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.

A solid phase synthesis of des-myo-inositol mycothiol disulfide and its alpha-configured methyl- and benzyl-glycoside derivatives has been developed. Kinetic characterisation of these compounds has demonstrated their viability as alternative substrates for use in mycothiol disulfide reductase enzyme assays.

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The divalent anionsodium symporter (DASS) family of transporters (SLC13 family in humans) are key regulators of metabolic homeostasis, disruption of which results in protection from diabetes and obesity, and inhibition of liver cancer cell proliferation. Thus, DASS transporter inhibitors are attractive targets in the treatment of chronic, age-related metabolic diseases. The characterisation of several DASS transporters has revealed variation in the substrate selectivity and flexibility in the coupling ion used to power transport. Here, using the model DASS co-transporter, VcINDY from Vibrio cholerae, we have examined the interplay of the three major interactions that occur during transport: the coupling ion, the substrate, and the lipid environment. Using a series of high-throughput thermostability-based interaction assays, we have shown that substrate binding is Na+-dependent; a requirement that is orchestrated through a combination of electrostatic attraction and Na+-induced priming of the binding site architecture. We have identified novel DASS ligands and revealed that ligand binding is dominated by the requirement of two carboxylate groups in the ligand that are precisely distanced to satisfy carboxylate interaction regions of the substrate binding site. We have also identified a complex relationship between substrate and lipid interactions, which suggests a dynamic, regulatory role for lipids in VcINDY’s transport cycle.

Members of the divalent anion sodium symporter (DASS) family (SLC13 in humans) play critical roles in metabolic homeostasis, influencing many processes including fatty acid synthesis, insulin resistance, adiposity, and lifespan determination. 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 VcINDY conformation using a site-specific alkylation strategy to probe the solvent accessibility of several broadly distributed positions in VcINDY in the presence and absence of substrates (Na + and succinate). Our findings reveal that accessibility to all positions tested can be modulated by the presence of substrates, with the majority becoming less accessible in the presence of Na + .Mapping these solvent accessibility changes onto the known structures of VcINDY suggests that Na + binding drives the transporter into an as-yet-unidentified intermediate state. We also observe substantial, separable effects of Na + and succinate binding at several amino acid positions suggesting distinct effects of the two substrates. Furthermore, analysis of a solely succinatesensitive residue indicates that VcINDY binds its substrate with a low affinity and proceeds via an ordered process in which one or more Na + ions must bind prior to succinate. These findings provide insight into the mechanism of VcINDY, which is currently the only structural-characterised representative of the entire DASS family.