Solvent accessibility changes in a Na+-dependent C4-dicarboxylate transporter suggest differential substrate effects in a multistep mechanism

Abstract

The divalent anion sodium symporter (DASS) family (SLC13) play critical roles in metabolic homeostasis, influencing many processes including fatty acid synthesis, insulin resistance, and adiposity. DASS transporters catalyse the Na+-driven concentrative uptake of Krebs cycle intermediates and sulfate into cells; disrupting their function can protect against age-related metabolic diseases and can extend lifespan. An inward-facing crystal structure and an outward-facing model of a bacterial DASS family member, VcINDY from Vibrio cholerae, predict an elevator-like transport mechanism involving a large rigid body movement of the substrate binding site. How substrate binding influences the conformational state of VcINDY is currently unknown. Here, we probe the interaction between substrate binding and protein conformation by monitoring substrate-induced solvent accessibility changes of broadly distributed positions in VcINDY using a site-specific alkylation strategy. Our findings reveal that accessibility to all positions tested are modulated by the presence of substrates, with the majority becoming less accessible in the presence of saturating concentrations of both Na+ and succinate. We also observe separable effects of Na+ and succinate binding at several positions suggesting distinct effects of the two substrates. Furthermore, accessibility changes to a solely succinate-sensitive position suggests that substrate binding is a low affinity, ordered process. Mapping these accessibility changes onto the structures of VcINDY suggests that Na+ binding drives the transporter into an as-yet-unidentified conformational state, involving rearrangement of the substrate binding site-associated re-entrant hairpin loops. These findings provide insight into the mechanism of VcINDY, which is currently the only structural-characterised representative of the entire DASS family.

Thermostability-based binding assays reveal complex interplay of cation, substrate and lipid binding in the bacterial DASS transporter, VcINDY

“…These data suggest that allosteric changes to nearby residues induced by Na + coordination, that cannot be replicated by coordination of Li + , are the trigger for succinate binding, and not the mere presence of positively charged ions in the binding site, as previously proposed (13). This suggestion of allosteric changes to nearby residues is supported by a recent study from our group revealing that VcINDY undergoes Na + -specific conformational changes to the hairpins loops that contribute to the binding site (57). However, a clear understanding of the role of Na + binding on substrate interactions requires an apo structure of VcINDY and further detailed characterisation of this interplay.…”

Topological analysis of a bacterial DedA protein associated with alkaline tolerance and antimicrobial resistance

“…Residues L53C and V55C are predicted to be positioned in the arm of HPout on the periplasmic side of the protein, but are only protectable by NEM (Figs 4e and 2b). While this may make the structural model and SCAM data seem incongruent, the exclusive accessibility of L53C and V55C to NEM can be explained by one of two possibilities: NEM, which is relatively hydrophobic compared to the negatively charged MTSES, is able to penetrate deeper into hydrophobic pockets on the periplasmic side of YqjA; or conformational changes occur in the re-entrant hairpin loops, as seen for hairpin-containing secondary active transporters [38][39][40][41], exposing that region of HPout to the cytoplasmic solution. The observation that S52C, which is obviously proximal to L53C and V55C due to its position in the primary sequence, is protectable by MTSES, which cannot penetrate the bilayer, very strongly supports the positioning of this region on the periplasmic side of the protein.…”

“…This structural feature has been identified in several integral membrane protein structures, most commonly associated with ion-driven secondary active transporters, including members of the excitatory amino acid transporter (EAAT) family and their prokaryotic homologue, Glt Ph [44,45], members of the divalent anion sodium symporter (DASS) family [46][47][48] and VcCNT [49], and without exception, the tips of these hairpin loops form a crucial site for substrate interactions. Re-entrant loops are also thought to be involved in gating access to the binding site and undergo conformational changes by which they control ingress and egress of the substrate(s) to and from the binding site [38][39][40][41]. Four functionally essential residues have been identified in YqjA: E39, D51, R130 and R136 [6,36,37], and while the exact role that these residues play in the function of YqjA is unknown, they coalesce at the tips of the re-entrant hairpins, demonstrating the importance of this structural motif in YqjA.…”

Topological analysis of a bacterial DedA protein associated with alkaline tolerance and antimicrobial resistance

“…Residues L53C and V55C are predicted to be positioned in the arm of HPout on the periplasmic side of the protein, but are only protectable by NEM (Fig 4E and 2B). While this may make the structural model and SCAM data seem incongruent, the exclusive accessibility of L53C and V55C to NEM can be explained by one of two possibilities; NEM, which is relatively hydrophobic compared to the negatively charged MTSES, is able to penetrate deeper into hydrophobic pockets on the periplasmic side of YqjA; or, conformational changes occur in the re-entrant hairpin loops, as seen for hairpin-containing secondary active transporters [32][33][34][35] , expose that region of HPout to the cytoplasmic solution. The fact that S52C, which is obviously proximal to L53C and V55C due to its position in the primary sequence, is protectable by MTSES, which cannot penetrate the bilayer, very strongly supports the positioning of this region on the periplasmic side of the protein.…”

“…This structural feature has been identified in several integral membrane protein structures, most commonly associated with ion-driven secondary active transporters, including members of the excitatory amino acid transporter (EAAT) family and their prokaryotic homologue, GltPh 38,39 , members of the divalent anion sodium symporter (DASS) family [40][41][42] , and VcCNT 43 , and without exception, the tips of these hairpin loops form a crucial site for substrate interactions. Re-entrant loops are also thought to be involved in gating access to the binding site and undergo conformational changes by which they control ingress and egress of the substrate(s) to and from the binding site 32,33,35,44 . Four functionally essential residues have been identified in YqjA; E39, D51, R130 and R136 6,30,31 , and while the exact role these residues play in the function of YqjA is unknown, they coalesce at the tips of the re-entrant hairpins, demonstrating the importance of this structural motif in YqjA.…”