PEER of REVIEW the DASS-mediated anion transport. Particularly, in all the known 9 of 12 structures of VcINDY, the substrate-binding site opens into the cytoplasm, i.e., adopting the inward-facing inward-facing conformation. Therefore, the molecular basis for the interconversion between the conformation. Therefore, the molecular basis for the interconversion between the inward- and inward- and outward-facing conformations, which lies at the heart of the transport mechanism outward-facing conformations, which lies at the heart of the transport mechanism [47,48], remains [47,48], remains unclear. Moreover, the structural comparison of the succinate- and citrate-bound unclear. Moreover, the structural comparison of the succinate- and citrate-bound VcINDY and its VcINDY and its humanized variant gave little insight into how a DASS selects between humanized variant gave little insight into how a DASS selects between di/tricarboxylates and sulfate. di/tricarboxylates and sulfate. Furthermore, although modulation of the function of DASS seems to Furthermore, although modulation of the function of DASS seems to be a viable therapeutic option for be a viable therapeutic option for battling metabolic and neurological disorders, the mechanism of battling metabolic and neurological disorders, the mechanism of such modulation is poorly understood. such modulation is poorly understood. To address these critical questions, future work should be aimed at deciphering the structure To address these critical questions, future work should be aimed at deciphering the structure of of an outward-facing DASS. Previous studies have implied that VcINDY undergoes an elevator-like an outward-facing DASS. Previous studies have implied that VcINDY undergoes an elevator-like movement during transport [49]. Such a structural mechanism appears to be widespread in a variety of movement during transport [49]. Such a structural mechanism appears to be widespread in a variety transporters with diverse physiological function, including the anion transporters SeCitS and GltPh [50]. of transporters with diverse physiological function, including the anion transporters SeCitS and In these transporters, the protein can be divided into the scaffold and transport domains, the latter of GltPh [50]. In these transporters, the protein can be divided into the scaffold and transport domains, which contains the substrate- and cation-binding sites. During transport, the scaffold domain, or the the latter of which contains the substrate- and cation-binding sites. During transport, the scaffold “hoistway”, remains stationary, whereas the transport domain, or the “cabin”, moves up and down, domain, or the “hoistway”, remains stationary, whereas the transport domain, or the “cabin”, moves thereby exposing the substrate- and cation-binding sites alternately to either side of the membrane. up and down, thereby exposing the substrate- and cation-binding sites alternately to either side of Based on this concept, a structural model of the outward-facing VcINDY (Figure8) can be built by the membrane. Based on this concept, a structural model of the outward-facing VcINDY (Figure 8) using the succinate-bound structure [25]. Despite its usefulness as a starting point for deciphering the can be built by using the succinate-bound structure [25]. Despite its usefulness as a starting point for transport mechanism, this outward-facing model of VcINDY needs to be further modified through deciphering the transport mechanism, this outward-facing model of VcINDY needs to be further additional biochemical and/or structural studies, since the transporter likely interacts with its substrate modified through additional biochemical and/or structural studies, since the transporter likely somewhat differently in distinct conformations [41]. interacts with its substrate somewhat differently in distinct conformations [41].

Figure 8. Structural model of the outward-facing VcINDY. Structure of the inward-facing VcINDY as viewed from the membrane bilayer (left panel). VcINDY is drawn as a ribbon diagram, with its scaffold (residues 18-128, 250-357) and transport (residues 129-249, 358-462) domains colored blue and Figure 8. Structural model of the outward-facing VcINDY. Structure of the inward-facing VcINDY as orange, respectively. Na+ ions (green) and succinate are shown as spheres. The structural model of the viewed from the membrane bilayer (left panel). VcINDY is drawn as a ribbon diagram, with its outward-facing VcINDY (right panel). The orientation and coloring scheme are both the same as in scaffold (residues 18-128, 250-357) and transport (residues 129-249, 358-462) domains colored blue panel A. As the transport domain traverses the membrane, VcINDY alternates between the inward- and orange, respectively. Na+ ions (green) and succinate are shown as spheres. The structural model and outward-facing conformations. of the outward-facing VcINDY (right panel). The orientation and coloring scheme are both the same Thisas in elevator-likepanel A. As the mechanism transport alsodomain predicts traver thatses the any membrane, compounds VcINDY that can alternates glue the between transport the and scaffoldinward- domains and outward-facing of DASS together conformations. can serve as effective inhibitors of the transporter. Thus, the structural model of outward-facing VcINDY alongside the inward-facing structure may be useful for designingThis elevator-like such inhibitors. mechanism These compounds also predicts likely that actany as compounds allosteric inhibitors that can glue of DASS the transport and they and are presumablyscaffold domains different of fromDASS what together have beencan serve studied as previously,effective inhibitors as prior work of th appearse transporter. to focus Thus, on those the chemicalsstructural that model target of theoutward-facing Na+- and substrate-binding VcINDY alongside sites in the the inward-facing human DASS transporters structure may [51,52 be]. usefulNeedless for todesigning say, these such allosteric inhibitors. inhibitors These can compounds vastly expand likely the act scope as allosteric for the development inhibitors of of DASS potentially and they useful are therapeuticspresumably different that target from the what human have DASS been proteins. studied Furthermore,previously, as toprior gain work further appears insights to focus into theon those chemicals that target the Na+- and substrate-binding sites in the human DASS transporters [51,52]. Needless to say, these allosteric inhibitors can vastly expand the scope for the development of potentially useful therapeutics that target the human DASS proteins. Furthermore, to gain further insights into the mechanism of the sulfate-transporting DASS proteins, including human NaS1 and NaS2, future work should also include the structure determination of a sulfate-transporting DASS. Although challenging, these studies will advance our understanding of how a DASS transports key

Int. J. Mol. Sci. 2019, 20, 440 10 of 12 mechanism of the sulfate-transporting DASS proteins, including human NaS1 and NaS2, future work should also include the structure determination of a sulfate-transporting DASS. Although challenging, these studies will advance our understanding of how a DASS transports key metabolites inside cells and how its function can be modulated. Such knowledge promises to inform the design of new pharmaceuticals to prevent and treat the DASS-associated diseases.

Funding: The research in my laboratory is in part supported by the US National Institutes of Health (R01-GM094195). Acknowledgments: The author thanks Jindrich Symersky for help with analyzing the Na+-binding sites in membrane proteins with available structures as well as generating the structural model of outward-facing VcINDY. Conflicts of Interest: The author declares no conflict of interest.

Abbreviations

DASS Divalent Anion/Sodium Symporter NaDC1 Na+-dependent DiCarboxylate symporter1 NaDC3 Na+-dependent DiCarboxylate symporter3 NaCT Na+-dependent Citrate Transporter INDY I’m Not Dead Yet NaS1 Na+-dependent Sulphate symporter1 NaS2 Na+-dependent Sulphate symporter2 VcINDY Vibrio Cholerae I’m Not Dead Yet (a divalent anion/sodium symporter from Vibrio cholerae)

References

1. Pajor, A.M. Sodium-coupled dicarboxylate and citrate transporters from the SLC13 family. Pflugers Arch. 2014, 466, 119–130. [CrossRef] 2. Markovich, D. Na+-sulfate SLC13A1. Pflugers Arch. 2014, 466, 131–137. [CrossRef][PubMed] 3. Prakash, S.; , G.; Singhi, S.; Saier, M.H. The ion transporter superfamily. Biochim. Biophys. Acta 2003, 1618, 79–92. [CrossRef][PubMed] 4. Wright, S.H.; Kippen, I.; Klinenberg, J.R.; Wright, E.M. Specificity of the transport system for tricarboxylic acid cycle intermediates in renal brush borders. J. Membrane Biol. 1980, 57, 73–82. [CrossRef] 5. Burckhardt, B.C.; Drinkuth, B.; Menzel, C.; Konig, A.; Steffgen, J.; Wright, S.H.; Burckhardt, G. The renal Na+-dependent dicarboxylate transporter, NADC-3, translocates dimethyl- and disulfhydryl-compounds and contributes to renal heavy metal detoxification. J. Am. Soc. Nephrol. 2002, 13, 2628–2638. [CrossRef] [PubMed] 6. Inoue, K.; Fei, Y.J.; Zhuang, L.; Gopal, E.; Miyauchi, S.; Ganapathy, V. Functional features and genomic organization of mouse NaCT, a sodium-coupled transporter for tricarboxylic acid cycle intermediates. Biochem. J. 2004, 378, 949–957. [CrossRef][PubMed] 7. Busch, A.E.; Waldegger, S.; Herzer, T.; Biber, J.; Markovich, D.; Murer, H.; Lang, F. Electrogenic cotransport + + 2− of Na and sulfate in Xenopus oocytes expressing the cloned Na SO4( ) transporter protein NaSi-1. J. Biol. Chem. 1994, 269, 12407–12409. 8. Markovich, D.; Regeer, R.R.; Kunzelmann, K.; Dawson, P.A. Functional characterization and genomic organization of the human Na+-sulfate cotransporter hNaS2 gene (SLC13A4). Biochem. Biophys. Res. Commun. 2005, 326, 729–734. [CrossRef] 9. Bergeron, M.J.; Clemencon, B.; Hediger, M.A.; Markovich, D. SLC family of Na+-coupled di- an dtri-carboxylate/. Mol. Asp. Med. 2013, 34, 299–312. [CrossRef] 10. Hall, J.A.; Pajor, A.M. Functional characterization of Na+-coupled dicarboxylate carrier protein from Staphylococcus aureus. J. Bacteriol. 2005, 187, 5189–5194. [CrossRef] 11. Hall, J.A.; Pajor, A.M. Functional reconstitution of SdcS, a Na+-coupled dicarboxylate carrier protein from Staphylococcus aureus. J. Bacteriol. 2007, 189, 880–885. [CrossRef][PubMed] 12. Youn, J.W.; Jolkver, E.; Kramer, R.; Marin, K.; Wendisch, V.F. Identification and characterization of the dicarboxylate uptake system DccT in Corynebacterium glutamicum. J. Bacteriol. 2008, 190, 6458–6466. [CrossRef][PubMed] Int. J. Mol. Sci. 2019, 20, 440 11 of 12

13. Strickler, M.A.; Hall, J.A.; Gaiko, O.; Pajor, A.M. Functional characterization of a Na+-coupled dicarboxylate transporter from Bacillus licheniformis. Biochim. Biophys. Acta 2009, 1788, 2489–2496. [CrossRef][PubMed] 14. Mulligan, C.; Fitzgerald, G.A.; Wang, D.N.; Mindell, J.A. Functional characterization of a Na+-dependent dicarboxylate transporter from Vibrio cholera. J. Gen. Physiol. 2014, 143, 745–759. [CrossRef][PubMed] 15. Knauf, F.; Rogina, B.; Jiang, Z.; Aronson, P.S.; Helfand, S.L. Functional characterization and immunolocalization of the transporter encoded by the life-extending gene Indy. Proc. Natl. Acad. Sci. USA 2002, 99, 14315–14319. [CrossRef][PubMed] 16. Inoue, K.; Fei, Y.J.; Huang, W.; Zhuang, L.; Chen, Z.; Ganapathy, V. Functional identity of Drosophila melanogaster Indy as a cation-independent, electroneutral transporter for tricarboxylic acid-cycle intermediates. Biochem. J. 2002, 367, 313–319. [CrossRef][PubMed] 17. Rogina, B.; Reenan, R.A.; Nilsen, S.P.; Helfand, S.L. Extended life-span conferred by cotransporter gene mutations in Drosophila. Science 2000, 290, 2137–2140. [CrossRef][PubMed] 18. Neretti, N.; Wang, P.Y.; Brodsky, A.S.; Nyguyen, H.H.; White, K.P.; Rogina, B.; Helfand, S.L. Long-lived Indy induces reduced mitochondrial production and oxidative damage. Proc. Natl. Acad. Sci. USA 2009, 106, 2277–2282. [CrossRef][PubMed] 19. Fei, Y.J.; Inoue, K.; Ganapathy, V. Structural and functional characteristics of two-sodium-coupled dicarboxylate transporters (ceNaDC1 and ceNaDC2) from Caenorhabditis elegans and their relevance to life span. J. Biol. Chem. 2003, 278, 6136–6144. [CrossRef] 20. Fei, Y.J.; Liu, J.C.; Inoue, K.; Zhaung, L.; Miyake, K.; Miyauchi, S.; Ganapathy, V. Relevance of NAC-2, an Na+-coupled citrate transporter, to life span, body size and fat content in Caenorhabditis elegans. Biochem. J. 2004, 379, 191–198. [CrossRef][PubMed] 21. Birkenfeld, A.L.; Lee, H.-Y.; Guebre-Egziabher, F.; Alves, T.C.; Jurczak, M.J.; Jornayvaz, F.R.; Zhang, D.; Hsiao, J.J.; Martin-Montalvo, A.; Fischer-Rosinsky, A.; et al. Deletion of the mammalian INDY homolog mimics aspects of dietary restriction and protects against adiposity and insulin resistance in mice. Cell Metab. 2011, 14, 184–195. [CrossRef][PubMed] 22. Bhutia, Y.D.; Kopel, J.J.; Lawrence, J.J.; Neugebauer, V.; Ganapathy, V. Plasma Membrane Na+-Coupled Citrate Transporter (SLC13A5) and Neonatal Epileptic Encephalopathy. Molecules 2017, 22, 378. [CrossRef] 23. Li, Z.; Li, D.; Choi, E.Y.; Lapidus, R.; Zhang, L.; Huang, S.M.; Shapiro, P.; Wang, H. Silencing of 13 member 5 disrupts energy homeostasis and inhibits proliferation of human hepatocarcinoma cells. J. Biol. Chem. 2017, 292, 13890–13901. [CrossRef] 24. Mancusso, R.; Gregorio, G.G.; Liu, Q.; Wang, D.N. Structure and mechanism of a bacterial sodium-dependent dicarboxylate transporter. Nature 2012, 491, 622–626. [CrossRef] 25. Nie, R.; Stark, S.; Symersky, J.; Kaplan, R.S.; Lu, M. Structure and function of the divalent anion/Na+ symporter from Vibrio cholerae and a humanized variant. Nat. Commun. 2017, 8, 15009. [CrossRef][PubMed] 26. Su, C.-C.; Bolla, J.R.; Kumar, N.; Radhakrishnan, A.; Long, F.; Delmar, J.A.; Chou, T.-H.; Rajashankar, K.R.; Shafer, W.M.; Yu, E.W. Structure and function of Neisseria gonorrhoeae MtrF illuminates a class of antimetabolite efflux pumps. Cell Rep. 2015, 11, 61–70. [CrossRef][PubMed] 27. Su, C.-C.; Delmar, J.A.; Radhakrishnan, A.; Chou, T.-H.; Rajashankar, K.R.; Yu, E.W.; Bolla, J.R.; Kumar, N.; Long, F. Crystal structure of the Alcanivorax borkumensis YdaH transporter reveals an unusual topology. Nat. Commun. 2015, 6, 6874. 28. Johnson, Z.L.; Cheong, C.G.; Lee, S.Y. Crystal structure of a concentrative nucleotide transporter from Vibrio cholerae at 2.4 Å. Nature 2012, 483, 489–493. [CrossRef] 29. Boudker, O.; Ryan, R.M.; Yernool, D.; Shimamoto, K.; Gouaux, E. Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter. Nature 2007, 445, 387–393. [CrossRef] 30. Yamashita, A.; Singh, S.K.; Kawate, T.; Jin, Y.; Gouaux, E. Crystal structure of a bacterial homologue of Na+/Cl−-dependent neurotransmitter transporters. Nature 2005, 437, 215–223. [CrossRef] 31. Gouaux, J.E.; Lipscomb, W.N. Three-dimensional structure of carbamoyl phosphate and succinate bound to aspartate carbamoyltransferase. Proc. Natl. Acad. Sci. USA 1988, 85, 4205–4208. [CrossRef][PubMed] 32. Leys, D.; Tsapin, A.S.; Nealson, K.H.; Meyer, T.E.; Cusanovich, M.A.; Van Beeumen, J.J. Structure and mechanism of the flavocytochrome c fumarate reductase of Shewanella putrefaciens MR-1. Nat. Struct. Biol. 1999, 6, 1113–1117. [PubMed] 33. Muller, I.; Stuckl, C.; Wakeley, J.; Kertesz, M.; Uson, I. Succinate complex crystal structures of the α-ketoglutarate-dependent dioxygenase AtsK. J. Biol. Chem. 2005, 280, 5716–5723. [CrossRef][PubMed] Int. J. Mol. Sci. 2019, 20, 440 12 of 12

34. Zhou, Y.-F.; Nan, B.; Nan, J.; Ma, Q.; Panjikar, S.; Liang, Y.-H.; Wang, Y.; Su, X.-D. C4-dicarboxylates sensing mechanism revealed by the crystal structures of DctB sensor domain. J. Mol. Biol. 2008, 383, 49–61. [CrossRef] [PubMed]

35. Cheung, J.; Hendrickson, W.A. Crystal structures of C4-dicarboxylate ligand complexes with sensor domains of histidine DcuS and DctB. J. Biol. Chem. 2008, 283, 30256–30265. [CrossRef][PubMed] 36. Dutzler, R.; Campbell, E.B.; Cadene, M.; Chait, B.T.; MacKinnon, R. X-ray structure of a ClC chloride channel at 3.0 Å reveals the molecular basis of anion selectivity. Nature 2002, 415, 287–294. [CrossRef][PubMed] 37. Hibbs, R.E.; Gouaux, E. Principles of activation and permeation in an anion-selective Cys-loop receptor. Nature 2011, 474, 54–60. [CrossRef][PubMed] 38. Dickson, V.K.; Pedi, L.; Long, S.B. Structure and insights into the function of a Ca2+-activated Cl− channel. Nature 2014, 516, 213–218. [CrossRef] 39. Geertsma, E.R.; Chang, Y.N.; Shaik, F.; Neldner, Y.; Pardon, E.; Steyaert, J.; Dutzler, R. Structure of a prokaryotic fumarate transporter reveals the architecture of the SLC26 family. Nat. Struct. Mol. Biol. 2015, 22, 803–808. [CrossRef] 40. Wöhlert, D.; Grötzinger, M.J.; Kühlbrandt, W.; Yildiz, Ö. Mechanism of Na(+)-dependent citrate transport from the structure of an asymmetrical CitS dimer. eLife 2015, 4, e09375. [CrossRef] 41. Kim, J.W.; Kim, S.; Kim, S.; Lee, H.; Lee, J.O.; Jin, M.S. Structural insights into the elevator-like mechanism of the sodium/citrate symporter CitS. Sci. Rep. 2017, 7, 2548. [CrossRef][PubMed] 42. Guskov, A.; Jensen, S.; Faustino, I.; Marrink, S.J.; Slotboom, D.J. Coupled binding mechanism of three sodium ions and aspartate in the homologue GltTk. Nat. Commun. 2016, 7, 13420. [CrossRef] [PubMed] 43. Zheng, H.; Wisedchaisri, G.; Gonen, T. Crystal structure of a nitrate/nitrite exchanger. Nature 2013, 497, 647–651. [CrossRef][PubMed] 44. Yan, H.; Huang, W.; Yan, C.; Gong, X.; Jiang, S.; Zhao, Y.; Wang, J.; Shi, Y. Structure and mechanism of a nitrate transporter. Cell Rep. 2013, 3, 716–723. [CrossRef][PubMed] 45. Fukuda, M.; Takeda, H.; Kato, H.; Doki, S.; Ito, K.; Maturna, A.D.; Ishitani, R.; Nureki, O. Structural basis for dynamic mechanism of nitrate/nitrite antiport by NarK. Nat. Commun. 2015, 6, 7097. [CrossRef] 46. Scopelliti, A.J.; Font, J.; Vandenberg, R.J.; Boudker, O.; Ryan, R.M. Structural characterisation reveals insights into substrate recognition by the glutamine transporter ASCT2/SLC1A5. Nat. Commun. 2018, 9, 38. [CrossRef] 47. Mitchell, P. A general theory of membrane transport from studies of bacteria. Nature 1957, 180, 134–136. [CrossRef] 48. Jardetzky, O. Simple allosteric model for membrane pumps. Nature 1966, 211, 969–970. [CrossRef] 49. Mulligan, C.; Fenollar-Ferrer, C.; Fitzgerald, G.A.; Vergara-Jaque, A.; Kaufmann, D.; Li, Y.; Forrest, L.R.; Mindell, J.A. The bacterial dicarboxylate transporter VcINDY uses a two-domain elevator-type mechanism. Nat. Struct. Mol. Biol. 2016, 23, 256–263. [CrossRef] 50. Drew, D.; Boudker, O. Shared molecular mechanisms of membrane transporters. Annu. Rev. Biochem. 2016, 85, 543–572. [CrossRef] 51. Colas, C.; Pajor, A.M.; Schlessinger, A. Structure-based identification of inhibitors for the SLC13 family pf Na+/dicarboxylate . Biochemistry 2015, 54, 4900–4908. [CrossRef][PubMed] 52. Huard, K.; Gosset, J.R.; Montgomery, J.I.; Gilbert, A.; Hayward, M.M.; Magee, T.V.; Cabral, S.; Uccello, D.P.; Bahnck, K.; Brown, J.; et al. Optimization of a dicarboxylate series for in vivo inhibition of citrate transport by the solute carrier 13 (SLC13) family. J. Med. Chem. 2016, 59, 1165–1175. [CrossRef][PubMed]

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