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

Sampson, Connor Dereck David, Stewart, Matthew J, Mindell, Joseph A, Mulligan, Christopher (2020) Solvent accessibility changes in a Na+-dependent C4-dicarboxylate transporter suggest differential substrate effects in a multistep mechanism. Journal of Biological Chemistry, . ISSN 0021-9258. E-ISSN 1083-351X. (doi:10.1074/jbc.RA120.013894) (KAR id:84472)

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http://dx.doi.org/10.1074/jbc.RA120.013894

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.

Item Type: Article
DOI/Identification number: 10.1074/jbc.RA120.013894
Uncontrolled keywords: Anion transport, conformational change, membrane transport, protein conformation, protein chemical modification, transmembrane domain, transporter, tricarboxylic acid cycle, Vibrio cholerae.
Subjects: Q Science > QP Physiology (Living systems) > QP517 Biochemistry
Divisions: Divisions > Division of Natural Sciences > School of Biosciences
Depositing User: Christopher Mulligan
Date Deposited: 01 Dec 2020 14:11 UTC
Last Modified: 02 Dec 2020 15:46 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/84472 (The current URI for this page, for reference purposes)
Mulligan, Christopher: https://orcid.org/0000-0001-5157-4651
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