ARTICLES

Crystal structure of a bacterial homologueof the kidney urea transporterElena J. Levin1, Matthias Quick2,3 & Ming Zhou1

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 evolvedto achieve rapid and selective urea permeation. Here we present the 2.3 A structure of a functional urea transporter from thebacterium Desulfovibrio vulgaris. The transporter is a homotrimer, and each subunit contains a continuousmembrane-spanning pore formed by the two homologous halves of the protein. The pore contains a constricted selectivityfilter that can accommodate several dehydrated urea molecules in single file. Backbone and side-chain oxygen atoms providecontinuous coordination of urea as it progresses through the filter, and well-placed a-helix dipoles provide furthercompensation for dehydration energy. These results establish that the urea transporter operates by a channel-likemechanism and reveal the physical and chemical basis of urea selectivity.

Urea is ubiquitous in nature. Bacteria take up urea and convert it toammonia for use as a nitrogen source, and in certain enteric patho-gens, a buffer for surviving the extreme acidic conditions in thestomach1,2. In higher organisms such as mammals, urea is producedas an end product of protein catabolism because it is less toxic thanammonia and more soluble than uric acid. As well as being a vehicle fornitrogen excretion, urea is used as an osmolyte. For example, sharksand rays use urea to maintain isosmotic with sea water3, and mammalsare able to concentrate urea by hundreds of fold in the kidney toprovide the osmotic gradient essential for water re-absorption4,5.

Urea has a stronger dipole moment (4.6 D) than water (1.8 D), andits unassisted diffusion across lipid bilayers is slow6. There are at leastfour families of transporters that facilitate selective permeation ofurea: an ATP-dependent ABC type urea transporter7; an ion-motiveforce-dependent urea transporter8; an acid-activated urea channelthat belongs to the urea/amide channel family2; and finally the ureatransporter (UT) family, which is the most widely distributed familyand the focus of this research. Since the first cloning of a UT frommammalian kidney by expression cloning9, UT members have beenfound in bacteria, fungi, insects and many vertebrates including allmammals.

Previous characterizations of mammalian and bacterial UTs1,9–12

have demonstrated that they facilitate the diffusion of urea and ureaanalogues along their concentration gradients at rates between 104

and 106 s21 (refs 10, 13), consistent with a channel-like mechanism.In the absence of structural information, how urea transporterscoordinate and stabilize an at least partially dehydrated urea toachieve selectivity and facilitate permeation remains unknown. Toaddress these questions, we focused on the functional and structuralcharacterization of a UT from the bacterium D. vulgaris, dvUT,which is homologous to mammalian UTs (Supplementary Fig. 1a).All UT sequences have two homologous halves that probably arosefrom the duplication of an ancient gene14, and are predicted to con-tain ten transmembrane helices. Identity between the transmem-brane domains of dvUT and human UTs is around 35%, andclusters of highly conserved residues are distributed throughout theprimary sequence. We demonstrate that dvUT is a functional urea

transporter, and present the atomic resolution structure of dvUT andthe molecular basis it reveals for selective permeation of urea.

Functional characterization of dvUT

The function of dvUT was examined in two assays. First, urea fluxthrough dvUT was measured in a tracer uptake assay2,9. dvUT wasexpressed in Xenopus laevis oocytes by injection of complimentaryRNA, and the uptake of 14C-labelled urea (,181 mM) by individualoocytes was measured. The uptake of labelled urea increased withtime and reached ,50 pmol per oocyte in about 60 min (Fig. 1a).Assuming the volume of an oocyte is ,0.25 ml (ref. 9), the estimatedintracellular concentration of labelled urea is ,200 mM, essentially inequilibrium with the extracellular urea. In contrast, urea accumula-tion in water-injected oocytes showed a much slower time course andhad not reached equilibrium even after 120 min (Fig. 1a).

Because urea flux through mammalian UTs is inhibited by phlo-retin9,15, we examined its effect on both human UT-B and dvUT(Fig. 1b). The uptake of urea by oocytes expressing UT-B reachedequilibrium in ,15 min, comparable to a previous report9 and fasterthan oocytes expressing dvUT. However, this does not necessary indi-cate that dvUT has a slower flux rate because the relative amounts ofthe two functional UT proteins expressed on the membrane areunknown. Phloretin (1 mM) inhibited facilitated urea transportthrough UT-B and dvUT (Fig. 1b) by 86% and 87%, respectively,suggesting a similar mode of interaction of phloretin with bothproteins. In fact, phloretin also inhibits a urea transporter fromActinobacillus pleuropneumoniae (apUT)11, indicating that a commonarchitecture is probably shared by prokaryotic and eukaryotic UTs.

Second, equilibrium binding of urea to dvUT was measured in ascintillation proximity assay (SPA)16,17. In this assay, purified His-tagged dvUT was immobilized on copper-coated scintillation beadsthat emit light only when a radioactive ligand stably binds to dvUT.The addition of 14C-labelled urea induced light emission from thescintillant, indicating urea binding to dvUT. Fitting the data to theHill equation revealed an apparent equilibrium dissociation constant(Kd) of 2.3 6 0.14 mM (mean 6 standard error) (Fig. 1c) with a Hillcoefficient of 3.4 6 0.7, indicative of cooperative binding of about

1Department of Physiology & Cellular Biophysics, 2Department of Psychiatry, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York, New York10032, USA. 3Division of Molecular Therapeutics, New York State Psychiatric Institute, 1051 Riverside Drive, New York, New York 10032, USA.

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three urea molecules per molecule of dvUT, and suggesting thatdvUT has several urea-binding sites.

Interaction of N,N9-dimethylurea (DMU)—a urea analogue thatwas used in the structural studies—with dvUT was measured in anSPA-based competition assay (Fig. 1d). The addition of increasingconcentrations of DMU to dvUT incubated with a fixed concentra-tion of labelled urea caused progressive loss of light emission, indi-cating that the bound urea was displaced. The concentration of DMUnecessary to achieve half-maximum inhibition (IC50) was1.4 6 0.2 mM. As a control experiment, unlabelled urea was usedfor isotopic dilution of the 14C-labelled urea. A 50% reduction of14C-urea binding was obtained at 2.3 6 0.7 mM urea, consistent withthe Kd measured in the previous equilibrium binding assay (Fig. 1c)and slightly higher than the IC50 of DMU (Fig. 1d). These resultsindicate that DMU and urea may occupy similar sites in dvUT.

Channel architecture

The structure of dvUT was determined by single-wavelength anoma-lous dispersion (SAD) using a mercury-derivatized crystal, andrefined to 2.3 A using a gold-derivatized crystal (Methods andSupplementary Table 1). The final structure contains one protomerin the asymmetric unit, which has residues 1–163 and 168–334 ofdvUT along with 9 gold atoms and 55 waters. Three residues from thecarboxy terminus and four residues from a long loop connecting thehomologous halves of the protein are disordered and omitted fromthe model.

The dvUT protomer contains two hemi-cylindrical domains of sixhelices each, and the two domains are related by a rotational pseudo-two-fold symmetry axis lying in the plane of the membrane (Fig. 2aand Supplementary Fig. 1b). The first helix of each domain, which wecall pore helix a or b (Pa or Pb), is tilted at a roughly 50u angle withrespect to the membrane norm and extends ,10 A into the mem-brane. The pore helices end in loops that turn sharply to exit on thesame side of the membrane. The next four helices from each domain,T1a–T4a and T1b–T4b, span the entire membrane. The remaininghelices, T5a and T5b, are perpendicular to the membrane andunwind at the middle of the membrane into an extended coil to crossthe membrane. The a-carbon atoms of the first half, Pa and T1a–T5a,can be superposed onto the second half, Pb and T1b–T5b, by arotation of 179.5u with a root mean square deviation (r.m.s.d.) of1.13 A.

The amino and C termini exit the membrane on the same side of theprotein and both end in short helical segments (Fig. 2a and Sup-plementary Fig. 1b). Although the orientation of dvUT in the plasmamembrane has not been experimentally determined, the excess ofpositive charge on the termini-containing face suggests that this sideorients towards the cytoplasm18, and this orientation is consistent withthe known topology of its mammalian homologues19–21.

dvUT crystallizes as a homotrimer (Fig. 2b), with its three-foldrotational axis coincident with a crystallographic symmetry axis.Helices T4a and T5a of one subunit interact with helices T4b andT5b of the neighbouring subunit. The interface between the subunitsis largely hydrophobic with a total interacting surface area of5,010 A2. On the three-fold symmetry axis is a large cavity(Supplementary Fig. 2a). Both ends of the cavity are sealed off fromthe bulk solvent by two layers of tightly packed hydrophobic sidechains circling the three-fold rotational axis: Leu 160 and Pro 287 onthe periplasmic face, and Phe 120 and Trp 124 on the cytoplasmicface (Supplementary Fig. 2b, c). The residues lining the walls of thecentral cavity are largely hydrophobic; tryptophan, phenylalanineand leucine residues predominate. Several large electron density

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Figure 1 | dvUT mediated urea flux and binding. a, Time course of 14C-ureauptake in oocytes injected with dvUT cRNA (squares) or water (circles).b, Uptake of radiolabelled urea in the presence (red) or absence (black) of1 mM phloretin. c, Saturation equilibrium urea binding by dvUT. The solidline represents fitting of the data to the Hill equation. d, SPA-based 14C-ureaequilibrium binding in the presence of increasing concentrations of urea(circles) and DMU (squares). The solid lines correspond to data fit with asingle-site binding isotherm. c.p.m., counts per minute. Error bars in allpanels are s.e.m. of 3–10 measurements.

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Figure 2 | Fold and oligomeric structure of dvUT. a, Cartoon representationof the dvUT protomer. The two-fold pseudo-symmetry axis, marked as ablack oval, is normal to the plane of the figure. Colour of helices matches that

in the topology diagram (Supplementary Fig. 1b). b, Cartoon representationof the full dvUT trimer. The crystallographic three-fold symmetry axis ismarked as a black triangle.

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peaks in the cavity probably correspond to partially ordered deter-gent or lipid molecules.

Several lines of evidence suggest that the observed trimer is not anartefact of crystal packing. In addition to the large area of the inter-face, chemical crosslinking experiments support that detergent-solubilized dvUT is a trimer (Supplementary Fig. 3a); also, the samehomotrimer was observed in a lower resolution structure obtainedfrom the native protein, which crystallizes in a lower symmetry spacegroup with different packing (Supplementary Fig. 3b, c).

Selectivity filter

Each dvUT protomer has a continuous solvent accessible permeationpathway that is formed between the two homologous halves of theprotein (Fig. 3a). This pore has a constricted region, ,16 A long, thatopens into two wide vestibules on either side. We define the con-stricted region as the selectivity filter. Highly conserved residues fromsix different regions of the protein, Pa and Pb, T3a and T3b, and T5aand T5b, are brought together to form the selectivity filter (Sup-plementary Fig. 6a). The residues that line the selectivity filter arecoloured in Supplementary Fig. 1a and shown in Fig. 3b. One side ofthe selectivity filter has two linear arrays of three oxygen atoms, whichwe call the oxygen ladders (Fig. 3b). Each oxygen ladder is flanked bytwo parallel and closely spaced phenylalanine side chains that com-press the filter into a slot-like shape. Hydrophobic phenylalanineand leucine residues complete the lining of the filter opposite to eachof the oxygen ladders. Between the two oxygen ladders, there is a gap(, 6 A) packed by two valine side chains, Val 25 and Val 188. A pairof leucine side chains, Leu 84 and Leu 247, constricts this part ofthe filter just like the phenylalanines above and below, and on theopposite side of the valines are two threonine side chains, Thr 130and Thr 294. Despite the relatively weak overall conservationbetween the two halves of the protein, the nature and positioningof the side chains forming the wall of the selectivity filter are remark-ably symmetrical: with the exception of a single Gln 24/Glu 187 pair,every residue in the filter has an identical symmetry-related partner(Fig. 3b). For each oxygen ladder, the distances between the oxygen

atoms are 3.4–3.6 A, except for the distance between the side chainand backbone oxygens of Gln 24, which is 4 A. However, the positionof this side chain may be perturbed in the crystal structure by itsinteraction with a gold atom, added during the crystallization pro-cess, sitting at the entrance of the pore (Fig. 3b).

The permeability of dvUT to water has not been determined experi-mentally; however, water permeation has been observed in apUT11 anda mammalian UT22, although there is controversy over the latter10. Inthe absence of ligand, three positive electron density peaks are visible inselectivity filter, which probably correspond to partially ordered watermolecules and raise the possibility that dvUT is water permeable. Twoof the peaks lie directly between the pairs of phenyalanines and arewithin hydrogen-bonding distance of the oxygen ladders. The third ishydrogen-bonded to the side-chain hydroxyl of Thr 294.

Potential urea-binding sites

To investigate how selective permeation of urea is facilitated bythe physical and chemical properties of the selectivity filter, we co-crystallized dvUT with urea. After initial attempts to obtain high-resolution data in the presence of urea were unsuccessful, weco-crystallized dvUT with DMU. A 2.4 A data set was collected ona crystal grown in the presence of 10 mM DMU.

The structure of dvUT–DMU was solved by molecular replace-ment, and two strong electron densities were observed in a differencemap. Each density is flat and triangular in shape, and is located closeto an oxygen ladder (Fig. 3c, e and stereo image in SupplementaryFig. 4). The triangle-shaped flat electron density makes the orienta-tion of DMU unambiguous, with its two amide nitrogen atomsfacing an oxygen ladder. We define the two sites as So and Si, for theirproximity to the outside or inside of a cell (Fig. 3b).

We propose that a partially dehydrated DMU in the So site is sta-bilized by four different interactions. First, the two amide nitrogenatoms on DMU are 2.9 and 2.6 A away from the side-chain and back-bone oxygen atoms of Glu 187, respectively, poised to form hydrogenbonds. Second, the two side chains of Phe 190 and Phe 243 sandwichDMU so that the two aromatic rings provide stabilization of the partial

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Figure 3 | Structure of the dvUT pore and DMU-binding sites. a, A surfacerepresentation of the dvUT pore with T1a, T1b and the N and C terminiremoved. Oxygen and nitrogen atoms are coloured in red and blue,respectively. b, Stereo view of residues lining the selectivity filter. Two DMUmolecules, coordinates taken from the dvUT–DMU complex, are shown inthe 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 filterfor dvUT–DMU complex. The dark blue mesh corresponds to 2Fo 2 Fc

electron density map contoured at 1.5s. The green mesh in the So and Si sitescorresponds to 3.0s Fo 2 Fc electron density calculated with DMU and thedisplayed water molecule omitted.

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positive charge on the amide nitrogen by amide–p stacking interac-tions23. Third, the carbonyl oxygen atom on DMU is 3.0 A away from awater molecule that in turn hydrogen bonds with a backbone nitrogenatom (Fig. 3c). Fourth, the oxygen ladder is at the very end of Pb(Figs 3a and 4), and this arrangement conveniently uses the partialnegative charge created by the a-helix dipole to further stabilize thepartial positive charge on the amide nitrogens. The use of helix dipolesto stabilize bound permeants has been noted previously in potassiumchannels24 and in chloride transporters25.

Binding of DMU in the Si site is similar to that at the So site, butshifted a few tenths of an angstrom towards the centre of the mem-brane so that both of the amide nitrogen atoms are 2.7 and 2.8 A awayfrom the centre oxygen in the ladder (Fig. 3e). The slight shift isprobably because the side-chain oxygen atom of Gln 24 is not avail-able because it is farther away to coordinate a nearby gold atom.

Given the similarities in shape and electrostatics between urea andDMU, urea probably occupies the So and Si sites in a similar manneras DMU. We therefore modelled urea into the two sites using theDMU coordinates (Fig. 4). For urea to complete its journey across thecell membrane, it must pass through the space between So and Si,which we define as the Sm site because it is in the middle of themembrane (Fig. 3b, d). We modelled a urea molecule into this siteby positioning the two amide nitrogen atoms within hydrogen-bonding distance to the two innermost oxygen atoms from the oxy-gen ladders (Fig. 4). This arrangement naturally places the carbonyloxygen atom of urea within hydrogen-bonding distance to thehydroxyls from both Thr 130 and Thr 294—two residues that are100% conserved in all known UT sequences. Unlike urea in the So

and Si sites, a urea in Sm is sandwiched by two leucine side chains,6.1 A apart that also exhibit strong conservation in mammalianUTs. We propose that the Sm site in the middle of the selectivity filteris superbly suited to screen a permeating ligand for the appropriateelectrostatic properties as well as a stringent test of planarity. This sitehowever, is probably not accessible to DMU because the methylgroups will clash with the two threonine side chains.

Summary

The structure of dvUT showed a continuous solvent accessible porein the structure, indicating that dvUT, and by extrapolation, UTs ingeneral probably operate by a channel-like mechanism. Selectivepermeation of urea is achieved by a long and narrow selectivity filterthat allows dehydrated urea molecules to permeate in single file.Compensation for dehydration energy is provided by continuouscoordination of urea with hydrogen bonds as it goes through theselectivity filter, and by well-placed a-helix dipoles and amide–p

interactions (Fig. 4). Furthermore, as a urea molecule progressesthrough the filter, its orientation is rigorously maintained by closelyspaced hydrophobic residues, so that optimal hydrogen bonding andsingle-file conduction can be achieved. This mechanism is consistentwith the ability of UTs to exclude molecules larger than urea, butallow smaller analogues such as formamide to pass through.

It is not immediately apparent how universal the trimeric complexis among urea transporters. A recent study on apUT argued that thatparticular homologue was a dimer11. The stoichiometry of mam-malian UT remains unknown. The existence of the mammalianUT-A1 isoform, which consists of two tandem copies of UT sequences,suggests thatUT-A1formsoligomerswithevennumbersofUTdomains.However, UT-A1 co-expresses in the same cells with UT-A3 (ref. 26),which only has one UT domain, thus raising the possibility that UT-A1and UT-A3 assemble to form a trimer-like complex, although attemptsto isolate a UT-A1–UT-A3 complex by co-immunoprecipitation havenot been successful27. Given that UTs seem to conduct urea constitu-tively, and that each subunit contains its own complete pore, it is entirelypossible that differences in native oligomeric state between homologueswould not have a large effect on their basic function.

Similar to other channels that conduct neutral molecules, suchas ammonia28,29, water30,31 or glycerol32, UT is constructed by twooppositely oriented homologous halves. The arrangement of the UTtransmembrane helices T1a–T5a and T1b–T5b bears a resemblanceto the fold of the AMT (ammonium transporter)/Rh (rhesus) familyof ammonia channels28,29 (Supplementary Fig. 5a, b). Together withthe weak sequence identity between the two proteins (22% betweendvUT and Escherichia coli AmtB), this would suggest that the twofamilies are evolutionarily related. However, there is little similaritybetween the two proteins in the structure of the permeation pathwayitself. The most significant difference is that the tilted helices Pa and Pbthat contribute the residues forming the oxygen ladders are notpresent in AmtB. Their place is taken in the AmtB structure by mostlyhydrophobic side chains from helices T1 and T6 (equivalent to T1aand T1b in dvUT, which are not involved in the pore) (Supplemen-tary Fig. 6a, b). The selectivity filter of AmtB is narrower overall thandvUT (Supplementary Fig. 6c), and is largely hydrophobic except for apair of central histidine residues that invite comparison to Thr 130and Thr 294 in dvUT (Supplementary Fig. 6d, e). Furthermore, dvUTlacks the 11th transmembrane segment possessed by AmtB, and thetrimer interface is constructed by different transmembrane helices(Supplementary Fig. 5c, d).

The structure of dvUT has revealed the physical and chemicalprinciples governing selective permeation of urea in UTs. The struc-ture also provides a framework for further studies to understand how

Pb

Pa T5b

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Figure 4 | Schematic view of the selectivity filter. The selectivity filter isshown from two angles. The predicted locations of three urea molecules andtheir hydrogen-bonding partners are on the left. In the perpendicular

direction, the filter is compressed by phenylalanine and leucine side chainslining the walls of the pore (right). Helices contributing residues to theselectivity filter are represented as grey cylinders.

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the rate of urea permeation is determined, and how the regulation ofUT function, non-covalently by small-molecule compounds9,33 andcovalently by phosphorylation34,35, is achieved.

METHODS SUMMARYdvUT was identified as a suitable homologue for structural studies in an initial

expression screen conducted by the New York Consortium on Membrane

Protein Structure (NYCOMPS, see Methods), and then adapted to the Smt3

system (Invitrogen) for enhanced yield. An N-terminal His-tagged SUMO–

dvUT fusion protein was expressed in E. coli BL21(DE3) cells and purified on

a cobalt affinity column. After cleavage of the affinity tag and SUMO domain

with SUMO protease, and a second round of chromatography on a Superdex 200

10/300 GL gel filtration column, the protein was concentrated to ,8 mg ml21 in

buffer containing 300 mM NaCl, 20 mM HEPES, pH 7.5, 5 mM b-mercaptoeth-

anol and 40 mM n-octyl-b-D-maltoside (OM). Crystals were obtained by sitting-

drop vapour diffusion in mother liquor containing 22% PEG1500, 100 mM

sodium cacodylate, pH 6.5 and 10 mM thiomersal or potassium gold cyanide.

A 2.5 A mercury-derivatized and a 2.3 A gold-derivatized data set were collectedon beamlines X25 and X29 at the National Synchrotron Light Source (NSLS) at

Brookhaven National Laboratory. The mercury data set was used for location of

heavy atom sites and SAD phasing. After density modification and automated and

manual model building, the resulting partial structure was used as a molecular

replacement model for the gold data set. Iterative rounds of building and refine-

ment produced a final model with 330 out of 337 residues resolved and R and Rfree

values of 17.9 and 20.4%, respectively. High resolution DMU–dvUT crystals were

obtained by co-crystallizing the protein with 10 mM DMU as well as potassium

gold cyanide, and the 2.4 A ligand-bound structure was solved by molecular

replacement. For the SPA binding assays, 100ml of 2.5 mg ml21 Cu21-coated

YSi SPA beads (GE Healthcare), 181mM 14C-urea (55 mCi mmol21), 50–250 ng

dvUT, and appropriate concentrations of cold urea or DMU were combined per

assay in clear bottomed/white-wall 96-well plates in buffer containing 150 mM

Tris/Mes, pH 7.5 with 50 mM NaCl, 20% glycerol, 1 mM Tris(2-carboxyethyl)-

phosphine (TCEP), and 0.1% n-dodecyl-b-D-maltoside (DDM). Reactions

performed in the presence of 400 mM imidazole served as negative controls for

all conditions tested.

Full Methods and any associated references are available in the online version ofthe paper at www.nature.com/nature.

Received 29 June; accepted 8 October 2009.Published online 28 October 2009.

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28. Khademi, S. et al. Mechanism of ammonia transport by Amt/MEP/Rh: structureof AmtB at 1.35 A. Science 305, 1587–1594 (2004).

29. Zheng, L., Kostrewa, D., Berneche, S., Winkler, F. K. & Li, X. D. The mechanism ofammonia transport based on the crystal structure of AmtB of Escherichia coli. Proc.Natl Acad. Sci. USA 101, 17090–17095 (2004).

30. Murata, K. et al. Structural determinants of water permeation through aquaporin-1. Nature 407, 599–605 (2000).

31. Sui, H., Han, B. G., Lee, J. K., Walian, P. & Jap, B. K. Structural basis of water-specific transport through the AQP1 water channel. Nature 414, 872–878 (2001).

32. Fu, D. et al. Structure of a glycerol-conducting channel and the basis for itsselectivity. Science 290, 481–486 (2000).

33. Levin, M. H., de la Fuente, R. & Verkman, A. S. Urearetics: a small molecule screenyields nanomolar potency inhibitors of urea transporter UT-B. FASEB J. 21,551–563 (2007).

34. Shayakul, C. & Hediger, M. A. The SLC14 gene family of urea transporters. PflugersArch. 447, 603–609 (2004).

35. Zhang, C., Sands, J. M. & Klein, J. D. Vasopressin rapidly increasesphosphorylation of UT-A1 urea transporter in rat IMCDs through PKA. Am. J.Physiol. Renal Physiol. 282, F85–F90 (2002).

Supplementary Information is linked to the online version of the paper atwww.nature.com/nature.

Acknowledgements We thank R. MacKinnon for advice and support throughoutthe project, C. Miller and E. Gouaux for comments on the manuscript, J. Love andNYCOMPS for cloning and the initial screen of protein expression levels, Y. Pan formessenger RNA preparation and oocyte injection, and J. Weng and Y. Cao forcrystal screening and data collection at the synchrotrons. Data for this study weremeasured at beamlines X4A, X4C, X25 and X29 of the NSLS and the NE-CAT24ID-E at the Advanced Photon Source. This work was supported by the USNational Institutes of Health (HL086392 to M.Z. and T32HL087745 to E.J.L.), theNYCOMPS that is supported by the NIH Protein Structure Initiatives PSI-II(GM075026 to W. A. Hendrickson), and the American Heart Association(0630148N to M.Z.). M.Z. is a Pew Scholar in Biomedical Sciences.

Author Contributions E.J.L. and M.Z. conceived and designed the experiments.E.J.L. purified and crystallized the protein; M.Q. performed and analysed theradiotracer flux and SPA binding assays; E.J.L. and M.Z. collected and processedthe X-ray data, solved the structure, and wrote the paper.

Author Information Atomic coordinates and structure factors have beendeposited with the Protein Data Bank under accession 3K3F and 3K3G. Reprintsand permissions information is available at www.nature.com/reprints.Correspondence and requests for materials should be addressed to M.Z.(mz2140@columbia.edu).

NATURE | Vol 462 | 10 December 2009 ARTICLES

761 Macmillan Publishers Limited. All rights reserved©2009

METHODSHomology screen, protein purification and crystallization. Human UT-B

(NCBI accession NM_015865) was nominated as a target to NYCOMPS, and

bacterial homologues were then selected36. A total of 14 UT genes were amplified

by PCR from the genomic DNA of the following bacteria: Actinobacillus pleur-

opneumoniae, Bacteroides fragilis, Colwellia psychrerythraea, D. vulgaris,

Nitrosomonas europaea, Ochrobactrum anthropi, Pseudomonas aeruginosa,

Pseudomonas fluorescens, Pseudomonas putida, Staphylococcus epidermidis,

Staphylococcus saprophyticus, Yersinia frederiksenii, Yersinia mollaretti and

Yersinia pseudotuberculosis. Each gene was first cloned into modified pET plas-mids (Invitrogen Inc.) that produced either an N- or a C-terminal His-tagged

protein. Small-scale (2 l) test purifications were conducted, and D. vulgaris UT

(dvUT) and Y. frederiksenii UT yielded stable detergent-solubilized protein as

judged by elution as a single mono-dispersed peak from a size-exclusion column.

The identity of the purified protein was verified by mass spectrometry. Only

dvUT yielded diffracting crystals, and was the focus of further experiments.

To increase the expression level, dvUT was cloned into a modified pET-

SUMO plasmid (Invitrogen) with an N-terminal polyhistidine tag and a

SUMO domain. The protein was expressed in E. coli BL21(DE3) cells, solubilized

with 30 mM DDM, and purified on a cobalt affinity column (Clontech Inc.).

After cleavage of the His tag and SUMO domain by incubation with SUMO

protease, the protein was exchanged into a buffer of 300 mM NaCl, 20 mM

HEPES, pH 7.5, 5 mM b-mercaptoethanol and 40 mM low-purity n-octyl-b-D-

maltoside (Sol-Grade from Anatrace) on a Superdex 200 10/300 GL gel filtration

column (GE Health Sciences). Each litre of cell culture yielded 0.2–0.3 mg of

dvUT after the gel-filtration step. The final protein concentration was

,8 mg ml21 as approximated by ultraviolet absorbance. Native crystals were

grown by vapour diffusion in unmixed sitting drops formed by combining2 ml of the protein solution with an equal volume of well solution containing

22% polyethylene glycol PEG1500 and 100 mM sodium cacodylate, pH 6.5.

Derivatized crystals were grown by including 10 mM thiomersal or potassium

gold cyanide in the drop solution. Before flash-freezing in liquid nitrogen, the

crystals were cryoprotected by gradually increasing the concentration of

PEG1500 in the well solution to 45% over a period of 30 h. The native crystals

diffracted to resolutions of up to 3.8 A and indexed to the P31 space group, with

unit cell dimensions a and c of 102.9 and 141.7 A, respectively. Curiously, crystals

grown from protein purified in high-purity OM (AnaGrade from Anatrace)

uniformly failed to diffract to better than 4.5–5 A. Inclusion of heavy atoms

changed the space group to P63 with unit cell dimensions a and c equal to

110.13 and 84.86 A, respectively, and improved resolution to up to 2.3 A. The

DMU-bound crystals were obtained by incubating the protein with 10 mM

ligand at 20 uC for 30 min before setting up drops in the same condition used

to obtain the gold-derivatized crystals.

Data collection and structure solution. Diffraction data were collected on

beamlines X25 and X29 at the NSLS and on beamline 24ID-E at the Advanced

Photon Source. Two data sets were used for structure solution: a 2.5 A mercurydata set collected at a wavelength of 1.01 A, and a 2.3 A gold data set collected at a

wavelength of 1.04 A. The data were indexed, integrated and scaled using the

HKL2000 software suite37. The mercury data set showed a stronger anomalous

signal and was therefore used to obtain the initial phases. Eight heavy atom sites

were located by SAD using the programs SHELX38 and phenix.hyss39. The pro-

grams Phaser40 and RESOLVE as run by phenix.autosol were used to calculate

experimental phases, carry out density modification, and build a partial model

containing 213 of the total 337 residues. A further 32 residues were added manu-

ally in Coot41, and the improved model was used to calculate an improved map

using combined experimental and model phases in PHASER. After density modi-

fication in DM42, ARP/wARP43 was able to dock 189 residues in sequence. Iterative

rounds of map calculation, density modification and manual building were used

until the model was roughly 85% complete, and then it was used as a molecular

replacement model for the gold data set with Molrep44. The structure was com-

pleted using Refmac5 (ref. 45) and Coot. For the later cycles of refinement, four

TLS groups identified by TLSMD46 were included, and the model geometry was

analysed using Molprobity. The final model was complete except for three

C-terminal residues and four residues from a disordered loop. The final structurewas then used as a model for molecular replacement for a 2.5 A data set collected

on the DMU-bound crystals. After rigid-body refinement of the molecular

replacement solution, the DMU-bound model was refined by simulated anneal-

ing in CNS47 and automatic and manual refinement in Refmac5 and Coot.

Tracer uptake in Xenopus laevis oocytes. dvUT and human UT-B (NCBI acces-

sion NM_015865) were cloned into a modified pBluescript vector for in vitro

transcription, and the mRNAs were purified by the Trizol reagent (Invitrogen).

For each measurement, ten oocytes were transferred to a transport vial that

contained 360ml transport buffer that completely covers the oocytes and is

composed of 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2 and 5 mM

HEPES, pH 7.6, and supplemented with 181mM of 14C-urea at 55 mCi mmol21.

A timer was started immediately after oocyte transfer. The reaction was stopped

at the desired time point by adding 4 ml of ice-cold buffer without radiotracer

and aspirated after a brief mixture by swirling the vial. The oocytes were washed

three times with the same buffer. After the last wash each oocyte was transferred

into a separate scintillation vial that contained 200ml of 10% SDS, and vortexedvigorously. After an oocyte was dissolved, 5 ml of scintillation cocktail was added

to each vial and vortexed. Radioactivity in each vial was determined by liquid

scintillation counting. For standards, 3 3 5ml of each transport buffer was used,

plus 200ml of 10% SDS and 5 ml scintillation cocktail.

SPA-based binding assay. Cu21-coated YSi SPA beads (GE Healthcare) were

diluted to 2.5 mg ml21 in 150 mM Tris/Mes, pH 7.5, with 50 mM NaCl, 20%

glycerol, 1 mM TCEP (Sigma), 0.1% n-dodecyl-b-D-maltopyranoside (Anatrace

Inc.) with purified His-tagged dvUT (50–250 ng per assay) and radiolabelled

urea. One-hundred microlitres of SPA-bead/protein/radiotracer solution was

added to individual wells of clear-bottom, white-wall 96-well plates. For isotopic

dilution and competition experiments depicted in Fig. 1e the final concentration

of 14C-urea was kept constant at 181mM (55 mCi mmol21), whereas the con-

centration of non-labelled urea or DMU was increased from 0–50 mM.

Saturation binding (as shown in Fig. 1d) was performed with increasing con-

centrations of 14C-urea at a specific activity of 2 mCi mmol21. Binding was

performed in the dark for 30 min at 4 uC with vigorous shaking on a vibrating

platform. To determine the non-specific background binding activity, 400 mM

imidazole was added to the wells because imidazole competes with the His-tag of

the recombinant protein. Plates were read in the SPA mode of a Wallac 1450

MicroBeta plate PMT counter. Total c.p.m. were corrected for non-specific

binding by subtracting the c.p.m. of the samples in the presence of imidazole,

yielding the specific c.p.m. To prevent radioligand depletion in the saturationbinding experiments 250 ng of purified dvUT, a protein amount substantially

below the capacity of the SPA beads, was used per assay. The transformation of

c.p.m. to the amount of urea was calculated with a standard curve of known

amounts of radioactivity (determined by adding scintillation cocktail to the

samples), and the slope of this linear relation was used to transform c.p.m. into

mol l21 (ref. 17). All experiments were performed at least in duplicate with

replicas of $3 and data are expressed as mean 6 standard error. Data fits of

kinetic analyses were performed using nonlinear regression algorithms in

Sigmaplot (SPSS Inc.), and error bars represent the s.e.m. of the fit.

36. Punta, M. et al. Structural genomics target selection for the New York Consortiumon Membrane Protein Structure. J. Struct. Funct. Genomics. doi:10.1007/s10969-009-9071-1 (in press).

37. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected inoscillation mode. Methods Enzymol. 276, 307–326 (1997).

38. Pape, T. & Schneider, T. R. HKL2MAP: a graphical user interface formacromolecular phasing with SHELX. J. Appl. Crystallogr. 37, 843–844 (2004).

39. Terwilliger, T. C. & Berendzen, J. Automated MAD and MIR structure solution.Acta Crystallogr. D 55, 849–861 (1999).

40. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40,658–674 (2007).

41. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. ActaCrystallogr. D 60, 2126–2132 (2004).

42. Collaborative Computational Project number 4.. The CCP4 suite: programs forprotein cystallography. Acta Crystallogr. D 50, 760–763 (1994).

43. Potterton, L. et al. Developments in the CCP4 molecular-graphics project. ActaCrystallogr. D 60, 2288–2294 (2004).

44. Lebedev, A. A., Vagin, A. A. & Murshudov, G. N. Model preparation in MOLREPand examples of model improvement using X-ray data. Acta Crystallogr. D 64,33–39 (2008).

45. Vagin, A. A. et al. REFMAC5 dictionary: organization of prior chemical knowledgeand guidelines for its use. Acta Crystallogr. D 60, 2184–2195 (2004).

46. Winn, M. D., Isupov, M. N. & Murshudov, G. N. Use of TLS parameters to modelanisotropic displacements in macromolecular refinement. Acta Crystallogr. D 57,122–133 (2001).

47. Brunger, A. T. et al. Crystallography & NMR system: A new software suite formacromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).

doi:10.1038/nature08558

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