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Updated: Oct 17, 2025
Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions
Published on: February 10, 2022
Thermostability-based binding assays reveal complex interplay of cation, substrate and lipid binding in the bacterial
Connor D D Sampson1, Cristina Fàbregas Bellavista1, Matthew J Stewart1
1School of Biosciences, University of Kent, Canterbury, Kent CT2 7NH, U.K.
Abstract:
The divalent anion sodium 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.
Insights
Divalent anion sodium symporter (DASS) inhibitors offer therapeutic potential for metabolic diseases. This study reveals substrate binding is Na+-dependent and influenced by ligand structure and lipids, impacting transporter function.
Area of Science:
- Biochemistry
- Molecular Biology
- Membrane Transport
Background:
- Divalent anion sodium symporter (DASS) family transporters (SLC13) regulate metabolic homeostasis.
- Disruptions in DASS function are linked to protection from diabetes, obesity, and liver cancer.
- Inhibitors of DASS transporters are promising therapeutic targets for metabolic diseases.
Purpose of the Study:
- To investigate the interplay of coupling ion, substrate, and lipid environment interactions in the model DASS co-transporter, VcINDY.
- To elucidate the mechanism of Na+-dependent substrate binding and identify novel DASS ligands.
- To explore the regulatory role of lipids in the VcINDY transport cycle.
Main Methods:
- Utilized high-throughput thermostability-based interaction assays.
- Examined the VcINDY co-transporter from Vibrio cholerae.
- Analyzed substrate and lipid interactions.
Main Results:
- Demonstrated that substrate binding to VcINDY is dependent on Na+ ions.
- Identified that Na+-dependence is mediated by electrostatic attraction and binding site priming.
- Discovered novel DASS ligands requiring specific carboxylate group positioning for binding.
- Revealed a complex interplay between substrate and lipid interactions, suggesting a regulatory role for lipids.
Conclusions:
- Substrate binding in DASS transporters is critically dependent on sodium ions.
- Ligand design for DASS inhibitors should consider the precise spatial arrangement of carboxylate groups.
- Lipids play a dynamic and regulatory role in the transport mechanism of VcINDY.
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