Crystal structure of a bacterial homologue of the kidney urea transporter

Abstract

Urea is highly concentrated in the mammalian kidney to produce the osmotic gradient necessary for water re-absorption. Free diffusion of urea across cell membranes is slow owing to its high polarity, and specialized urea transporters have evolved to achieve rapid and selective urea permeation. Here we present the 2.3 A structure of a functional urea transporter from the bacterium Desulfovibrio vulgaris. The transporter is a homotrimer, and each subunit contains a continuous membrane-spanning pore formed by the two homologous halves of the protein. The pore contains a constricted selectivity filter that can accommodate several dehydrated urea molecules in single file. Backbone and side-chain oxygen atoms provide continuous coordination of urea as it progresses through the filter, and well-placed alpha-helix dipoles provide further compensation for dehydration energy. These results establish that the urea transporter operates by a channel-like mechanism and reveal the physical and chemical basis of urea selectivity.

Figure 1. dvUT mediated urea flux and binding

a. Timecourse of ¹⁴C-urea uptake in oocytes injected with dvUT cRNA (squares) or water (circles). b. Uptake of radio-labelled urea in the presence (red) or absence (black) of 1 mM phloretin. c. Saturation equilibrium urea binding by dvUT. The solid line represents fitting to the Hill equation. d. SPA-based ¹⁴C-urea equilibrium binding in the presence of increasing concentrations of urea (circles) and N,N′-dimethylurea (squares). The solid lines correspond to data fit with a single-site binding isotherm. Error bars in all panels are standard errors of the mean of 3–10 measurements.

Figure 2. Fold and oligomeric structure of dvUT

a. Cartoon representation of the dvUT protomer. The two-fold pseudo-symmetry axis, marked black as a black oval, is normal to the plane of the figure. Color of helices matches that in the topology diagram (Fig. S1b). b. Cartoon representation of the full dvUT trimer. The crystallographic three-fold symmetry axis is marked as a black triangle.

Figure 3. Structure of the dvUT pore and dimethylurea binding sites

a. A surface representation of the dvUT pore with T1a, T1b and the N- and C-termini removed. Oxygen and nitrogen atoms are colored in red and blue, respectively. b. Stereo view of residues lining the selectivity filter. Two dimethylurea, coordinates taken from the dvUT-dimethylurea complex, are shown in the So and Si sites. A gold atom that co-crystallizes with dvUT is shown in gold. c-e. Views of the So (c), Sm (d) and Si (e) regions of the selectivity filter for dvUT-dimethyl urea complex. The dark blue mesh corresponds to 2Fo-Fc electron density map contoured at 1.5 σ. The green mesh in the So and Si sites corresponds to 3.0 σ Fo-Fc electron density calculated with dimethylurea and the displayed water molecule omitted.

Figure 4. Schematic view of the selectivity filter

The selectivity filter is shown from two angles. The predicted locations of three urea molecules and their hydrogen bonding partners are on the left. In the perpendicular direction, the filter is compressed by phenylalanine and leucine side chains lining the walls of the pore (right). Helices contributing residues to the selectivity filter are represented as gray cylinders.