Fig. 4: The potential proton-binding sites and proposed transport mechanism of DUR3.

a Titratable residues (aspartates, glutamates and histidines) throughout a DUR3 protomer. Aspartates and glutamates are shown as red spheres, and histidines are shown as pink spheres. D312 and E402 are positioned around the central pocket and labeled. b Structural comparison between DUR3 (red) and vSGLT (green). Na2 sites of vSGLT are shown as spheres. The residue H222 of DUR3 is depicted as sticks and labeled. His222^TM5 is located between TM1 and TM8. c Key sites for conformational transformation, residue P474^TM4 are shown as sticks. d, e Mutational Analysis of Critical Residues involved in proton-binding and conformational transitions. The bars represent the mean ± s.e.m (n = 3), biological independent samples. Two-tailed unpaired t-test, ****P < 0.0001. f Proposed Proton-Driven Transport Mechanism of DUR3. In the outward-facing conformation, urea enters the binding site with Asp312 and Glu402 protonated. The protonation of Asp312 and Glu402 then promotes isomerization to an inward-facing conformation, where the urea is released. The schematic illustrates three distinct states of DUR3: outward-open, occluded, and inward-open. Transmembrane helices TM1, TM6, TM8 and TM10 are depicted as cylinders, highlighted in red, blue, gray, and brown, respectively. The residues D312^TM6, Y315^TM6, E402^TM8 and P474^TM10 are shown as sticks. Urea molecules are represented by blue triangles, and hydrogen ions are shown as purple circles.