PROTEINS: Structure, Function, and Genetics 25510-513 (1996) Crystallization of the Purine Salvage Enzyme Adenine Phosphoribosyltransferase Cynthia L. Phillips, Buddy Ullman, and Richard G. Brennan Department of Biochemistry and Molecular Biolog.y, L-224, Oregon Health Sciences University, Portland, Oregon 97201 -3098 ABSTRACT Adenine phosphoribosyltransferase from the protozoan parasite Leishmania domvani has been crystallized in the presence of the substrate Mg2+-a-~-5-phosphoribosyl-1-pyrophosphate(PRPP)or the product adenosine-5-monophosphate,as well as in the absence of ligand. These crystals belong to the space group P6,22 or its enantiomorph P6,22, with unit cell dimensions of a = b = 64.0 A, c = 240.5 A, (Y = p = go",and y = 120".The crystals diffract to 1.9 A. o 1996 Wiley-Liss, Inc. A high-resolution crystal structure of the L. donovani APRT would be an important first step in the design of new reagents, providing an atomic framework for structure-based inhibitor or subversive substrate design?-'' In addition to drug design, the structure of the L. donovani APRT will permit structural comparison with the recently solved crystal structures of the hypoxanthine/guanine PRT (HGPRT) from humans, the orotate PRT (OPRT) from Salmonella typhimurium, and the glutamine PRPP amidotransferase from Bacillus subtil~s."-'~ Despite their divergent primary sequences, the tertiary structures of these Key words: Leishmania, parasite metabolism, PRTs show a highly conserved core structure that selenomethionine, X-ray diffracextends to their nearby nucleotide binding f01ds.l~ tion However, their structural similarities fade toward the solvent-accessible surfaces, at the oligomerizaINTRODUCTION tion interfaces, and at the interacting N- and C-terAdenine phosphoribosyltransferase (APRT), a minal regions and loops, which include their active member of the phosphoribosyltransferase (PRT) sites. The structure of L. donovani APRT, which family, is responsible for recycling the purine base shares only 9% sequence identity with human adenine by catalyzing the Mg2+-dependent converHGPRT, for example, will define more clearly the sion of adenine and a-~-5-phosphoribosy1-l-pyrostructural requirements for catalysis of members of phosphate (PRPP) to adenosine-5-monophosphate the PRT family and delineate which regions of the and pyrophosphate.' In mammals and other organunrelated primary sequence comprise these funcisms that can synthesize purines de novo, recycling tionally related structures. Moreover, a structure of of purines is primarily important during periods of an APRT will likely lend insight into the structural rapid growth, such as for rapidly dividing cancer basis for the sequence divergence found between the cell^.^,^ Although APRT is not essential for human human and L. donovani APRTs, which are only 27% cells, a genetic defect in APRT activity is not withidentical, yet catalyze identical reactions. out consequence, causing a 2,8-dihydroxyadenine Finally, the structure of APRT should contribute urolithiasis (kidney stone^).^ In contrast, protozoan to an understanding of the function of the varied parasites lack a de novo biosynthetic pathway for oligomeric states in which PRTs exist and the role of purines and must scavenge purines from their host.5 oligomerization in catalysis. Studies on S. typhimuThese differences in purine metabolism of protozoan rium OPRT show that this enzyme functions as a parasites, as well as tumor cells, relative to their dimer in which Lysine 103 of one monomer is rehuman hosts suggest that enzymes of the purine salquired for catalytic activity of the other monovage pathway, such as APRT, are judicious targets mer.15,16 Human HGPRT is also a dimer, but the for chemotherapeutic intervention. structural relationship between the oligomeric state The protozoan parasite Leishmania donovani and catalysis is unknown. The glutamine PRPP causes visceral leishmaniasis, a fatal disease if unamidotransferase is a cooperative tetramer. Gel filtreated. Unfortunately, therapies for leishmaniasis tration experiments on L. donovani APRT show that are far from ideal and employ highly toxic and nonselective drugs. APRT appears to be important, if not indispensible, for purine salvage by the infective form of the parasite.6 The L. donovani aprt gene has Received January 19, 1996; accepted February 2, 1996. Address reprint requests to Richard G. Brennan, Dept. of been cloned, sequenced, and overexpressed in EsBiochemistry and Molecular Biology, L-224, Oregon Health cherichia coli, and the recombinant APRT protein Sciences University, 3181 SW Sam Jackson Park Road, Porthas been purified in large amounts to h~mogeneity.~ land, OR 97201-3098. 0 1996 WILEY-LISS,INC. 511 CRYSTALS OF ADENINE PHOSPHORIBOSYLTRANSFERASE it behaves as a monomer in the presence of either Mg2+ -PRPP or AMP.7 Whether there is an internal catalytic lysine in APRTs that functions analogously to the lysine of S. typhimurium OPRT is not known. High-resolution structures will be valuable for a more complete understanding of the general structure of PRTs both before and after catalysis. MATERIALS AND METHODS AND RESULTS Expression and Purification of L. donovani APRT The L. donouani APRT gene encodes a 238 amino acid protein and has been subcloned into the pBAce expression vector and overexpressed in the E . coli strain ,54446, which lacks the bacterial APRT.7 Minor modifications were made to the single-step chromatography purification from Leishmania cell culture described by Allen et al.17 The E . coli cells were inoculated from a frozen glycerol stock and harvested after 15 hours of growth at 37°C in low phosphate media. The cells were lysed and sonicated in TMD2O buffer (20 mM Tris, pH 7.4, 5 mM MgCl,, 1 mM dithiothreitol), with 1 mM phenylmethylsulfonyl fluoride and 1pg/ml DNase. The lysate was centrifuged at 20,OOOg for 20 minutes and the supernatant loaded onto an AMP-agarose affinity column (Sigma, catalog #A-3019) and eluted with 1 mM PRPP or 2 mM AMP in TMD2O buffer. A typical yield was 5 mg of purified protein per liter of bacterial cells, and the purity was > 95% as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The selenomethionine-incorporated APRT was overexpressed by transforming the pBAce expression vector directly into the E . coli strain DL41, an auxotroph for methionine (Yale Genetic Stock Center, strain 7177;h-, metA28). There are six methionine residues in L. donouani APRT, including the amino-terminal methionine. Growth in modified "Hail Mary" media (defined media with each amino acid added separately") was exceedingly slow, with a doubling time of more than 2 hours. The media included 0.1 mM K2HP04as the sole source of phosphate, and D/L-selenomethionine at 75 mg/L was substituted for methionine. A 5 ml culture containing 100 pg/ml methionine was used to inoculate each liter of selenomethionine-containing media, resulting in a less than 1%dilution of the selenomethionine by methionine. The purification was otherwise identical to that for unlabeled APRT, with a final yield of 1 mg of APRT per liter of bacterial culture. Crystallization and Preliminary X-ray Characterization of L. donovani APRT Purified APRT was concentrated to 11 mg/ml in TMD2O buffer containing either 1 mM PRPP or 2 mM AMP. Protein concentration was determined by ultra-violet absorbance using a calculated extinction coefficient of = 16,100 cm-' M-l. Crystals of native APRT were obtained by vapor diffusion using both hanging and sitting drops. Initial crystallization screens using Hampton Research Crystal Screens yielded crystals in 30% of the conditions in Crystal Screen I and 14% of the conditions in Crystal Screen 11. All but two of these crystal forms appeared to be hexagonal bipyramids. To produce the highest quality diffraction crystals, the APRT was mixed 1:l (v:v) with the crystallization reagent [1.2 to 1.6 M (NH,),SO, in 0.1 M Na-Acetate, pH 4.901 and equilibrated in 12 pl drops over 1ml reservoirs a t 4°C for 2 weeks t o 2 months. Crystals of APRT were enzymatically active by radioactive assay." The crystals of selenomethionine-incorporated APRT grew similarly. The L. donouani APRT crystals were grown reproducibly to dimensions of 0.5 mm x 0.5 mm x 0.8 mm in the presence of M 8 -PRPP or AMP or in the absence of ligand. Although the protein had to be used for crystallization within 1 week of purification, once grown, the crystals were stable for months and diffract beyond 1.9 A. Precession photographs of the hkO and Okl planes revealed hexagonal symmetry and a 6, or 6, screw axis. Both substrate and product crystal forms have unit cell dimensions of a = b = 64.0A,c = 240.5A,cw = p = 90",andy = 120" and assume space group P6,22 or its enantiomorph P6,22. One monomer per asymmetric unit yields a VM of 2.73 A3fDa and a solvent content of + 55%.,O Data Collection Initial intensity data of the native apo-APRT crystals or crystals grown in the presence of either Mg2+-PRPPor AMP were collected a t room temperature using an Area Detector Systems Corporation (ADSC) multiwire area detector and processed with software provided by ADSC. The rotating anode X-ray source (Rigaku RU2OOH) operated a t 40 kV and 150 mA. Data for APRT crystals grown in the presence of M8+-PRPP had an R, of 3.89% at 3.6-21.5 resolution (3,775 unique reflections, 99.0% complete), an R,, of 5.25% at 2.6-5.0 A resolution (7,475 unique reflections, 91.0% complete), and an Ray, of 7.66% a t 1.95-3.15 A (7,297 unique reflections, 43.7% complete). Data on APRT crystals grown in the presence of AMP had an R, of 4.66% a t 3.6-21.5 A resolution (3,612 unique reflections, 94.7% complete) and an Ray, of 6.57% a t 2.6-5.0 A resolution (7,959 unique reflections, 96.8% complete). Data for the apo-APRT crystals had an Rsym of 3.71% a t 2.3-4.9 A resolution (9,816 unique reflections, 80.2% complete) and an R,, of 4.09% at 3.0-20.0 A resolution (2,681 unique reflections, 95.5% complete). Merged intensity data for the APRT crystals grown in the presence of Mg2+-PRPP had an Rsymof 7.8% (21.5-2.6 A); for crystals grown 5 12 C.L. PHILLIPS ET AL. TABLE I. Derivative Statistics HgC1, Resolution (A) Unique reflections Completeness (%) R (%)* PCMBS 14-3.6 3,648 96.9 3.53 0.206 14-3.6 3,674 97.6 3.39 “7 0.236 RIB0 :R,. on intensities = Z,, Zi(Ii(hk 1) - I(h k 111 / Z, Zi I,(h k 1). 4, = L l IFderi.Ahk 1)l - IF&i”Jh k 1)l I 1 Thimerosal CH3HgC1 Selenomethionine 14-3.6 3,679 97.7 3.72 0.232 14-2.6 3,110 82.6 5.03 0.215 14-3.6 3,729 99.0 2.03 0.097 mdivel. in the presence of AMP, an Rsymof 7.8% (21.5-2.6 A); and apo-APRT had an Rsymof 7.0% (20.0-2.3 A). It is interesting that, as observed for 0PRT,l5 substrate or product binding does not alter noticeably the unit cell dimensions. Heavy Atom Derivatives Four mercurials, HgCl,, thimerosal, CH,HgCl, and p-chloromercuriphenylsulfonic acid (PCMBS), have shown promise as derivatives. Precession photographs of each of these potential derivatives showed intensity differences from native APRT. Similar intensity changes for the HgCl, and the thimerosal-soaked crystals suggested derivatization a t the same site($. Three-dimensional intensity data from 15.0-3.6 A have been collected for each of the mercurial soaked crystals, and all of these appeared to be isomorphous with the native crystals. The selenomethionine-labeled APRT crystals also appeared to be isomorphous and were as stable in the X-ray beam as native APRT. Some statistics relevant to these derivatives are given in Table I. The mercurials and selenomethionine derivatives are good candidates for the solution of the APRT structure by standard multiple isomorphous replacement methods. SUMMARY The excellent diffraction quality of the L. donouani APRT crystals suggests that the ensuing structure will provide a high-resolution framework for structure-based inhibitor and design. Furthermore, the structural determination of the L. donouani APRT will contribute to our understanding of its catalytic mechanism and more generally to PRT structure both before and after catalysis. Finally, the L. donouani APRT structure may reveal differences between PRTs from organisms capable of de novo purine synthesis anti those that rely upon purine salvage. ACKNOWLEDGMENTS We thank Darrick Carter for determining the catalytic activity of crystallized APRT and Patrick Jimenez for initial crysta.llizations. 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