The Crystal Structure and Activity of a Putative Trypanosomal Nucleoside Phosphorylase Reveal It to...

doi:10.1016/j.jmb.2010.01.013 J. Mol. Biol. (2010) 396, 1244–1259

Available online at www.sciencedirect.com

The Crystal Structure and Activity of a PutativeTrypanosomal Nucleoside Phosphorylase Reveal It tobe a Homodimeric Uridine Phosphorylase

Eric T. Larson1,2, Devaraja G. Mudeppa3, J. Robert Gillespie4,Natascha Mueller1,2, Alberto J. Napuli1,2, Jennifer A. Arif4,Jenni Ross1,2, Tracy L. Arakaki1,2, Angela Lauricella5, George DeTitta5,Joseph Luft5, Frank Zucker1,2, Christophe L. M. J. Verlinde1,2,Erkang Fan1,2, Wesley C. Van Voorhis1,4, Frederick S. Buckner1,4,Pradipsinh K. Rathod3, Wim G. J. Hol1,2 and Ethan A. Merritt1,2⁎
1Medical Structural Genomicsof Pathogenic ProtozoaConsortium (MSGPP),University of Washington,Seattle, WA 98195, USA2Department of Biochemistry,University of Washington,Seattle, WA 98195, USA3Department of Chemistry,University of Washington,Seattle, WA 98195, USA4Department of Medicine,University of Washington,Seattle, WA 98195, USA5The Center for HighThroughput Structural Biology(CHTSB), Hauptman-Woodward Institute, Buffalo,NY 14203, USA
Received 3 November 2009;received in revised form4 January 2010;accepted 5 January 2010Available online11 January 2010

*Corresponding author. E-mail addAbbreviations used: UP, uridine p

phosphorylase; RNAi, RNA interferphosphorylase 1; PDB, Protein DataUK, uridine kinase; SeMet, selenomGenomics of Pathogenic Protozoa Cbis(β-aminoethyl ether)N,N′-tetraace

0022-2836/$ - see front matter © 2010 E

Purine nucleoside phosphorylases (PNPs) and uridine phosphorylases(UPs) are closely related enzymes involved in purine and pyrimidinesalvage, respectively, which catalyze the removal of the ribosyl moiety fromnucleosides so that the nucleotide base may be recycled. Parasitic protozoagenerally are incapable of de novo purine biosynthesis; hence, the purinesalvage pathway is of potential therapeutic interest. Information aboutpyrimidine biosynthesis in these organisms is much more limited. Thoughall seem to carry at least a subset of enzymes from each pathway, thedependency on de novo pyrimidine synthesis versus salvage varies fromorganism to organism and even from one growth stage to another. We havestructurally and biochemically characterized a putative nucleoside phos-phorylase (NP) from the pathogenic protozoan Trypanosoma brucei and findthat it is a homodimeric UP. This is the first characterization of a UP from atrypanosomal source despite this activity being observed decades ago.Although this gene was broadly annotated as a putative NP, it was widelyinferred to be a purine nucleoside phosphorylase. Our characterization ofthis trypanosomal enzyme shows that it is possible to distinguish betweenPNP and UP activity at the sequence level based on the absence or presenceof a characteristic UP-specificity insert. We suggest that this recognizablefeature may aid in proper annotation of the substrate specificity of enzymesin the NP family.

© 2010 Elsevier Ltd. All rights reserved.

Keywords: nucleoside phosphorylase; pyrimidine salvage; nucleotidemetabolism; sleeping sickness; gene annotation
Edited by I. Wilson
ress: [email protected]; PNP, purine nucleoside phosphorylase; NP, nucleosideence; MAD, multiwavelength anomalous dispersion; hUPP1, human uridineBank; NH, nucleoside hydrolase; UPRT, uracil phosphoribosyltransferase;ethionine; NCS, noncrystallographic symmetry; MSGPP, Medical Structuralonsortium; TbUP, Trypanosoma brucei uridine phosphorylase; EGTA, ethylene glycoltic acid; PEG, polyethylene glycol; SSRL, Stanford SynchrotronResearch Lightsource.

lsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.jmb.2010.01.015

1245Trypanosomal Uridine Phosphorylase

Introduction

All living cells are dependent on purine and pyri-midine nucleotides to carry out a plethora ofbiochemical processes. These nucleotides may besynthesized completely de novo and/or be salvagedfrom the cell's environment. Both pathways requiremultiple enzymes, but the salvage pathway is lesscostly to the cell energetically. Though manyspecies, including mammals, utilize both de novosynthesis and salvage, most parasitic protozoa relyon one pathway or the other to fulfill their purineand pyrimidine requirements.1–3 For instance, para-sitic protozoa lack de novo purine synthesis, thusmaking purine salvage enzymes potentially attrac-tive drug targets. The story for pyrimidine biosyn-thesis is not as straightforward and, in general,pyrimidine biosynthetic pathways have not beenstudied to the extent of their purine counterpartsamong parasitic protozoa. Many parasitic protozoacontain at least a subset of the enzymes involved inboth de novo synthesis and salvage, though they mayrely more heavily on one pathway versus the other invarious life stages to meet their pyrimidine needs.1–4

These differing dependencies on de novo synthesis orsalvage with respect to purines and pyrimidinesunderscore the importance of correctly annotatingthe function of the gene products involved in thesepathways as they are identified through the variousgenome projects of protozoan pathogens.Because of the importance of nucleoside biosynthe-

sis and salvage in protozoa, a putative nucleosidephosphorylase (NP) from Trypanosoma brucei(GeneDB5 accession number Tb927.8.4430), the caus-ative agent of African sleeping sickness, was selectedfor investigation as a possible drug target by theMedical Structural Genomics of Pathogenic ProtozoaConsortium (MSGPP)†.6 NPs are ubiquitous enzymesinvolved in nucleotide salvage pathways fromorganisms in all domains of life. They catalyze thereversible cleavage of the glycosidic bond in purineand pyrimidine nucleosides or deoxynucleosidesusing inorganic phosphate to yield the purine orpyrimidine base and α-ribose-1-phosphate. The freebases can then be used for nucleotide formation in lieuof costly de novo biosynthesis. The phosphorylasesuperfamily (Pfam7 01048) is subdivided into twofamilies based primarily on structure (reviewed inRef. 8). Each family encompasses many sequences oflow identity and a broad substrate range.Members ofthe NP-I family are single-domain proteins thatdisplay an α/β-fold and may adopt a hexameric(trimer of dimers) or trimeric quaternary structure.Though there are exceptions, hexameric enzymes aremore typical in bacteria while the trimeric enzymesare typically found in mammals. NP-I family mem-bers act on a variety of purine or pyrimidinesubstrates and include purine nucleoside phosphory-lase (PNP, EC 2.4.2.1), uridine phosphorylase (UP;EC 2.4.2.3), and 5′-deoxy-5′-methylthioadenosine

†www.msgpp.org

phosphorylase (EC 2.4.2.28). The NP-I fold is alsocommon to 5′-methylthioadenosine/S-adenosylho-mocysteine nucleosidase (EC 3.2.2.9) and AMPnucleosidase (EC 3.2.2.4). NP-II family members aretwo-domain proteins with an α/β-fold domain,unrelated to that of the NP-I family, connected to asmaller α-helical domain. NP-II enzymes are specificfor pyrimidines, namely, uridine and thymidine, andtypically function as homodimers.Though the targeted T. brucei gene is annotated

generally as a putative NP, it was widely inferred tobe a PNP because the majority of proteins returnedfrom a BLAST9 search are annotated as such. Here,we report, however, that close inspection of theresults of this search, ignoring sequence annotationsof uncharacterized gene products and comparingonly to enzymes of characterized activity, suggeststhat it is more similar to UP. Further, when searchingthe conserved domain database,10,11 the sequencereturns UP (COG2820) as the top hit followed by themore broad pfam01048 (PNP_UDP_1, phosphory-lase superfamily). However, since PNPs and UPs arequite similar in structure and sequence, we did notappreciate this apparently greater similarity to UP insequence-based searching until after characteriza-tion of the actual activity of the T. brucei geneproduct. Since parasitic protozoa have differingdependencies upon purine and pyrimidine salvagedue to differing capacity for de novo synthesis of thenucleotides, the true substrate specificity of thisputative NP from T. brucei is of intrinsic biologicaland potential therapeutic interest.To this end, we have solved the crystal structure of

a putative NP from the pathogenic protozoa T. bruceiin complex with uracil and α-ribose-1-phosphate,confirming that it is a member of the hexamericfamily of NP-I NPs. Interestingly, the T. bruceienzyme is not observed to form the canonicalhexameric trimer of dimers characteristic of otherfamilymembers but rather exists only as a functionaldimer that is stabilized by an intermolecularlycoordinated calcium ion. To determine the preferredactivity of the enzyme, we performed crystal soakingand cocrystallization experiments as well as activityassays using a series of purine and pyrimidine basesor nucleosides. The results of the activity assayssupport the crystallographic evidence that thisenzyme is a functional UP. This constitutes the firstcharacterization of a specific trypanosomal UPdespite its activity being suggested in studies ofseveral trypanosomatids decades ago.12–14 Further,since the essentiality of this gene product has notbeen evaluated, we have used RNA interference(RNAi) to determine its potential as an anti-trypanosomal drug target.

Results

Structure of T. brucei UP (TbUP)

The x-ray crystallographic structure of the puta-tive NP from T. brucei, the causative agent of African

Table 1. Data collection statistics

Data set Se peak Se remote Uridine cocrystal

Beamline SSRL 9-2 SSRL 9-2Space group P21 P21Unit cell parametersa, b, c (Å) 63.1, 95.6, 63.5 63.1, 95.4, 63.5α, β, γ (°) 90, 105.9, 90 90, 105.9, 90Resolution (Å) 50–2.27 (2.35–2.27) 40–1.44 (1.50–1.44)Wavelength (Å) 0.9791 0.9116 0.917Unique reflections 31,735 22,922 123,263Completeness (%) 95.1 (77.8) 71.0 (65.1) 95.4 (73.4)Rmerge 0.11 (0.30) 0.09 (0.38) 0.08 (0.64)Mean I/σ(I) 10.0 (2.8) 8.1 (1.8) 8.4 (1.2)Redundancy 3.3 (3.0) 2.3 (2.4) 3.4 (2.4)Wilson B-factor (Å2) 26.7 15.8

Values in parentheses are for the highest-resolution shell.

1246 Trypanosomal Uridine Phosphorylase

sleeping sickness, was solved using multiwave-length anomalous dispersion (MAD) and refinedto 1.45 Å resolution (Tables 1 and 2). The asymmetricunit is composed of two copies of the polypeptidethat form the biologically relevant homodimer.The final model consists of residues 15 to 341 (theC-terminus of the full-length protein) of eachmonomer, bound uracil and ribose-1-phosphateoccupying each active site, an intermolecularlycoordinated calcium ion, and 509 water molecules(Fig. 1a). The N-terminal His-tag and the first 14residues of each monomer are not modeled due todisorder in this region of the proteins.

Table 2. Model refinement statistics

Data set Uridine cocrystal

Resolution (Å) 35.4–1.44Rwork 0.155Rfree 0.184No. of reflections:

refinement/test set123,193/6191

RMSD bonds (Å) 0.01RMSD angles (°) 1.3Protein atoms 5047Nonprotein atoms 554Residues in favored

regions (%)a97.9

Residues in allowedregions (%)a

100

Unmodeled residues −7 to 14 for both chainsTLS groups (residues)b A chain: 15–63, 64–106, 107–118,

119–145, 146–174, 175–196, 197–227,228–257, 258–309, 310–319, 320–341;B chain: 15–21, 22–105, 106–113,

114–145, 146–175, 176–191, 192–224,225–253, 254–283, 284–313, 314–341

Mean Biso+BTLS proteinatoms (Å2)

23.9

Mean Biso catalytic productatoms (Å2)

22.0

Mean Biso metalatom (Å2)

13.0

Mean Biso solventatoms (Å2)

32.4

PDB code 3BJEa Determined using the MolProbity Server.15b Selected with the aid of the TLSMD server.16

Eachmonomer is composed of a central 11-strandedmixed β-sheet surrounded by 14 α-helices and twoperipheral short, 2-stranded antiparallel β-sheets(Fig. 1a). This fold is characteristic of the NP-I familyof NPs. Indeed, with the exception of a few unusualstructural features that will be discussed below, thestructure of the TbUP dimer is very similar to that ofall other NP-I structures currently available in theProtein Data Bank (PDB20), particularly those thatutilize (deoxy)uridine as the primary substrate. Twomonomers form a tight dimer with approximately2765 Å2, just over 18%, of the solvent-exposed surfacearea per monomer buried in the dimer interface asdetermined by the PISA server.21 The dimer interfaceis additionally stabilized by a tightly coordinatedmetal ion near the center of mass of the molecule(discussed below). The dimer present in the asym-metric unit is equivalent to the dimer unit seen inmembers of the hexameric NP-I subfamily; however,it is not possible to assemble three TbUP dimers intothe canonical hexamer, aswill bedescribedbelow. Therecent structural characterization of human uridinephosphorylase 1 (hUPP1) has shown that it is also ahexameric NP-I family member that assembles onlyinto a functional dimer,22 but the structural featuresthat prevent hexamer formation are quite differentfrom those of the T. brucei enzyme. Because residuesfrom neighboring monomers of the dimer contributeto the active site of NP-I NPs, the dimeric assembly ofthe T. brucei and human enzymes constitutes theminimal catalytic unit necessary for activity.Comparison of the sequence and structure of the

T. brucei enzyme with other hexameric NP-I familyenzymes explains why it does not assemble into theprototypical hexamer. As apparent in a sequencealignment (Fig. 1b), the T. brucei enzyme containstwo large inserts greater than 25 amino acids andtwo smaller inserts. Two of these inserts createadditional secondary structural elements that wouldprotrude into the neighboring dimer of the canonicalNP-1 hexamer, thus sterically blocking trimerizationof the dimers (Fig. 1c). The first is a long, largelyhydrophilic, helical insert (α6–α7) located immedi-ately upstream of a typically relatively shorthydrophobic patch (FPAV in Escherichia coli UP)important for the dimer–dimer interaction of thehexamer, which have been replaced by morehydrophilic residues in TbUP (199YTSM202). Thisregion is also responsible for preventing hexamerformation in hUPP1, but the structural details arequite divergent. The human enzyme lacks the largehelical insert (Fig. 1b) and instead has substituted ashort insert and a two-stranded antiparallel β-sheetin the vicinity of TbUP helix α5. It is actually theshort insert upstream of this new β-sheet along withthe substitution of the typically hydrophobic patchfor hydrophilic residues that block hexamer forma-tion of the human enzyme, while the novel β-sheetprojects towards the neighboring monomer andeffectively increases the buried surface area of thedimer.22 The second hexamer-blocking insert ofTbUP is a short two-stranded antiparallel β-sheet(β11–β12) that lies adjacent to helix α6 (Fig. 1c).


Intermolecular metal-binding site

A very strong difference density peak, alsocorresponding to the largest peak in the anomalousdifference Fourier map, was present near the center

Fig. 1 (legend o

of mass of the dimer and right between the activesite of each monomer (Fig. 1a and d). This peak wasobserved in the electron density from all data sets ofthis protein despite there being no extraneousmetalsadded during expression, purification, or crystalli-

n next page)

Fig. 1. Structure of homodimeric UP from T. brucei. (a) One monomer is shown as a semitransparent white surfacewith the UP-specificity region orange and the hexamer-blocking inserts magenta. The other monomer is a ribbon diagramwith α-helices colored red, β-strands colored blue, and loops colored green with the exception of the UP-specificity regionand the hexamer-blocking inserts, which are colored orange and magenta, respectively, as for the surface monomer.Uracil and α-ribose-1-phosphate products highlight the active sites and are shown as ball-and-sticks. The intermolecularcalcium ion is shown as a sphere. The view for the left-hand panel is into the active sites down the 2-fold axis and the right-hand panel has been rotated 90° along the depicted axis. (b) Structure-based sequence alignment of representative UPsand PNPs. Amino acid numbering and secondary structural elements of TbUP are mapped at the top of the alignment.Trypanosome-specific inserts that block hexamer formation are labeled by magenta bars and the region of human UPP1that is responsible for blocking hexamer formation is shaded pink. The blue box marks the hydrophobic patch importantfor dimer–dimer interactions that allow for the trimer of dimer organization of the prototypical hexameric NP-Isubfamily. This region has been substituted by more hydrophilic residues in the trypanosomal and human homodimers.The UP-specificity region is marked by an orange bar, and the key uridine recognition residues are highlighted in cyan.The metal coordinating residues of TbUP are marked with an “M” at the bottom of the sequences. The key substrate/product interacting residues are labeled “U” for uracil, “R” for ribose, and “P” for phosphate. The residues contributed bythe neighboring monomer are lowercase and colored green rather than red. Structure-based sequence alignment wascreated using Expresso (3D-Coffee)17 with some manual editing and the figure was prepared using TEXshade.18 Tb, T.brucei; Hs, Homo sapiens; Ec, E. coli; Pf, Plasmodium falciparum; PDB codes used for the structural alignment follow thespecies-enzyme name. (c) Superposition of the TbUP dimer on the prototypical hexameric form of the NP-I family, in thiscase represented by the PNP from P. falciparum (PDB code: 2bsx),19 to highlight the consequence of the TbUP hexamer-blocking inserts. The view is equivalent to that in the right-hand picture of (a), looking parallel with what would be the3-fold symmetry axis of the trimer of dimers. The TbUP dimer is red with an orange UP-specificity region and magentahexamer-blocking inserts. The equivalent P. falciparum dimer unit is green and the neighboring dimer of the hexamer isgray. The TbUP inserts that prevent hexamer formation sterically clash with the neighboring hexameric subunit. (d)Square antiprism coordination environment of the intermolecular calcium ion. The coordination environment is shownas gray dashes and the coordination bonds are black lines with distances in angstroms. The coordinating residues areMet87, Asp90, and Asn91 contributed by each monomer, one colored green and the other cyan.


zation with the exception of sodium chloride. Thesite is coordinated by four noncrystallographicsymmetry (NCS)-related oxygen atoms from eachmonomer for a total of eight ligands: the carbonyloxygen of Met87, Oδ1 and Oδ2 of Asp90, and Oδ1 ofAsn91 from each chain across the NCS 2-fold axis.The eight oxygen ligands form a slightly distortedsquare antiprism around the metal with the biden-tate interactions of the two Asp90 side-chaincarboxylates forming one square face and theMet87 carbonyl oxygen atoms/Asn91 side-chainoxygen atoms forming the other square face (Fig. 1d).This electron density peak was modeled as a

calcium ion based on the coordination geometrydescribed above with metal-to-ligand distances thatare characteristic of calcium to oxygen.23 The assign-

ment of calcium is also supported by the calciumbond-valence sum method,24 a method of using therefined metal-to-ligand distances and the geometricenvironment to aid in assignment of appropriatemetal ions in crystal structures that is most reliablewhen the resolution exceeds 1.5 Å, which is the casefor the TbUP structure. Importantly, the assignmentof calcium is supported by calcium bond-valencesum independent of whether the structure is refinedwith the metal modeled as calcium, potassium,sodium, or zinc, which should have a slight effect onthe resulting metal-to-ligand distances due to thevarying restraints on these distances for the differentmetal ions. The average metal-to-ligand distance forthe final refined calcium ion is 2.5 Å, which is a bitlonger than the calcium–donor target distance.23


However, if each Asp90 is treated as a monodentateligand rather than bidentate by considering only theoxygen of closer proximity to the metal, the averagecalcium–donor distance is 2.4 Å, which is muchcloser to the target distance. This still leaves sixligands to the metal in a slightly distorted trigonalprismatic coordination geometry. Both this and thesquare antiprism coordination geometry describedabove are consistent with calcium. The final metal-to-ligand distances are also consistent with thoseexpected for sodium but sodium was rejected as theidentity of the metal for two primary reasons. First,there is still a considerable peak in the differenceFourier map after refinement as sodium, suggestingthat the true metal contains more electrons, andsecond, a significant peak is present in the anoma-lous difference Fourier map and the f″ of sodium isessentially zero at the wavelengths used for datacollection; thus, an anomalous peak larger than thatobserved for sulfurs in the structure would not beexpected for sodium. Since Ca2+ was not added inany step of protein production to crystallization, theenzyme likely acquired this metal ion during itsbacterial expression.This is not the first observation of a UP containing

an intramolecular metal ion. Some structures of theE. coli25,26 and the Salmonella typhimurium27 enzymescontain a potassium ion that is situated betweenactive sites in the dimer interface but not at thestructurally equivalent location observed for thecalcium ion in the T. brucei enzyme. Comparison ofstructures with and without the potassium ionsuggests that the metal plays a structural role,stabilizing tighter dimers within the hexamer that,in turn, stabilizes the phosphate-binding pocketswithin the active sites, which accordingly leads to anapproximately twofold enhancement of activitywith increasing potassium concentration.25 It isplausible that TbUP is more dependent on thestabilizing effect of an inter-monomer metal thantypical NP-I family NPs because it exists only as adimer rather than the higher-order hexameric state,which is suggested to contribute to the overallstability of the enzyme.27 Along these lines, thehuman enzyme, hUPP1, also stabilizes its homo-dimeric structure, but via an alternate strategy. Itlacks the residues required for metal coordination(Fig. 1b) and rather has greatly increased the size ofthe dimer interface by reconfiguring the structuralelements of the would-be hexamerization interfaceso that they interact with its monomeric partnerinstead of another dimer.22

Active-site pockets and bound products

In common with the dimer unit of all hexamericNP-I family members, the T. brucei dimer comestogether so that the two active-site pockets areapproximately 20 Å apart and resemble boots withthe toes oriented towards the dimer interface. Thecatalytically required phosphate binds in the toe ofeach boot-shaped active-site pocket while theuridine substrate binds with the uracil at the boot's

heel and the ribose in the arch region, adjacent to thephosphate (Fig. 2a). Most of the amino acids thatcompose the active-site pocket are contributed by asingle monomer, but several important residues arecontributed by the neighboring monomer, includ-ing two that make crucial interactions with thesubstrate/products, namely, His26 and Arg66(Figs. 1b and 2b). The side chain of His26 hydrogenbondswith the 5′ hydroxyl of the ribose,whileArg66is a key residue in the phosphate pocket, formingtwo hydrogen bonds with phosphate oxygen atoms.The phosphate is further bound by the nitrogen ofGly42 and the side chains of Arg46, Arg137, andThr140. Residues Leu25, His26, and Arg66 togethercomprise roughly half of the accessible surface at theopening of the active-site pocket. The amino acidsresponsible for binding the phosphate and ribosemoieties of the substrates and products are muchmore highly conserved between UPs (Fig. 2c) andPNPs than are the residues responsible for bindingthe respective bases (Figs. 1b and 3a). In fact, two ofthe key UP residues that interact with the uracil,Gln246 and Arg248 (TbUP numbering), have noanalogous amino acids in PNPs and reside on a UP-specific insert. These discriminating residues inconjunction with the insert they are located on maybe used to distinguish between the catalytic activitiesof UP and PNP enzymes at the level of the primarysequence and thus may aid in proper annotation(Figs. 1b and 3; discussed below).In the initial structure that was solved without the

addition of organic ligands, phosphate, present inthe crystallization solution, was apparent in thephosphate-binding pocket of the active site for eachmonomer along with an adjacent oddly shaped butindistinct difference density peak. This peak waslocated at the position that would be expected forthe nucleobase of the substrate or product and waspresent despite not having added any extraneouspotential substrates or products during purificationor crystallization of the enzyme. Neither purine norpyrimidine base could be modeled convincinglyinto the density, and a preference for one class ofbase over the other was not clear; hence, we set outto determine enzyme specificity by soaking crystalswith or cocrystallizing with various purine andpyrimidine bases and nucleosides. These experi-ments did not allow this mysterious density to beresolved any more readily than the original struc-ture with the exception of a uridine cocrystal. In thiscrystal, it was immediately obvious from strongdifference density peaks of definable shape in theactive site of each monomer that the products of thecatalytic reaction, ribose-1-phosphate and free uracil,were present (Fig. 2b). The phosphate moiety ofribose-1-phosphate is essentially in the identicallocation as the phosphate observed in the structuresthat were not cocrystallized with the nucleotidesubstrate, and clear density for the ribose moietyextends towards the heel of the boot-shaped activesite. The ribose is in the C1′-exo conformation, but thestereochemistry of the anomeric C1′ carbon isopposite what it would be when linked to the

Fig. 2. Active-site environment of TbUP. (a) The boot-shaped active-site pockets are shown as a transparent surfaceand the products are shown as sticks. α-Ribose-1-phosphate and uracil are bound in the heel and the toe of the boot,respectively. Residues within 4 Å of the products or the intermolecular Ca2+ ion are shown as lines, colored cyan for onemonomer and green for the second, with the exception of those in the UP-specificity region that are orange for bothmonomers. The view is rotated 100° around the y-axis with respect to the view in the right-hand picture of Fig. 1a. (b)Stereo view of the active-site environment around the products, uracil and α-ribose-1-phosphate, and calcium ion. TheσA-weighted28 Fo−Fc electron density map (mesh) was calculated with products and the calcium ion omitted from themodel and is contoured at +5 σ (green mesh) and −5 σ (red mesh). The view is zoomed in on the products from the right-hand monomer of (a). Amino acids within 4 Å of the products are shown as sticks and colored according to the monomerthey come from as in (a). Hydrogen bonds are shown as broken pink lines. (c) Superposition of the ligand-bound activesites of TbUP with EcUP (PDB code: 1tgy) and HsUPP1 (PDB code: 3euf). The side chains of residues (or whole Glyresidues) within 4 Å of the bound uracil and ribose-1-phosphate are shown as sticks with TbUP colored cyan, EcUPcolored magenta, and HsUPP1 colored yellow. The ligands (uracil and ribose-1-phosphate for TbUP and EcUP and5-benzylacyclouridine for HsUPP1) are shown as ball-and-sticks; metals and waters are shown as spheres and arethe same color as the enzyme for which they are bound. Structures were superimposed by SSM29 in Coot.


Table 3. Substrate specificity

SubstrateActivity

(μmol/mg/min)aRelative

activity (%)

Uridine 2.81±0.30 100Deoxyuridine 0.40±0.03 14Thymidine b0.04 b1.4Cytidine b0.001 b0.03Deoxycytidine b0.001 b0.03Inosine b0.001 b0.03Adenosine b0.15 b5.0Deoxyadenosine b0.2 b7Guanosine b0.3 b10Deoxyguanosine b0.2 b7

a Reactions at 25 °C, pH 7.5.

Fig. 3. Comparison of NPs in the vicinity of the UP-specificity region. (a) Sequence alignment of severalcharacterized UP and PNP sequences with severaluncharacterized putative NP sequences from pathogenicprotozoa of interest to MSGPP. The UP-specificity regionis present in known UPs and is absent in known PNPs.The presence of this insert with the two key substratediscrimination residues (Gln and Arg) in many uncharac-terized sequences strongly suggests that they possess UPactivity. Alignment was created using Expresso (3D-Coffee)17 with manual editing using the alignment inFig. 1b as a guide. (b) Structural comparison of the back-bone trace of the UP-specificity region. TbUP (PDB code:3bje) is colored orange, and the structurally alignedcounterpart from P. falciparum PNP (PDB code: 2bsx) iscolored green. The key substrate-discriminating residues,Gln246 and Arg248, are shown as sticks for TbUP, andPfPNP has no equivalent residues.


nucleobase because of the attack of the phosphatefrom the opposite face of the sugar ring. The nowclear uracil density occupies the approximate loca-tion of the previously unfittable density. The active-site environment is essentially the same as seen in theproduct-bound E. coli structures [PDB codes: 1rxc25

and 1tgy (Bu et al., unpublished results)] and theinhibitor-bound human UPP1 structure (PDB code:3euf22), which is not surprising given the high

structural and sequence conservation of the activesites (Fig. 1b), particularly the residues involved ininteractions with the products/substrates (Fig. 2c).When comparing the isomorphous “ligand-free”

and ligand-bound structures of TbUP, we do not seethe large conformational changes in the active sitepreviously described for the bacterial enzyme.25 Thebacterial structures exhibit a highly flexible “flap”loop that is often disordered and undergoes a largeconformational change upon substrate binding.25The equivalent TbUP residues, approximately resi-dues 312Val-Gly320, are well ordered and retainessentially the same conformation in the “ligand-free” and ligand-bound structures. This region ofTbUP is slightly longer and conformationally differ-ent from that of EcUP and is in a position somewhatintermediate between the open and closed bacterialstates. The lack of a conformational change may bebecause the TbUP “ligand-free” active site is not fullydevoid of ligands, as described above, and thus, wehave not captured the true ligand-free conformation.On the other hand, it is also possible that this rigidityis a characteristic of eukaryotic UPs since the equi-valent region in hUPP1 remains essentially in thesame intermediate conformation between ligand-bound and unbound states.22

Enzyme activity

Since NPs that prefer either purine or pyrimidinesubstrates may still display weak activity against theother, the crystal structure may not be a reliablemeans of determining specificity. Thus, to identifythe actual substrate specificity of TbUP, and toconfirm that the preferred substrate is uridine assuggested by the crystallographic experiments, wetested purified enzyme for activity against a series ofpurine and pyrimidine nucleoside and deoxynu-cleoside substrates. Among the potential substratestested for activity, only uridine and deoxyuridineshowed detectable cleavage in the presence of theenzyme (Table 3). The strict specificity for pyrimi-dines by TbUP is in agreement with previous studiesof bacterial and mammalian UPs.8,30–32 TbUP doesnot, however, possess a low activity against thymi-dine, which is often observed. Also in line with theactivity of other UPs, deoxyuridine is a much less

Fig. 4. Effect of Ca2+ on TbUP stability. TbUP activitywas monitored over time at elevated temperature in thepresence of EGTA or CaCl2 to assess if the intermolecularlybound Ca2+ observed in the crystal structure contributes toenzyme stability. The reaction buffer was not supple-mented with EGTA or CaCl2 for the control experiment.The rapid loss of enzyme activity in the presence of EGTAcompared to the gradual loss of activity in the presence ofCa2+ suggests that the metal ion stabilizes the catalyticallyactive form of the TbUP. Activity is plotted as a percentageof the initial activity at time point 0.


efficient substrate of TbUP, being cleaved at a rateonly approximately 14% of that seen for uridine.Interestingly, it has been reported for human UPthat the activity against deoxyuridine is alsoabout 15% that of uridine.33 In common withEcUP, three hydrogen bonds are formed with theribose 2′-hydroxyl group (Fig. 2b), providing a likelyexplanation for the substrate preference of uridineover deoxyuridine.25 The inability of thymidine toform these hydrogen bonds may contribute to theinability of TbUP to catalyze its phosphorolysis.These biochemical results confirm the crystallo-graphic evidence that TbUP is actually a UP.It has been reported that there are two groups of

uridine-cleaving enzymes distinguished by their pHoptima: one with an optimum at pH 6.5–6.7 andanother with an optimum at pH 7.9–8.1.30 Thisprompted us to further test TbUP activity for pHdependence using a pH range of 6.0–8.5 (Table 4).Maximal activity with uridine as the substrate is atpH 7.5, which is in agreement with the optimal pHof 7.3 found for activity of EcUP.31 Curiously, themaximal activity with deoxyuridine as the substrateis much lower at pH 6.5, and the activity againstdeoxyuridine is much more sensitive to pH change.Approximately 40% decrease in activity is observedon either side of optimum pH when uridine is thesubstrate but the activity increases about 350% asthe pH decreases from 7.5 to 6.5 when deoxy-uridine is the substrate. The significance of theseobservations is not clear, but it is possible that thethree hydrogen bonds between the enzyme and the2′-hydroxyl group and/or the loss of the observedintramolecular hydrogen bond (2.95 Å) between the2′-hydroxyl and the O1′ of ribose-1-phosphate thatwould be lost when deoxyuridine is the substratemay play a role in this pH effect.As mentioned above, maximal activity of bacterial

UP is dependent on the presence of an intermolecu-larly coordinated K+ ion that stabilizes the activesite.25 To test the possibility that activity of TbUP islikewise influenced by the potentially stabilizingintermolecular Ca2+ ion, we monitored uridinecleavage at elevated temperature, 55 °C, in thepresence of up to 2 mM ethylene glycol bis(β-aminoethyl ether)N,N′-tetraacetic acid (EGTA) or0.5 mM CaCl2 (Fig. 4). In the presence of EGTA,there is almost a complete loss of activity after about50 min. When the enzyme is supplemented withCaCl2, 60% of the initial activity remains after

Table 4. Effect of pH on UP activity

Activity (μmol/mg/min)a

pH Uridine Deoxyuridine

6.0 1.70±0.46 1.44±0.066.5 1.86±0.29 1.55±0.277.0 1.91±0.39 0.64±0.107.5 2.75±0.32 0.44±0.078.0 2.68±0.34 0.22±0.158.5 1.55±0.24 0.13±0.08

a Reactions at 25 °C.

90 min. In control experiments, when no EGTA orCaCl2 was added, the enzyme loses about 40% of itsinitial activity after 20 min but then only experiencesan additional 10% loss over the next 70 min. Due tolimitations with the experiment, the precise rela-tionship between Ca2+ and TbUP activity is notclear, but it is clear that the intermolecularlycoordinated metal ion does contribute to the overallstability of the enzyme.

RNAi of TbUP

To evaluate the potential of TbUP as a drugtarget against T. brucei, we subjected the gene tosilencing by overexpression of stem–loop RNA fromtetracycline-regulated constructs. Messenger RNAlevels at 72 h post-induction were decreased byapproximately 90%asmeasured by quantitative PCR(Fig. 5). Bloodstream-form parasite growth for fiveseparate cloneswasmeasured over a period of 7 daysfollowing induction of RNAi. No growth inhibitionwas observed over this time period for any of theclones compared to the uninduced controls (Fig. 5).

Can UPs and PNPs be annotated correctly fromtheir sequences?

Though annotated with the very broad label“putative nucleoside phosphorylase,” the Tb927.8.4430 gene product was largely assumed to be aPNP based on BLAST searches and inferred from thelinks that existed within the gene's records inGeneDB‡, TargetDB, and TDR Targets.34 As men-

‡The gene is now annotated as “uridine phosphor-ylase, putative” in the current beta version of thenext generation of GeneDB (http://beta.genedb.org/NamedFeature?name=Tb927.8.4430).

Fig. 5. Gene knockdown of TbUP using RNAi.Cumulative cell densities are shown on a log scale as theproduct of the cell number and the total dilution. T. bruceiundergoing RNAi of UP (TCN+) is compared to cellswithout RNAi induction (TCN−). The growth rate wasalso compared with the ‘single marker’ (SM) strain of T.brucei that was not transfected with the RNAi expressionvector (semilogarithmic plot). Knockdown of T. bruceimRNA expression 72 h after induction of RNAi withtetracycline was demonstrated by PCR of cDNA at cycles23, 26, and 29 and visualized by ethidium bromide stain ofagarose gel (lower inset). Even loading was demonstratedby PCR of the β-tubulin gene (upper inset).


tioned previously, digging deeper into the sequencecomparisons and including consideration for con-served domains seemed to point towards thepossibility that the enzyme was really a UP. How-ever, since PNPs and UPs are quite similar insequence and structure, this marginally greatersimilarity to UP in sequence-based searching onlystood out after the high-resolution uridine cocrystalstructure had been solved, which suggested UPactivity that was later confirmed by biochemicalassays. These results then emphasized the existenceof a UP-specific insert that was alluded to in earlystructural studies of EcUP by Morgunova et al.35 andlater emphasized by Caradoc-Davies et al.25

Realizing that sequence-based searches may haveactually been able to correctly identify the activity ofthe T. brucei gene product, we looked at thesequences of homologous proteins from otherorganisms of interest to the MSGPP1 that have notyet been characterized to see if their functions maybe clarified. These protein sequences include the“putative nucleoside phosphorylase” in Trypano-soma cruzi (accession numbers XP_814980.1 andXP_811342.1), “nucleoside phosphorylase-like pro-tein” in several species of Leishmania (L. major:XP_001681435.1, L. infantum: XP_001463753.1 andXP_001464547.1, L. braziliensis: XP_001562879.1),“purine nucleoside phosphorylase” in Entamoebahistolytica (XP_652740.1, XP_654874.1, andXP_655398.1), and “UPL-1” in Giardia lamblia

(XP_001707342.1; this Giardia gene product ispossibly responsible for the UP activity previouslystudied36,37). Indeed, alignment of these sequenceswith the T. brucei gene product characterizedherein and other known UPs or PNPs (Fig. 3a)shows that all contain the UP-specificity region(amino acids 243–264 in TbUP) that bears thesubstrate-discriminating glutamine and arginineresidues (Gln246 and Arg248 in TbUP), stronglysuggesting that all are bona fide UP enzymes. Thedivergence in UP and PNP sequences in this regionleads to differences in structure (Fig. 3) that allow forselection of one type of substrate over the other andis suggested to have led to the evolutionary split ofhexameric NPs with purine or uridine cleavageactivity.25 The presence or absence of this insert mayhelp guide the annotation process. A cursory look atmany other NP-like genes obtained in a BLASTsearch shows thatmany annotated PNPs are likely tobe UPs or vice versa and that it may be possible toassign more specific annotation to many that arecurrently annotated generally as NP.The alignment additionally suggests that the

probable UPs from the MSGPP target organisms,all eukaryotic parasites, likewise function as homo-dimers (not shown). All of these protozoan geneproducts have a large insert corresponding to thelarge hexamer-blocking insert 1 of TbUP. In addition,the other trypanosomatid sequences possess thesecond, smaller hexamer-blocking insert and thecalcium-coordinating motif found in TbUP, furthersupporting a homodimeric quaternary structure. Asmentioned previously, human UPP1, the only othereukaryotic UP present in the PDB, also is present as ahomodimer but has utilized a slightly different,moresubtle structural mechanism to prevent a hexamericquaternary structure and to stabilize the homo-dimeric form. These features are not as immediatelyobvious from the sequence alone because the largehexamer-blocking insert seen in many protozoansequences is not present but the sequence differencesare recognizable nonetheless, particularly with thestructural context of human UPP1 as a reference.Using this information, it appears that the UPs frommany other higher eukaryotes are also dimers. Thesenon-protozoan UP sequences look similar to thedimeric human UPP1 and generally have a smallinsert and a greater number of hydrophilic residuesin the vicinity of the prototypical hexameric dimer–dimer interface.

Discussion

Here, we show crystallographically and bio-chemically that a putative NP from T. brucei shouldbe classified more specifically as a UP (EC 2.4.2.3).The first evidence we saw for uridine cleavageactivity of this enzyme was from inspection of acrystal structure following cocrystallization with thesubstrate uridine in the presence of phosphatebuffer. Strong electron density was observed ineach active site for the products uracil and ribose-1-


phosphate. Although the equivalent complexes withvarious purines were not obtained despite signifi-cant effort, this complex structure alone was notsufficient to assign the function as UP since manyPNPs also show low activity against uridine (e.g.,the Plasmodium PNP19,38). We thus initiated activityassays with purified recombinant enzyme against apanel of pyrimidine and purine (deoxy)nucleosidesand found that it is indeed specific for uracil-containing substrates. Interestingly, UP activityshows significant pH dependence with deoxyuri-dine as the substrate but uridine cleaving activity isnot substantially affected in the tested pH range of 6to 8.5. At pH 7.5, the maximum for uridine cleavage,the activity against uridine is more than fivefoldgreater than the turnover of deoxyuridine. AtpH 6.5, the maximum for deoxyuridine cleavage,the cleavage of uridine is still slightly greater thanthat of deoxyuridine. This demonstrates that theenzyme has greater specificity for the ribosyl moietyover the deoxyribosyl moiety and is understandablegiven the hydrogen-bonding interaction betweenthe enzyme and the 2′ hydroxyl group of the ribose.All pyrimidine biosynthesis, whether de novo or

via salvage, utilizes UMP. Through the salvagepathway, UMP can be formed directly from uridineby the activity of uridine kinase (UK) or indirectlyby first being broken down to uracil by either UP orby a nucleoside hydrolase (NH). The uracil is thenconverted to UMP by uracil phosphoribosyltrans-ferase (UPRT).3 It is noteworthy that no UK activityhas yet been detected in any life stage of anytrypanosomatid tested.12,39 It thus appears that allUMP formation resulting from the salvage pathwaymust go through UPRT, the enzyme immediatelydownstream of UP. As mentioned above, there ispotentially overlapping activity between UP andNH, which irreversibly removes the base from thenucleoside using water as the nucleophile, insupplying uracil to UPRT. No specific uridinehydrolases are known in the trypanosomatids buturidine hydrolase activity has been detected asbeing distinct from UP activity in cell extracts ofseveral trypanosomatids.13 Also, an inosine–uridine-preferringNHhas been characterized inCrithidia andLeishmania,40,41 and there are genes annotated as“putative nucleoside hydrolase” in various trypano-somal genomes. Thus, there may be an alternatemeans to supply uracil for UPRT on the path to UMPproduction in these parasites that apparently lackUKactivity.12,39

To determine the essentiality of this UP to T. bruceiin light of its potentially redundant activity, weperformed RNAi experiments on bloodstream-formparasites in vivo. UP mRNA levels were knockeddown N90%; however, no growth effect was ob-served, indicating that the gene function is notessential in this stage of the parasite's life cycle,with the caveat that the approximately 5–10% ofremaining expression may be sufficient to satisfy thecell requirements. Cellular pools of pyrimidines inT. brucei are likely maintained by the combination ofde novo synthesis and salvagemechanisms.42 It is also

possible that an as-yet uncharacterized NH that iscapable of acting on uridine exists in T. brucei and isable to compensate for the loss of UP activity.Regardless of the reason, these results suggest thatUP is not likely to be a good drug target againstT. brucei.The characterization of the true substrate speci-

ficity and activity of this T. brucei enzyme is still ofinterest biologically, however, because of theapparent variation in utilization of pyrimidinesalvage or de novo synthesis in different morpho-logical stages of many trypanosomatids.3,43 To thebest of our knowledge, this is the first actualcharacterization of a trypanosomal UP despiteuridine cleavage activity being observed in wholecell extracts of blood stage T. brucei and in all fourmajor morphological forms of various trypanoso-matids over 20 years ago12,13 and suggested fromstudies over 45 years ago.14 The newly definedactivity of this enzyme warrants further study of itsrole in pyrimidine salvage and in the balance ofuracil and uridine concentrations in the cells ofthese pathogenic human parasites in their diversemorphological forms and host environments.Through structural and biochemical studies of

EcUP, Caradoc-Davies et al. suggested that theemergence of a UP-specificity region may havegiven rise to distinct UP and PNP activities fromwithin an equivalent hexameric ancestral structuralframework.25 Indeed, the UP structures from allfour species that are currently in the PDB (bacterial:E. coli and S. typhimurium; eukaryotic: human andthe T. brucei enzyme described here) contain ahomologous sequence insert that exists in a similarstructural conformation with the Cα atoms of thekey uridine-discriminating residues, Gln246 andArg248 (TbUP numbering), superimposing to with-in 1.5 Å and with similarly oriented side chains.Neither this sequence insert nor the key residues arepresent in any of the non-uridine-specific sequencesor structures of NP-I enzymes of characterizedactivity that we are aware of. We came to appreciatethe implication of this observation through thecourse of this work and applied it to the uncharac-terized NP sequences from several other MSGPPtarget organisms in hopes of gleaning additionalinsight into their functions. This includes homolo-gous sequences from T. cruzi and several species ofLeishmania, as well as three sequences fromE. histolytica and one from G. lamblia. All of thesesequences contain the UP-specificity insert and thebase-discriminating Gln and Arg residues, leadingus to predict that they are likely to possess UPactivity. Beyond the sequences of interest to MSGPP,we suggest that the presence or absence of the keyuridine-discriminating Gln and Arg amino acids inthe context of the UP-specificity region wouldgreatly aid in cleaning up NP gene (mis)annotationsthat have propagated through sequence databasesand in properly annotating current genericallyannotated NPs and new sequences that arise asmore and more genomes are sequenced. Forexample, the PNP sequence from several species of


Plasmodium, an active antimalarial drug target, isstill misannotated as UP in several sequencedatabases despite an abundance of publishedresearch (e.g., Refs. 19, 38, 44, and 45) establishingit as a PNP. Even in the absence of this experimentalbasis, however, it should be possible to correctlyannotate these Plasmodium gene products as PNPbecause it is clear from a sequence alignment (Figs.1b and 3a) that it does not possess the UP-specificityregion or the discriminating Gln and Arg residues.Delving more deeply into the potential evolu-

tionary consequence of the acquisition of the UP-specificity region, it is interesting to note that all UPstructures currently available in the PDB aremembers of the hexameric/dimeric NP-I subfamilywhile PNP structures are drawn from both hexa-meric and trimeric subfamilies. This is in agreementwith the suggestion that hexameric and trimericNP-I subfamilies diverged from a common PNPancestor and then the hexameric subfamily furtherdiverged into bacterial PNPs and UPs.8 We specu-late that it is not coincidental that UPs divergedonly from the hexameric subfamily and that this is astructural correlate of the presence of the sequenceinsert that confers uridine specificity. The residuesresponsible for purine base recognition in the PNPslie in the sequence regions flanking the site of theUP-specificity insert. The conformation of theseflanking regions is compatible with both thehexameric and trimeric quaternary assemblies.However, uridine recognition is primarily conferredby Gln246 and Arg248 displayed by helix α10 of theUP-specificity insert (Figs. 1b and 3), and theobserved position of the adjacent helix α11 of thisinsert would sterically block formation of themonomer–monomer interface seen in trimericPNPs. Thus, the trimeric assembly may haveimposed an evolutionary restraint that preventedthe acquisition of the insert necessary for recogni-tion of the uridine substrate while the framework ofthe hexameric/dimeric enzyme, which lacks thisstructural restraint, was able to diverge into a classwith uridine specificity.In summary, we have structurally and biochemi-

cally characterized a putative NP from T. brucei andfind that it is a UP with high specificity for uracil-containing (deoxy)nucleosides. Though the structureis very similar to other members of the hexamericNP-I family, it has some unusual features. It isdimeric rather than hexameric, which is the result ofseveral trypanosome-specific sequence inserts thatprevent the dimer–dimer interaction required forhexamerization. Also, it contains an intermolecularcalcium ion that helps stabilize the smaller quater-nary assembly of TbUP. Comparison of the sequenceand structure of TbUP with other UP and PNP NP-Ifamily members has highlighted a UP-specificsequence insert with conserved Gln and Arg aminoacids that has two major consequences to theenzyme. It confers the ability to efficiently utilize(deoxy)uridine as a substrate, and it appears to beresponsible for the observation that UPs belong onlyto the hexameric/dimeric NP-I subfamily and not to

the trimeric NP-I subfamily commonly seen forPNPs among higher eukaryotes. We suggest that thepresence of this distinguishing feature that isidentifiable in the primary sequence may aid inproper annotation of the gene products of thisimportant enzyme family.

Materials and Methods

Target cloning, protein expression, and purification

The putative NP gene from T. brucei (GeneDB:5

Tb927.8.4430) was selected as a target (TargetDB:46

Tbru017883AAA) for the MSGPP.6 The full-length genewas cloned from genomic DNA of T. brucei strainTREU927 GUTat 10.1 into the E. coli expression vectorBG1861, a modified version of pET14b that includes an N-terminal noncleavable hexahistidine tag,47 using ligation-independent cloning.48 Protein was expressed in E. coliBL21 (DE3) and purified using immobilized metal affinitychromatography on a Ni-NTA column followed by size-exclusion chromatography on a HiLoad Superdex 75column (Amersham Pharmacia Biotech). Protein waseluted in SGPP buffer (0.5 M NaCl, 2 mM DTT, 0.025%NaN3, 5% glycerol, and 25 mM Hepes at pH 7.5),concentrated to 20 mg/ml, flash frozen in liquid nitrogen,and stored at 80 °C. Selenomethionine (SeMet) proteinwas produced according to the protocols of Studier49 andSGPP,50 and then purified and stored as described for thenative protein except that it was concentrated to 12 mg/mlprior to freezing.

Protein crystallization

Purified native TbUP (20 mg/ml) was screened forcrystallization leads at the high-throughput facility at theHauptmanWoodward Institute.51 Several leads were thenoptimized in-house using sitting-drop vapor diffusion toproduce crystals suitable for data collection. Initialdiffraction-quality crystals were grown at 4 °C from acrystallization drop composed of 0.4 μl native protein(20 mg/ml) mixed with 0.4 μl well solution consisting of39% polyethylene glycol (PEG) 8000, 0.1 M NaH2PO4,5 mM DTT, and 0.1 M Tris–HCl (pH 8.0). This yielded acrystal that produced a poor-quality data set from whichwe were unable to solve the structure by molecularreplacement. Assuming that this was due to the lowsequence identity (near 25%) with the closest availablestructure, we pursued crystallization of SeMet protein.Diffraction-quality SeMet crystals were obtained bysetting up a fine screen of pH versus (PEG 8000) aroundthe initial condition both with and without the DTTadditive using protein at 12 mg/ml. Most drops immedi-ately precipitated while setting up this screen; hence, itwas repeated with the addition of an equal amount ofMilli-Q H2O to the drop, thereby decreasing the initialconcentrations of all components by one-third. Thisproduced Se-Met crystals suitable for data collection.The mother liquor of the crystal that led to the initialphases was 37% PEG 8000, 0.1 M NaH2PO4, and 0.1 MTris–HCl (pH 7.9). No cryoprotectant was necessary; thus,crystals were mounted in cryoloops and directly frozen inliquid nitrogen in preparation for diffraction experiments.To shed light on the substrate specificity of the enzyme,

we soaked native and SeMet crystals with various purineand pyrimidine nucleosides, deoxynucleosides, and bases.


Soaking experiments were performed with substrateconcentrations varying from 10 to 33 mM and for timesranging from 1 to 72 h. Soaking failed to yield aninterpretable complex structure; hence, we turned tococrystallization. Cocrystallization experiments were setup as described above for SeMet protein, except that bothnative and SeMet protein (each diluted to 10 mg/ml withSGPP buffer) were used and an equal volume of 10 mMnucleoside, deoxynucleoside, or base was added to thedrop rather than water. Only native protein was used tofurther optimize cocrystals. Cocrystals of the phosphory-lase:uridine structure described herein grew from aninitial mixture of 1 μl native protein solution (15 mg/ml)+1 μl 10 mM uridine+1 μl well solution [39% PEG 8000,0.1 M unbuffered NaH2PO4, and 0.1 M Tris–HCl (pH 8.5)]equilibrated over 80 μl well solution at 4 °C.

Data collection and structure determination

Crystals of TbUP were screened at the StanfordSynchrotron Research Lightsource (SSRL) on beamline9-2 using the SSRL automated mounting system.52 Datawere collected at 100 K on a MarMosaic-325 CCDdetector using the Blu-Ice software package.53 MADdata were collected at the Se peak and remote wave-lengths, as determined from a fluorescence scan of asingle crystal of SeMet protein. One hundred sixtydegrees of data with 1° oscillation/image were collectedat the Se-peak wavelength, but only 80° of data werecollected at the remote wavelength due to crystal decay.One hundred eighty degrees of single-wavelength datawere collected from crystals or cocrystals of nativeprotein. All data were processed using HKL2000.54 Thecrystals belong to space group P21 with unit celldimensions of approximately 63.1 Å×95.5 Å×63.5 Åand a β angle of 105.9°. The Matthews coefficient55 isapproximately 2.4 Å3/Da with two protein molecules,forming a homodimer, present in the asymmetric unit.Data in the highest-resolution shell of the uridinecocrystal suffered from high mosaicity, leading to manyoverlapping reflections in some portions of reciprocalspace. Data collection statistics are presented in Table 1.Using the two-wavelength MAD data set, SOLVE56

found 22 Se sites out of 26 expected and produced initialphases to 2.3 Å. Of the sites identified, 18 subsequentlyproved to be true selenium sites, 1 was a metal ion, and 3were questionable. The remote wavelength data wereincluded in the initial phase determination steps to helpbreak the ambiguity despite the low completeness, and nosubsequent effort was made to solve the phase problem viasingle-wavelength anomalous dispersion since MAD wassuccessful. The results from SOLVE were then fed intoRESOLVE56 for densitymodification and automatedmodelbuilding. RESOLVE identified the 2-fold NCS from theheavy-atom positions and was able to build 571 residues,placing 554 side chains, out of 698 residues expected for twocopies of the full-length construct. Iterated manual modelbuilding and restrained refinement continued using the Se-peak data set with Coot57 and REFMAC5.58

When higher-resolution data (1.45 Å) were laterobtained from a crystal of native protein cocrystallizedwith uridine, subsequent cycles of building and refine-ment used this data set. Based on the difference densitypresent in the active site of the cocrystal structure, it wasimmediately clear that the enzyme had converted theuridine to the products uracil and ribose-1-phosphate.Ideal coordinates for these products were obtained fromthe HIC-Up database,59 and they were placed into the

difference density peaks using Coot. Refinement restraintsfor use in REFMAC5 and Coot were obtained from thePRODRG server.60 In the final cycles of refinement,perturbational displacement of the protein was describedby 11 TLS groups per chain as identified by the TLSMDserver,16,61 and TLS parameters were refined for eachgroup prior to restrained refinement in REFMAC5. Modelquality was monitored and validated using Coot andMolProbity.15 The CCP4 suite of programs62,63 was usedextensively for all steps from data preparation throughrefinement. Model refinement statistics are presented inTable 2. Molecular figures were created and rendered withPyMOL.64

Assays of catalytic activity

To determine the substrate specificity of TbUP, wetested purified protein for activity against different purineand pyrimidine nucleoside and deoxynucleoside sub-strates as described below. All enzymatic assays werecarried out in assay buffer [50 mM potassium phosphatebuffer (pH 7.5) containing 5% glycerol and 2mMDTT] at aprotein concentration of 2 μg/ml (determined by Bradfordassay). Control experiments were carried out in theabsence of either enzyme or substrate in the assay mixture,unless specified. All assays were performed at 25 °C usinga Beckman DU 530 UV/visible spectrophotometer.Inosine as substrate:A coupled enzymatic assay was used

to monitor the hydrolysis of inosine to hypoxanthine.19

Subsequent oxidation of the product hypoxanthine intouric acid by xanthine oxidase was monitored at 293 nm(E293=12.9 mM−1 cm−1). Inosine was used at 0.2 mM.Guanosine/deoxyguanosine as substrate: Hydrolysis of

guanosine/deoxyguanosine was monitored directly at262.5 nm (E262.5=−3.6 mM−1 cm−1).65 Guanosine/deoxy-guanosine was used at 0.2 mM.Adenosine/deoxyadenosine as substrate: Two methods

were used: (1) Hydrolysis of adenosine/deoxyadenosinewas monitored directly at 274 nm (E274=−1.9 mM−1

cm−1).44 Adenosine/deoxyadenosine was used at 0.2 mM.(2) A coupled enzymatic assay was used to monitor thereverse reaction, synthesis of adenosine from D-(3′-deoxy)ribose-1-phosphate and adenine. Subsequent deaminationof the product adenosine by adenosine deaminase wasmonitored at 265 nm (E265=−6.4 mM−1 cm−1).66 Adenineand D-ribose-1-phosphate were used at 80 and 240 μM,respectively.Uridine/deoxyuridine as substrate: Hydrolysis of uridine/

deoxyuridine was monitored directly at 282 nm (E282=−1.37 mM−1 cm−1).67 The reaction requires inorganicphosphate; thus, 50 mM Tris–HCl, pH 7.5, buffer wassubstituted for the phosphate buffer in the reactionmixture as a control. Uridine/deoxyuridine was used at0.4 mM.Thymidine as substrate: Hydrolysis of thymidine was

monitored directly at 290 nm (E290=−1.0 mM−1 cm−1).67

Thymidine was used at 1.0 mM.Cytidine/deoxycytidine as substrate:Hydrolysis of cytidine/

deoxycytidine was monitored directly at 270 nm (E270=−3.0 mM−1 cm−1).66 Cytidine/deoxycytidine was used at0.2 mM.Effect of pH on UP activity: The reaction was essentially

performed and monitored as described above for uridine/deoxyuridine as substrate but was carried out in 50 mMphosphate buffer containing 5% glycerol and 2 mM DTTat a pH range of 6.0–8.5.Effect of calcium on UP activity/stability: thermal inacti-

vation studies were carried out in the presence of EGTA or


CaCl2 similarly to those previously published for calcium-dependent peroxidase.68 For each reaction, 200 μg/ml ofenzyme in 50 mM phosphate buffer (pH 7.5) containing1 mM DTT was incubated at 55 °C in the presence of 0, 1,or 2 mM EGTA or 0.5 mM CaCl2. The control reaction didnot contain EGTA or CaCl2. At various time intervals, a5-μl sample was drawn from the reaction mixture, anduridine hydrolysis activity was monitored as describedabove except that the reaction buffer additionallycontained EGTA or CaCl2 if needed. No difference inactivity was observed for 1 or 2 mM EGTA, and it wasnot possible to raise the CaCl2 concentration above0.5 mM because of severe interference with spectropho-tometric readings as time progressed.

RNAi knockdown of TbUP gene product

A region of the TbUP gene sequence (Tb927.8.4430) wasselected for RNAi using the program RNAit.69 Bases 599–910 of the gene were amplified from T. brucei 927 genomicDNA using the primers 5′-ATACCAATGTGATGGCAT-CAATGGCGCATCCCAATA-3′ and 5′-ATACCATAGA-GTTGGCGGCTCCAGCTTGATAACC-3′. The ampliconwas ligated using TA cloning into the vector pGEM-T(Promega, cat #A3600) and then excised with the enzymeBstXI. This insert was ligated into the stem–loop RNAivector, pQuadra3, as previously described.70 The con-struct was sequenced to verify the identity of the insertand then linearized with NotI in preparation for electro-poration. T. brucei bloodstream-form parasites expressingthe T7 RNA polymerase and the Tet repressor under asingle selection marker were provided by G. Cross(Rockefeller University).71 Electroporation and culturemethods were done as previously described.42 Individualclones were selected for subsequent RNAi studies. Theexpression of the stem–loop RNA was induced byaddition of 1 μg/ml tetracycline to the cultures dilutedto 1×105 cells/ml. Cultures were passed at a 1:10–1:20dilution daily and cell concentrations were monitoredusing an ATPLite Luminescence ATP detection AssaySystem (PerkinElmer, cat#6016941). cDNA was preparedfrom messenger RNA collected 72 h post-RNAi inductionwith tetracycline. mRNA signal knockdown was analyzedby quantitative PCR of the cDNA using primers 5′-ATGGCTGCATCCGCTAATGG-3′ and 5′-GGGGAACC-GACTCAGCAGG-3′, which amplified a separate region ofthe gene than that which was used for the RNAi construct.The amplified products from different PCR cycles werequantified by densitometry (normalized to β-tubulin).

Accession numbers

Atomic coordinates and structure factors for TbUP havebeen deposited in the PDB§,20 with accession number3BJE.

Acknowledgements

We thank Isolde Le Trong for assistance withcrystal screening, Christine Stewart for bioinfor-matics and database expertise, and the rest of the

§http://www.pdb.org

MSGPP team for exceptional support and fruitfuldiscussions. Financial support from the ProteinStructure Initiative award NIGMS GM64655 andfrom NIAID award AI067921 is gratefully acknowl-edged. Portions of this research were carried out atthe SSRL, a national user facility operated byStanford University on behalf of the U.S. Depart-ment of Energy, Office of Basic Energy Sciences. TheSSRL Structural Molecular Biology Program issupported by the Department of Energy, Office ofBiological and Environmental Research, and by theNational Institutes of Health, National Center forResearch Resources, Biomedical Technology Pro-gram, and the National Institute of General MedicalSciences.

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