Thermostability-based binding assays reveal complex interplay of cation, substrate and lipid binding in the bacterial DASS transporter, VcINDY

Connor D D Sampson,Matthew J Stewart,Cristina Fàbregas Bellavista,Christopher Mulligan

doi:10.1042/bcj20210061

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.

Highlights

The SLC13 transporter family catalyses the transport of dicarboxylates, such as succinate, the tricarboxylate citrate, and sulfate across the plasma membrane of human cells
Ligand-induced stabilisation has been observed for several proteins, including transporters and globular proteins, and manifests itself as an increase in the melting temperature (Tm) of the protein, which is measurable by several methods, including circular dichroism (CD), differential scanning fluorimetry (DSF), and radioactive ligand binding, among many others [44,45,46]
We have used a series of thermostability-based binding assays to reveal insight into the interactions of cations, substrates and lipids with VcINDY, which is the structural model for the divalent anion sodium symporter (DASS) transporter family

Summary

Introduction

The SLC13 transporter family catalyses the transport of dicarboxylates, such as succinate, the tricarboxylate citrate, and sulfate across the plasma membrane of human cells. The five members of the SLC13 family, NaDC1, NaDC3, NaCT, NaS1 and NaS2, share ∼50% identity with each other, and have distinct functional properties in an array of tissues in the human body including the liver, kidney, brain, and placenta [1]. Functional disruption of DASS transporters in several model organisms has revealed their activity profoundly influences metabolism. Disrupting a DASS transporter (NaCT) in a mouse model resulted in resistance to adiposity and insulin insensitivity when fed a high fat diet [7], reinforcing the link to metabolism and unveiling DASS family members as extremely attractive therapeutic targets in the treatment of age-related metabolic diseases

Methods

Results

Conclusion