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Structural insights into the elevator-like mechanism of the sodium/citrate symporter CitS.

ABSTRACT: The sodium-dependent citrate transporter of Klebsiella pneumoniae (KpCitS) belongs to the 2-hydroxycarboxylate transporter (2-HCT) family and allows the cell to use citrate as sole carbon and energy source in anaerobic conditions. Here we present crystal structures of KpCitS in citrate-bound outward-facing, citrate-bound asymmetric, and citrate-free inward-facing state. The structures reveal that the KpCitS dimerization domain remains stationary throughout the transport cycle due to a hydrogen bond network as well as extensive hydrophobic interactions. In contrast, its transport domain undergoes a ~35° rigid-body rotation and a ~17?Å translocation perpendicular to the membrane to expose the substrate-binding site alternately to either side of the membrane. Furthermore, homology models of two other 2-HCT proteins based on the KpCitS structure offer structural insights into their differences in substrate specificity at a molecular level. On the basis of our results and previous biochemical data, we propose that the activity of the 2-HCT CitS involves an elevator-like movement in which the transport domain itself traverses the lipid bilayer, carrying the substrate into the cell in a sodium-dependent manner.

SUBMITTER: Kim JW

PROVIDER: S-EPMC5451387 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Structural insights into the elevator-like mechanism of the sodium/citrate symporter CitS.

Kim Ji Won JW Kim Subin S Kim Songwon S Lee Haerim H Lee Jie-Oh JO Jin Mi Sun MS

Scientific reports 20170531 1

The sodium-dependent citrate transporter of Klebsiella pneumoniae (KpCitS) belongs to the 2-hydroxycarboxylate transporter (2-HCT) family and allows the cell to use citrate as sole carbon and energy source in anaerobic conditions. Here we present crystal structures of KpCitS in citrate-bound outward-facing, citrate-bound asymmetric, and citrate-free inward-facing state. The structures reveal that the KpCitS dimerization domain remains stationary throughout the transport cycle due to a hydrogen b ...[more]

PMID: 28566738

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Project description:Background: The ability of thyroid follicular epithelial cells to accumulate iodide via the sodium/iodide symporter (NIS) is exploited to successfully treat most thyroid cancers, although a subset of patients lose functional NIS activity and become unresponsive to radioiodide therapy, with poor clinical outcome. Our knowledge of NIS regulation remains limited, however. While numerous membrane proteins are functionally regulated via dimerization, there is little definitive evidence of NIS dimerization, and whether this might impact upon radioiodide uptake and treatment success is entirely unknown. We hypothesized that NIS dimerizes and that dimerization is a prerequisite for iodide uptake. Methods: Coimmunoprecipitation, proximity ligation, and Förster resonance energy transfer (FRET) assays were used to assess NIS:NIS interaction. To identify residues involved in dimerization, a homology model of NIS structure was built based on the crystal structure of the dimeric bacterial protein vSGLT. Results: Abundant cellular NIS dimerization was confirmed in vitro via three discrete methodologies. FRET and proximity ligation assays demonstrated that while NIS can exist as a dimer at the plasma membrane (PM), it is also apparent in other cellular compartments. Homology modeling revealed one key potential site of dimeric interaction, with six residues <3Å apart. In particular, NIS residues Y242, T243, and Q471 were identified as critical to dimerization. Individual mutation of residues Y242 and T243 rendered NIS nonfunctional, while abrogation of Q471 did not impact radioiodide uptake. FRET data show that the putative dimerization interface can tolerate the loss of one, but not two, of these three clustered residues. Conclusions: We show for the first time that NIS dimerizes in vitro, and we identify the key residues via which this happens. We hypothesize that dimerization of NIS is critical to its trafficking to the PM and may therefore represent a new mechanism that would need to be considered in overcoming therapeutic failure in patients with thyroid cancer.

Dataset Information

Structural insights into the elevator-like mechanism of the sodium/citrate symporter CitS.

Publications

Structural insights into the elevator-like mechanism of the sodium/citrate symporter CitS.

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