Fig. 6: The mechanism of inhibition of hUT-A2 by 25a.

a The concentration dependent blockade of urea transport of hUT-A2 by 25a using urease assay. Data are shown as mean ± SEM of three independent experiments (n = 3). b The cryo-EM density (orange) of 25a in hUT-A2 homotrimer from top view or side view. The surfaces of three hUT-A2 monomer are represented by orange, green and blue, respectively. c, d The interactions between hUT-A2 and 25a in extracellular blocker binding pocket (EBBP) (c) or cytoplasmic blocker binding pocket (CBBP) (d). The hUT-A2 are represented in blue cartoon and the key amino acids are represented by blue sticks. The 25a was represented by orange stick-ball. e, f The cutaway view of binding pockets of 25a in extracellular side (e) or cytoplasmic side (f). In the extracellular side, the 25a occupied two subpockets, the EBBP1 (extracellular blocker binding pocket 1) and EUBP (extracellular urea binding pocket), represented by red or blue dashed line respectively. In the intracellular side, the 25a occupied CBBP1 (violet) and CUBP (blue). Residues forming polar interactions or hydrogen bonds with 25a are depicted using triangles. Residues only forming hydrophobic or van der Waals interactions are depicted as solid round circles. The 25a are represented by stick-ball and the hUT-A2 is represented by surface. g Sequence alignment of different UTs in the EBBP1 and CBBP1 regions. The nonconserved amino acids are highlighted with blue background. h The diagram of 25a engaging with the urea binding pocket or potential selective binding pocket with the interfaces represented by blue or violet dashed lines, respectively. Key residues interacting with 25a in these two pockets are shown in blue and violet rectangles, respectively. i, j Effects of hUT-A2 mutants F120L, F120V and L202V on the 25a activity toward hUT-A2 using urease assay. Values are mean ± SEM from three independent experiments (n = 3). The fold changes of IC₅₀ are calculated by dividing the mean value of the mutants by the mean value of the wild type.