Steered Molecular Dynamics Simulations And Free Energy Calculations Of Urea Transporter DvUT

Urea transporter has a profound role in the urinary concentrating process which is crucial for the survival of mammals. Considerable studies have been performed on urea transporters in the recent twenty years. Besides mammals, members of UT family are widely distributed in other domains of life, for example, birds, frogs, fishes, and even bacteria. Recently, the X-ray structure of Desulfovibrio vulagaris urea transporter, dvUT, a bacterial homologue of mammalian UTs, was reported with bound dimethyl urea at a resolution of 2.4 A, which revealed homotrimeric architecture with each subunit containing a continuous membrane-spanning pore. The channel contains a constricted region named selectivity filter (SF),～16 A long, that opens two wide vestibules on either side.Although the static structure of dvUT, the first X-ray structure of UT family, had been successfully solved, the detailed dynamics of conduction in dvUT is poorly understood. Here, a comprehensive mechanism underlying the conduction strategies of dvUT in atomatic details was explored through steered molecular dynamics (SMD) simulations. Both the constant-force SMD and the constant-velocity SMD approaches, which complement each other, were used. The direct calculation of electrostatic interaction of urea with its environment permitted us to identify the molecular determinants of conduction. Potential of mean force (PMF) for urea conduction in dvUT was successful reconstructed from multiple trajectories of cv-SMD simulation.Multiple sites contribute to the stabilization of urea in the SF. First of all, the oxygen ladders comprised by Gln24:Oε, Gln24:O,Val25:O, Val188:O, Glu187:O and Glu187:Oεpave a hydrogen bonding way for urea sliding along the channel axis. The displacements of urea through the SF were involved in the breakup of one series of hydrogen bonds between urea and oxygen ladders and the formation of another series. Moreover, in the central region of the SF, two hydroxyl groups of Thr130 and Thr294 make very strong and steady hydrogen bonds with urea molecule. Just like two arms, the two threonines optimize their hydroxyls'orientations to make hydrogen bonds with oxygen atom of urea, which stabilize urea molecule all the time in the central region. In addition, electrostatic field created by the a-helices dipole Pa and Pb provide the further stabilization of urea in the SF.There are two largest energetic barriers for urea permeation along the channel axis. They are located in the "slot" sandwiched by two aromatic rings in either side of the SF. Due to the narrow pore, and because urea is stabilized by only one oxygen atom from oxygen ladders, it is hard for urea molecule to reach the next oxygen atom of oxygen ladders.When urea moves into the SF, it is gradually dehydrated, and the electrostatic interaction energy between urea and water decreases accordingly. However, urea is hydrated and stabilized by water in either vestibule. So, water molecules serve like a vehicle which transport urea between the vestibules of dvUT and the bulk water. When urea comes in the vestibules, dvUT would attract urea to the entrance of the SF, which is revealed by the two minima in the PMF profile.Overall, the subtle balance between strong binding sites, energetic barriers of dvUT together with water molecules ensures the conduction and selectivity of urea through dvUT. Considering the high conservation, the conduction mechanism of dvUT can be applied to other members of the urea transporter family.