Fig. 2: Oxalate-bound occluded OxlT structure.

a Close-up of the binding site in oxalate-bound OxlT (PDB ID: 8HPK). Dashed lines indicate potential hydrogen bonds and salt bridges. Also shown a magnified view of the bound oxalate molecule with atom labels. b Superposition of the substrate-binding site structures of OxlT (with the underlined labels) and NarK (green, with normal labels; PDB ID: 4U4W) based on the topological similarity of the amino acid residues interacting with the substrates. c Oxalate binding assay by GFP-TS. Data represent means ± SEM of the increases in melting temperatures caused by the addition of 3 mM potassium oxalate in three independent experiments. WT: wild-type. d Oxalate uptake assay using recombinant E. coli cells. The relative transport activities of the mutant OxlT to that of the WT OxlT measured on the same day set as 100%, while that of the cells without expressing OxlT³⁶ was set as 0%, are displayed. The bars represent the means of the technically duplicated measurements from a single experiment for each mutant. The results of the R272A, and K355Q mutants are reposted from the previous study³⁶ for comparison. e Proteoliposome oxalate uptake assay. The resultant oxalate concentrations in liposome lysates were measured and normalised to those of the WT OxlT at 60 min. The results of the liposomes without OxlT are shown as “empty.” The bars represent the means of the results in three (data at 0 min and 60 min for empty, WT, and R272A) or two (others) independent experiments. au: arbitrary unit. In panels c and e, the data were analysed by a two-sided one-way analysis of variance with Dunnett’s test with the WT OxlT as a control, and the exact P values are provided in Supplementary Table 6. *P < 0.05, **P < 0.01, ***P < 0.001. The red bars denote the mutants (or mutations at the same residue) exhibiting a loss of activity in previous studies^31,32,34. f Interdomain interactions closing the cavity to cytoplasm. g Ionic interaction network at the cytoplasmic side of OxlT. h Interdomain interactions closing the cavity to periplasm.