Fig. 4: Urea permeation mechanism of the human urea transporter hUT-A2.

a The urea transport channel of hUT-A2 is highlighted by blue dotted line with the inner urea molecules displayed as stick-balls. b The hydrogen bonding and hydrophobic interactions of urea in the urea transport channel of hUT-A2. The black dashed lines are used to separate So, Sm, and Si regions of the channel. The H-bond are shown as red dashed line and the cation-π interaction are shown as purple dashed lines. c Free energy surface (FES) of urea within the hUT-A2 channel. The simulation reveals three distinct low-energy states, which are characterized by prolonged urea residence times across defined regions within the channel and corresponds to the So, Sm, and Si regions respectively. d Simplified one-dimensional energy landscape highlighting the So, Sm, and Si states of urea within the hUT-A2 channel. e Metadynamics analysis depicts urea dynamically transitioning between three states-So, Sm, and Si-within the hUT-A2 channel, with distribution probabilities of 37%, 19%, and 34%, respectively. f–h Metadynamics simulations elucidate the passage of urea through the Sm region. This process involves a rotation of approximately 40°in the side chains of T176^5a and T338^5b, facilitating the rearrangement of T176^5a–T338^5b side chains (f). Notably, hydrogen bond formations between urea and specific residues, including T338^5b and T176^5a, are depicted (g). The rotation of side chains of T338/T176 and structural rearrangement of L127/L291 form a hydrophobic cavity in the Sm region, guiding urea transportation across the channel (h). The H-bond are shown as red dashed line. The blue arrows indicate the direction of movement of the urea molecule. i Urea permeability shown by the detection of remaining ¹⁴C-labeled urea in cell transfected with different hUT-A2. plasmids are shown as gray and green columns respectively, while cells transfected with pcDNA3.1 blank plasmids for control are shown as blank column. Data are represented as mean ± SEM from 3 independent experiments (n = 3). **P < 0.01, ***P < 0.001, statistical differences were determined by the two-sided unpaired Student’s t-test (compared with wild type). j Schematic diagram of urea permeation mechanism of hUT-A2.