Fig. 9: Mechanism of noncompetitive blocking hUT-A2 by HQA2.

a The concentration dependent blockade of urea transport of wild type hUT-A2 and S279D, C285W mutants by HQA2. Values are mean ± SEM from three independent experiments (n = 3). b The HQA2 bound in the extracellular side of hUT-A2 without occupying the urea binding pocket. The hUT-A2 are represented in blue cartoon and the HQA2 shows as orange stick-ball. c The cryo-EM density (blue mesh) of HQA2 at the 4.0 σ contour level is located in the cavity surrounded by helices 3a, 3b–4b, and Pb, and the LECL, and ECL2b. d The key residues of the HQA2 binding pocket of hUT-A2 are represented by sticks and the residues involving in the “SCG” pocket are colored green. The H-bond are shown as red dashed line. e The cutaway view of the binding pocket of HQA2 (orange stick-ball). The EUBP, EBBP1-2 and the noncompetitive “SCG” pocket are represented by blue, red and green dashed line respectively. Residues forming polar interactions or hydrogen bonds with HQA2 are depicted using triangles. Residues only forming hydrophobic or van der Waals interactions are depicted as solid round circles. The HQA2 are represented by orange stick-ball and the hUT-A2 are represented by surface. f Sequence alignment of the structurally equivalent residues in the noncompetitive SCG pocket of hUT-A2 compared with other UT members including hUT-A3, rUT-A4 (rat UT-A4), mUT-A5 (mouse UT-A5), hUT-B, bUT-B (bovine UT-B) and zf-UT. Key residues of SCG pocket in hUT-A2 are shown in red font on the top line. Sequences with the same amino acids as the SCG pocket of hUT-A2 are represented in red, while those with non-conserved amino acids are represented in light blue. g The diagram of HQA2 engaging with the extracellular urea binding pocket (EUBP), L-P pocket and the noncompetitive SCG pocket with the interfaces represented by blue, violet and green dashed lines, respectively. Key residues interacting with L-P pocket and SCG pocket are shown in violet and green rectangles, respectively. The H-bond between HQA2 and residues S279 is shown as red dashed line.