Mapping Functionally Important Residues in the Na+/Dicarboxylate Cotransporter, NaDC1
Claire Colas1, Avner Schlessinger1, Ana M Pajor2
1Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai , New York, New York 10029, United States.
Biochemistry
|July 22, 2017
Summary
Researchers modeled outward-facing conformations of sodium-dependent dicarboxylate cotransporters (NaDC1) to identify key residues for ion and substrate binding. Mutagenesis revealed critical sites, enhancing understanding of SLC13 transporter mechanisms.
Area of Science:
- Structural biology and membrane transport
- Biochemistry and molecular mechanisms of transporters
Background:
- SLC13 family transporters mediate Na+ and dicarboxylate cotransport.
- Previous models of NaDC1 (SLC13A2) were based on inward-facing bacterial homologues.
- Understanding conformational changes is crucial for transporter function.
Purpose of the Study:
- To model the outward-facing conformation of rabbit and human NaDC1 (rbNaDC1 and hNaDC1).
- To identify key residues involved in Na+ and dicarboxylate binding sites.
- To elucidate the mechanism of substrate and ion recognition in SLC13 transporters.
Main Methods:
- Homology modeling using outward-facing bacterial VcINDY as a template.
- Site-directed mutagenesis of predicted cation and substrate binding site residues in rbNaDC1.
- Cysteine accessibility assays using MTSEA-biotin labeling.
Main Results:
- Mutagenesis of T474 (Na2 site) resulted in an inactive transporter.
- Mutagenesis of M539 (putative Na3 site) affected Na+ and Li+ affinity.
- Mutations at Y432 and T86 (substrate binding site) increased succinate K_m, with Y432C accessibility sensitive to Na+.
Conclusions:
- Structural models guided successful identification of functionally important residues in NaDC1.
- Specific residues T474, M539, Y432, and T86 are critical for ion and substrate binding.
- Findings provide a basis for developing conformationally specific inhibitors for SLC13 transporters.
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