The ups and downs of elevator-type di-/tricarboxylate membrane transporters

Abstract

The divalent anion sodium symporter (DASS) family contains both sodium-driven anion co-transporters and anion/anion exchangers. The family belongs to a broader Ion Transporter Superfamily (ITS), which comprises 24 families of transporters, including those of AbgT antibiotic efflux transporters. The human proteins in the DASS family play major physiological roles and are drug targets. We recently determined multiple structures of the human sodium-dependent citrate transporter (NaCT) and the succinate/dicarboxylate transporter from Lactobacillus acidophilus (LaINDY). Structures of both proteins show high degrees of structural similarity to the previously-determined VcINDY fold. Conservation between these DASS protein structures and those from the AbgT familiy indicates that the VcINDY fold represents the overall protein structure for the entire ITS superfamily. The new structures of NaCT and LaINDY are captured in the inward- or outward-facing conformations, respectively. The domain arrangements in these structures agree with a rigid-body elevator-type transport mechanism for substrate translocation across the membrane. Two separate NaCT structures in complex with a substrate or an inhibitor allowed us to explain the inhibition mechanism and propose a detailed classification scheme for grouping disease-causing mutations in the human protein. Structural understanding of multiple kinetic states of DASS proteins is a first step towards the detailed characterization of their entire transport cycle.

Graphical Abstract

Liver cells import citrate as an energy source and a precursor for fatty acid synthesis. This uptake is accomplished via the Na⁺-driven citrate transporter (NaCT) in the plasma membrane. Inhibition of uptake can reduce fat storage. We determined the structure of human NaCT in complex with an inhibitor. The structure reveals how the compound inhibits transport and why it inhibits NaCT selectively over other homologous dicarboxylate transporters in humans.

Introduction

Citrate and various dicarboxylates are TCA cycle intermediates and important energy sources for the cell. Mammalian cells import citrate and dicarboxylates across the plasma membrane via the sodium-dependent citrate transporter (NaCT) and sodium-dependent dicarboxylate transporters 1 and 3 (NaDC1 and NaDC3). These proteins belong to the solute carrier 13 (SLC13) family. SLC13 proteins, together with homologs in bacteria, yeast, plants and flies, are part of the larger ubiquitous divalent anion sodium symporter (DASS) family [1–3]. Although substrate transport of most DASS proteins is sodium-gradient driven, some members of the family from bacteria and plants are exchangers of di- and tricarboxylates, which do not depend on a sodium gradient [4]. Finally, in the broader sense, DASS, along with the other 23 families of transporters for proton, lactate, gluconate, drugs and short-chain fatty acids, together form the Ion Transporter Superfamily (ITS, Pfam: CL0182) [5–7] with 86,180 identified proteins (as of May 21, 2021) (https://pfam.xfam.org/clan/CL0182).

In addition to being TCA cycle intermediates, citrate and dicarboxylates play multiple physiological roles in the cell. Not only is cytosolic citrate a major precursor for fatty acid synthesis, it also activates the first committed enzyme of this synthesis pathway [8] and inhibits a rate-limiting enzyme in glycolysis [9]. Therefore, in liver and adipose cells the cytosolic citrate concentration directly modulates the rate of fatty acid synthesis [10, 11]. In addition to its role in metabolism, citrate also functions as a precursor for the acetyl-CoA synthesis needed for histone acetylation, regulating DNA transcription and replication [12].

In addition to citrate released from mitochondria, cytosolic citrate is imported from the blood across the plasma membrane by the citrate transporter NaCT (SLC13A5) [13–15]. Clinically, elevated NaCT expression is observed in patients with obese, non-alcoholic fatty liver disease. Knocking down NaCT expression significantly decreased the lipid content in hepatic cell lines [16]. NaCT-knockout mice have increased hepatic mitochondrial biogenesis and reduced lipogenesis, which taken together protect the mice from obesity and insulin resistance [17]. Finally, in human fetuses and newborns, citrate import via NaCT is particularly crucial for nutrient uptake and brain development [18, 19]. Over fifty mutations in NaCT are found to cause a type of early-onset epileptic encephalopathy named SLC13A5-Epilepsy [20–22].

NaCT’s central role in fatty acid biosynthesis makes it a particularly attractive drug target for the treatment of obesity, diabetes and cardiovascular diseases through the reduction of fat storage [17, 23]. Several novel small molecules with a dicarboxylate moiety have been developed as NaCT inhibitors [24, 25]. In particular the compound PF-06649298 (PF2) exhibits both high affinity and specificity for NaCT. The location of the PF2 binding site in NaCT and the nature of the inhibition mechanism, however, are both controversial. Resolving this ambiguity in PF2 binding and inhibition, and explaining its specificity toward NaCT over other human dicarboxylate transporters, requires high-resolution structural information on NaCT.

Extensive functional and biochemical studies have been carried out on both mammalian and bacterial DASS proteins. Available evidence indicates that Na⁺-driven DASS cotransporters operate via an ordered sequence, with Na⁺ binding preceding substrate binding and Na⁺ release following substrate release (Fig. 1) [26–30].

Fig. 1.

Kinetic cycle and outward- and inward-facing conformations of DASS proteins. A. Kinetic cycle of Na+-dependent DASS co-transporters. C_o: outward-facing state. C_i: inward-facing state. S: substrate. B. Schematic drawing showing the outward- and inward-facing conformations of DASS proteins. The proteins form homodimers, with each protomer comprising a scaffold domain (green) and a transport domain (pink). For substrate translocation across the membrane, the transport domain undergoes elevator-type movements, exposing the substrate binding site alternatively to the extracellular space and the cytosol. C. Outward-facing structure of LaINDY [4]. Although the protein is a succinate/dicarboxylate exchanger, independent of the Na⁺-gradient, its structure is homologous to the Na⁺-dependent dicarboxylate transporter VcINDY. D. Inward-facing structure of Na⁺-dependent dicarboxylate transporter from Vibrio cholerae VcINDY [4]. E. Representative MD structure from the simulation of the LaINDY C_o-S state. F. Representative MD structure from the simulation of the LaINDY C_i-S state. Transition between the C_o-S state to the C_i-S state in LaINDY has been visualized in MD simulations [4]. The scaffold and transport domains in C – F are colored similarly as in B. Figure reproduced from [4], and the figure was generated using PyMol and VMD.

VcINDY structure in its inward-facing conformation

For mechanistic understanding, the first high-resolution structure solved from any member of the DASS family or the ITS superfamily was that of the Na⁺-driven dicarboxylate transporter VcINDY from Vibrio cholerae, published in 2012 [31]. The structure captured VcINDY in an inward-facing (C_i-Na⁺-S) state, with its substrate-binding site open to the cytosol (Fig. 1; Fig. 2). The protein forms a homodimer and each protomer consists of both a scaffold and a transport domain (Fig. 1D). Of the 11 transmembrane helixes in a protomer, TM1–4 and TM7–9 form the scaffold domain, while the transport domain contains TM5, 6, 10, 11, as well as two hairpins, HP_in and HP_out (Fig. 2A,B). The N-terminal half of the protein TM2–6 and the C-terminal half TM7–11 are related by an inverted pseudo-twofold symmetry. In the transport domain, two carboxylate moieties of the bound substrate are coordinated by two conserved Ser-Asn-Thr (SNT) sequences, which are signature motifs for di-/tricarboxylate DASS transporters and also form part of the Na⁺-binding sites Na1 and Na2.

Fig. 2.

Structure conservation of the ITS superfamily and the VcINDY fold. A. Transmembrane topology of VcINDY. The N-terminal half (except TM1) is related to the C-terminal half by an inverted repeat symmetry. Each half contains a hairpin helix and a broken helix with a long loop in the middle. The tip of the hairpin helix and the long loop form a sodium binding site. The substrate binding site is located between these two sodium sites [31]. B. Inward-facing structure of VcINDY, viewed from the membrane plane (left) and from the extracellular space (right) [4, 31, 51]. The protein is a member of the DASS family. C. Structure of antimicrobial efflux protein YdaH [41]. This protein, along with the MtrF protein, are members of the AbgT family. DASS and AbgT families all belong to the Ion Transporter Superfamily. It is proposed that all members of the ITS superfamily adopts the VcINDY fold. D. Structure of the bacterial concentrative nucleotide transporter VcCNT [42, 43]. Although not a member of the ITS superfamily, the transport domain of VcCNT is similar to that of VcINDY. All structures shown in B – D are in their inward-facing conformation, and the helices are colored by the rainbow scheme. Panels A and B of the figure reproduced from [31], and the figure was generated using PyMol.

The height of the scaffold domain is insufficient to span the entire lipid bilayer of the membrane, which necessitates the homodimerization of the protein. Structural information, along with cross-linking and computer modeling using an inverted repeat symmetry in the protein sequence [31, 32], suggests that DASS proteins operate through an elevator-type transport mechanism. In this mechanism, the transport domain moves as a rigid body across the membrane, exposing the substrate and sodium binding sites alternatively to the two sides of the membrane to realize substrate transport. However, the conformational changes associated with DASS family transport remained unknown (Fig. 1B).

LaINDY structure in its outward-facing conformation

As a first step in understanding the conformational changes needed to translocate the substrate between the two sides of the membrane in DASS proteins, we determined the structure of another bacterial member of the transporter family, succinate/dicarboxylate exchanger from Lactobacillus acidophilus (LaINDY) (Fig. 1C) [4]. Although a sodium-independent exchanger, LaINDY nevertheless forms a dimer with the same fold as VcINDY. Importantly, the LaINDY structure we captured is in an outward-facing, C_o conformation. The individual domains exhibit strong structural homology to VcINDY, with backbone r.m.s.d.s of 2.8 Å and 2.0 Å for the scaffold and transport domains, respectively. Comparison of domain positions between the LaINDY C_o structure with the VcINDY C_i structure confirms the elevator-type of rigid-body conformational changes needed for substrate translocation in DASS proteins.

Unlike Na⁺-dependent cotransporters in the DASS family, the two “Na⁺ binding sites” in the DASS exchanger subfamily are each occupied by a conserved, positively-charged residue that acts as Na⁺ surrogate [4]. This observation strongly suggests that charge compensation [33, 34] is crucial for reducing the energy barrier in DASS proteins for translocating charged substrates across the hydrophobic lipid bilayer. Like Brownian motion [35], such charge compensation may be important to the transport of charged substrates in other membrane transporters, both co-transporters and exchangers [36].

Conformational changes between outward- and inward-facing states

To further characterize the conformational changes of DASS proteins during substrate translocation, our collaborators Trebesch and Tajkhoshid carried out biased molecular dynamics (MD) simulations on LaINDY [4] (Fig. 1E,F). The simulations revealed a rigid body movement of the transport domain between the C_o-S and C_i-S states through a 39° rotation and an 8.3 Å translation (Fig. 1C), agreeing well with the differences in transport domain position between VcINDY and LaINDY. Two horizontal helixes, H4c and H9c, are rigidly attached to the scaffold domain in order to support this rotation and translation. Such conformational changes are believed to be conserved among DASS family members, including human NaCT.

The VcINDY fold represents the overall structure for the entire Ion Transport Superfamily

The structural conservation between the VcINDY and LaINDY proteins provides strong evidence that the VcINDY fold is representative of the entire DASS family. Based on observations on other membrane transporter superfamilies [37, 38], it follows that other families in the ITS superfamily may adopt the same VcINDY fold [31, 39]. Furthermore, this suggests that members of the ITS superfamily bind substrate at the same location as seen in VcINDY and use an elevator-type transport mechanism for substrate translocation across the membrane.

In addition to the DASS family, another group of proteins in the ITS superfamily that has been structurally characterized is the AbgT family. The structures of MtrF from Neisseria gonorrhoeae and YdaH from Alcanivorax borkumensis were reported in 2015 [40, 41]. Both AbgT proteins are believed to function as antimicrobial efflux pumps. Like VcINDY, both MtrF and YdaH exist as homodimers, with the scaffold domains forming the dimer interface (Fig. 2C). Extensive bioinformatics analysis has shown structures of these AbgT proteins are highly similar to that of VcINDY [39]. The backbone α-carbon r.m.s.d.s of MtrF and YdaH versus VcINDY are 4.3 Å and 4.6 Å, respectively. If only the transport domains are considered, the differences drop to 3.3 Å and 2.9 Å respectively. At the equivalent position of the Na2 in VcINDY, a density for sodium is found in the YdaH structure. Such structural conservation between distantly related proteins supports the notion that the VcINDY fold represents the entire ITS superfamily.

While the fold and oligomeric state of VcINDY represent the members of the ITS superfamily, the structural conservation of its transport domain alone actually extends beyond the ITS superfamily. The transport domain of the concentrative nucleotide transporters (CNTs) [42, 43] share high structural similarity with that of VcINDY (Fig. 2D). The Vibrio cholerae CNT (VcCNT) and VcINDY proteins have a backbone α-carbon r.m.s.d. of 6.2 Å over their transport domains (HP1, TM4, TM5 from the N-terminal half, and HP2, TM7, TM11 from the C-terminal half in VcCNT; HP_in, TM5, TM6, and HP_out, TM10, TM11 in VcINDY). However, when the N- and C-terminal halves are compared separately, the r.m.s.d.s drops to 3.8 Å and 6.1 Å, respectively, indicating varied assembly of the N- and C-terminal halves in the transport domains on the two transporters. The substrate translocation in VcCNT is driven by one Na⁺, and the Na⁺ site is structurally equivalent to the Na1 site in VcINDY. Interestingly, the scaffold domain architecture of VcCNT is completely different, and the protein forms a homotrimer. Three long horizontal helixes, one from each protomer, provide a backbone framework for the movement of the transport domains. Additionally, structural modeling and mutagenesis analysis have shown that the transport domain of the Na⁺-dependent phosphate transporter NaPi-II, a member of the SLC34 family, is similar to the transport domain of VcINDY [44, 45].

Finally, it is worth comparing the structures of the bacterial Na⁺-driven citrate transporter CitS [46, 47] to that of VcINDY [48]. The two proteins have an insignificant amino acid sequence identity of ~14%. Whereas CitS also forms a dimer, with each protomer consisting of a scaffold and a transport domain, the r.m.s.d. of the backbone α-carbons between CitS and VcINDY is ~20 Å. In contrast to the VcINDY transport domain that consists of two HP-TM-TM motifs (Fig. 2A), in the CitS motifs the hairpin is sandwiched between two transmembrane helices (i.e. TM-HP-TM). As a result, the hairpins insert into the membrane in opposite directions between the two transporters. Interestingly, two hairpins in CitS intersect in the middle of the transporter and form the first Na⁺ binding site, with a second Na⁺ located toward the cytosol. This arrangement is distinct from VcINDY, where two hairpins each interact with a long loop to form two separate sodium sites at the same depth from the membrane surface.

Human NaCT structure in its inward-facing conformation

We recently determined an inward-facing structure of the human citrate transporter NaCT in complex with citrate and Na⁺, the first structure of a mammalian DASS protein (Fig. 3A) [49]. Requiring a specialized tilted cryo-EM data collection strategy [50], the NaCT structure resembles its bacterial homologs (Fig. 1) [4, 31, 51]. The dimer organization, the domain architecture and the transmembrane topology are all similar. In the transport domain, the two SNT signature motifs, the locations of the substrate-binding site and the Na⁺ sites Na1 and Na2 are all conserved with VcINDY. Due to weak scattering to electrons by the lithium ions needed for stabilizing the protein for cryo-EM structure determination, the locations of the two remaining Na⁺ sites have yet to be identified. Similarly, as the triple-negatively charged citrate is particularly sensitive to electron radiation damage, the quality of the substrate density is insufficient for detailed description on how the protein discriminates various di/-tricarboxylates.

Fig. 3.

Structural basis of the inhibition mechanism and selectivity of PF2 in NaCT [49]. A. Cryo-EM structure of the NaCT-PF2 complex, viewed from within the membrane plane. This structure is highly homologous to that of the protein bound to the citrate substrate. B. PF2 binding site viewed from within the membrane. The dicarboxylate moiety of PF2 binds at the citrate site in the transport domain (pink), whereas PF2’s benzene ring and isobutyl group interact with the scaffold domain (green) of NaCT. C. Schematic drawing showing the proposed inhibition mechanism of NaCT by PF2. In each protomer, the dicarboxylate moiety of PF2 (red circle) binds to the citrate site of NaCT in its inward-facing, C_i-Na⁺-S state, and blocks sodium release from the Na1 and Na2 sites. At the same time, the modified benzene ring of PF2 (yellow triangle) interacts with the scaffold domain, preventing the transition of the transport domain to an outward-facing conformation. Together, the two types of interactions arrest the transporter in its C_i-Na⁺-S state and inhibit transport. D. PF2 binding site shown as electrostatic surface, colored by electrostatic potential of the protein atomic model. Figure and accompanying legend reproduced from [49], and the figure was generated using PyMol.

Inhibition mechanism of NaCT by Pfizer compounds

Building on the NaCT-Citrate structure, we also examined the specificity and inhibition mechanism of the Pfizer compounds by determining the structure of NaCT in the presence of sodium and Pfizer compound PF2 (Fig. 3) [49]. Notably, within the NaCT-PF2 complex structure was an additional density, easily identified as the inhibitor.

The PF2 compound binds at the same location on the cytosolic side of the protein as the substrate in our NaCT-citrate structure (Fig. 3B). Such overlapping binding sites supports the previously proposed mechanism of competitive inhibition [24, 25, 52]. Interactions of PF2 with the cytosolic surface of the scaffold domain block the sliding movement of the transport domain towards the extracellular direction (Fig. 3C). Simultaneously, PF2 binding with the transport domain prevents sodium release and stops the C_i-Na⁺-S to C_i-Na⁺ state transition (Figs. 1A,B). Unable to undergo structural or chemical transitions, NaCT bound to PF2 is thus inhibited from citrate transport.

PF2 is highly specific for human NaCT, with significantly weaker inhibition of the homologous dicarboxylate transporters, NaDC1 and NaDC3 [24, 25, 52]. The NaCT structures reveal this selectivity is caused by the presence of Gly409 in NaCT’s scaffold domain, which directly interacts with the isobutyl group of PF2 (Fig. 3D). In contrast, asparagine is found at the equivalent position in both NaDC3 and NaDC1. This larger side chain would sterically clash with PF2, reducing its binding and inhibition to the two dicarboxylate transporters. Finally, we observed that the large inhibitor binding pocket can be utilized to increase the affinity of the inhibitor and its selectivity towards NaCT.

NaCT structure allowed detailed classification of SLC13A5-Epilepsy mutations

The locations of the SLC13A5-Epilepsy mutations within the NaCT structure allowed us to explain how each abolish NaCT’s transport activity [20–22]. Previously, cellular localization and biochemical analyses of clinical loss-of-function mutants [18, 19] had suggested two types of pathogenesis [22]: Type I mutations affect the localization and stability of the protein, while Type II directly abolishes transport activity. Now we can further classify each type based on their location in the three-dimensional structure of the protein. Among the Type I mutations, Type Ia are nonsense or frame shift mutations that result in incomplete protein molecules, while Type Ib mutations are hypothesized to destabilize the transporter. Within the fully expressed but inactive Type II mutants, Type IIa are close to the citrate and Na⁺ binding sites and are therefore expected to alter substrate or cation binding. Finally, mutations located at the interface between the scaffold and transport domains (Type IIb) likely obstruct the movement of the transport domain during substrate translocation. This better understanding of each type of mutation suggests that separate therapeutic strategies will be most effective in treating SLC13A5-Epilepsy.

Unanswered mechanistic questions

The VcINDY structure represents not only that of the DASS family, but also represents a general fold found across the entire ITS superfamily of membrane transporters. Structural determination of DASS proteins in both inward- and outward-facing conformations represent significant advances in the mechanistic understanding of this family of transporters. The human NaCT structures provide a solid foundation for understanding the mechanism of inhibition and molecular pathogenesis of SLC13A5-Epilepsy. These recent structures pose new questions for the field: Where are the two additional bound sodium ions in NaCT? And what is the structural basis for substrate – sodium coupling in DASS proteins [53]? How does the transporter convert between the outward-facing and inward-facing states? Perhaps, structural biology and biochemical techniques, along with molecular dynamics simulations and experimental techniques for studying protein dynamics such as single molecular fluorescence spectroscopy, electron paramagnetic resonance and atomic force microscopy [54], will allow us to better understand the entire transport cycle of DASS proteins. Instead of looking at snapshots, we will be watching molecular movies.

Abbreviations

AbgT

the p-aminobenzoyl-glutamate transporter family

CitS

bacterial sodium-citrate symporter

CNT

concentrative nucleotide transporter

cryo-EM

cryo-electron microscopy

DASS

divalent anion sodium symporter

HP

hairpin helix

ITS

Ion Transporter Superfamily

LAINDY

succinate/dicarboxylate exchanger from Lactobacillus acidophilus

MtrF

antimicrobial efflux protein from Alcanivorax borkumensis

NaCT

mammalian sodium-dependent citrate transporter

NaDC1

mammalian sodium-dependent dicarboxylate transporter 1

NaDC3

mammalian sodium-dependent dicarboxylate transporter 3

PF2

Pfizer compound PF-06649298

r.m.s.d.

root-mean-square deviation

SLC13

solute carrier family 13

TCA cycle

tricarboxylic acid cycle

TM

transmembrane α-helix

VcCNT

concentrative nucleotide transporter from Vibrio cholerae

VcINDY

sodium-dependent dicarboxylate transporter from Vibrio cholerae

YdaH

antimicrobial efflux protein from Alcanivorax borkumensis

Data Accessibility Statement

The unaligned electron micrographs used for the NaCT-citrate structure have been deposited in EMPIAR (ID: EMPIAR-10728).