2.24Å and 2.13Å structures of divalent anion sodium symporter (DASS) member LaINDY from L. acidophilus reveal highly-coordinated structural waters and a lipid at the dimer interface

Thesis

The divalent anion sodium symporter (DASS) family are low affinity dicarboxylate phosphate transporters found across all domains of life. The DASS family contains two distinct clades: cotransporters, which utilise a sodium gradient to drive transport across the plasma membrane; and exchangers which utilise dicaboxylate antiport. The cotransporter clade is most well characterised, and contains the human SLC13 family which are involved in the disease pathologies of diabetes, cancer and epilepsy. In contrast, the exchanger clade DASS members are exclusively found exclusively in plants and prokaryotes. Better understanding of the DASS-E clade may have implications for bacterial infections, bioproduction and agriculture. Through the model DASS exchanger LaINDY from Lactobacillus acidophilus this project aims to characterise the substrate binding, and the transport mechanism of the DASS exchangers.

Here I present 2.13Å and 2.24Å structures Cryo-EM structures of LaINDY, with the improved resolution revealing highly coordinated waters within the elevator domain that I hypothesise may have functional roles, and a phosphatidyl glycerol molecule modelled at the dimer interface. While the Cryo-EM maps were determined in the presence of ligands, no density was observed in the LaINDY binding site. This is consistent with in vitro binding experiments by tryptophan fluorescence quenching, which revealed LaINDYs low affinity for substrate at pH7.5. Ultimately my results reveal further complexity LaINDYs, transport mechanism, and substrate binding which may apply to the DASS exchanger clade as a whole.

Authors: Speedman, D

• DOI: 10.5287/ora-6rbe8onjz
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Lactobacillus acidophilus; cryo electron microscopy; structural water molecules; divalent anion sodium symporter; antiporter; phosphatidylglycerol
• Subjects: Lactobacillus acidophilus; Membrane proteins; Structure