Proc. Natl. Acad. Sci. USA Vol. 94, pp. 7162–7165, July 1997 Biochemistry

X-ray analysis of azido-thymidine diphosphate binding to nucleoside diphosphate kinase (antiviral agents͞phosphorylation͞azido-thymidine͞crystallography)

YINGWU XU*, OLIVIER SELLAM†,SOLANGE MORE´RA*, SIMON SARFATI‡,RICARDO BIONDI†,MICHEL VE´RON†, AND JOE¨L JANIN*§

*Laboratoire d’Enzymologie et de Biochimie Structurales, Unite´Propre de Recherche 9063, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette, France; †Unite´deRe´gulation Enzymatique des Activite´s Cellulaires, Unite´Mixte de Recherche 321, Centre National de la Recherche Scientifique, Institut Pasteur, 75724 Paris Cedex 15, France; and ‡Unite´de Chimie Organique, Institut Pasteur, 75724 Paris Cedex 15, France

Communicated by Max F. Perutz, Medical Research Council, Cambridge, United Kingdom, May 14, 1997 (received for review January 25, 1997)

ABSTRACT To be effective as antiviral agent, AZT (3؅- structure, especially at the active site where all residues are azido-3؅-deoxythymidine) must be converted to a triphos- conserved and make the same interactions with nucleotide phate derivative by cellular kinases. The conversion is inef- substrates (6, 7). The structure of the complex shows that the ficient and, to understand why AZT diphosphate is a poor analog binds at the same site and in the same way as natural substrate of nucleoside diphosphate (NDP) kinase, we deter- substrates. It brings strong support to the conclusion that the mined a 2.3-Å x-ray structure of a complex with the N119A 3ЈOH group of the sugar, missing in dideoxy compounds and point mutant of Dictyostelium NDP kinase. It shows that the replaced with a N3 azido group in AZT, is a major component analog binds at the same site and, except for the sugar ring of the mechanism of phosphate transfer catalysis by NDP pucker which is different, binds in the same way as the natural kinase (4). substrate thymidine diphosphate. However, the azido group that replaces the 3؅OH of the deoxyribose in AZT displaces a lysine side chain involved in catalysis. Moreover, it is unable MATERIALS AND METHODS to make an internal hydrogen bond to the oxygen bridging the Protein Preparation and Crystallization. Preparation of the ␤- and ␥-phosphate, which plays an important part in phos- N119A mutant of Dictyostelium NDP kinase has been de- phate transfer. scribed (8). The wild-type and N119A enzymes were expressed in Escherichia coli cells and purified as described (8) from the AZT (3Ј-azido-3Ј-deoxythymidine) and the dideoxy analogs of flow-through fraction of a DEAE-Sephacel (pH 8.4) column cytosine and inosine are major drugs against retroviral dis- (Pharmacia) by adsorption on Blue Sepharose (Pharmacia) at eases. Upon infection by human immunodeficiency virus, these pH 7.4 followed by elution in 0–2 M NaCl. Fractions were nucleoside analogs are incorporated by the reverse transcrip- concentrated by ultrafiltration on Diaflo PM10 membranes tase copying the viral genome, and DNA synthesis terminates, (Amicon) and equilibrated in 50 mM Tris⅐HCl buffer (pH 7.4). because they lack the 3ЈOH needed for elongation of the The proteins were pure as judged by SDS͞PAGE. polynucleotide chain. Because substrates for reverse transcrip- Commercially available nucleotides were obtained from tase are deoxynucleoside triphosphates, the analogs must Pharmacia, and [␥-32P]GTP (5,000 Ci͞mmol; 1 Ci ϭ 37 GBq) undergo phosphorylation before incorporation. This reaction from Amersham. The diphosphate (AZT-DP) and triphos- is performed in several steps by cellular phosphokinases, and phate derivatives of AZT were synthesized as described (4). it is very slow beyond the first step. Thus, lymphocytes The purity of AZT-DP was checked by 31P NMR. incubated with AZT accumulate inactive intermediates (1). Crystals of the N119A–AZT-DP complex were grown in With natural nucleotides, the conversion of the di- to the hanging drops over 32% PEG 550 as described for the wild- triphosphate is efficiently carried out by nucleoside diphos- type NDP kinase–ADP–AlF3 complex (9), with 10 mM phate (NDP) kinase, an enzyme present in all organisms (2). AZT-DP replacing ADP; 20 mM Mg2ϩ was present. They Human cells have two isotypes, NDP kinases A and B, the belong to space group P3121 (Table 1) with one-half of the products of the nm23-H1 and nm23-H2 genes (3). When the 100-kDa hexamer in the asymmetric unit. Attempts to crys- capacity of human NDP kinase B to phosphorylate derivatives tallize the wild-type protein–AZT-DP complex under similar of AZT and dideoxy-nucleosides was tested, it was found to be conditions were unsuccessful. several orders of magnitude less than for natural substrates (4). Enzyme Assay. Assays of NDP kinase activity were per- This may make triphosphate production rate-limiting in viral formed as described (4) with either dTDP or AZT-DP as the inhibition by the drugs, and suggests that analogs that are phosphate acceptor and 1 mM [␥-32P]GTP as the donor. To better substrates of NDP kinase should also be better drugs. prevent inhibition by the GDP reaction product, a regenerating To understand why NDP kinase is so unefficient in activat- system was included consisting of pyruvate kinase (0.3 unit) ing AZT, we determined a 2.3-Å x-ray structure of a complex and phosphoenolpyruvate (1 mM). The test included 10 pg of with AZT diphosphate (AZT-DP) and compared it with a enzyme when the substrate was dTDP, and 5 ng when it was previously determined complex with thymidine diphosphate AZT-DP. It was started by adding 3 ␮lenzymeina10␮l (dTDP) (5). The enzyme was a point mutant of the NDP reaction mixture containing 50 mM Tris⅐HCl (pH 7.4) and 5 kinase of the slime mold Dictyostelium discoideum, a 100-kDa mM MgCl2 and the substrates at 37°C; 3 ␮l aliquots were hexamer that is highly similar to the human enzyme (57% sequence identity). It has a very similar three-dimensional Abbreviations: NDP, nucleoside diphosphate; AZT, 3Ј-azido-3Ј- deoxythymidine; AZT-DP, AZT diphosphate; dTDP, thymidine The publication costs of this article were defrayed in part by page charge diphosphate. Data deposition: The atomic coordinates have been deposited in the payment. This article must therefore be hereby marked ‘‘advertisement’’ in Protein Data Bank, Chemistry Department, Brookhaven National accordance with 18 U.S.C. §1734 solely to indicate this fact. Laboratory, Upton, NY 11973 (reference 1LWX). © 1997 by The National Academy of Sciences 0027-8424͞97͞947162-4$2.00͞0 §To whom reprint requests should be addressed. e-mail: janin@ PNAS is available online at http:͞͞www.pnas.org. lebs.cnrs-gif.fr.

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Table 1. Statistics on crystallographic analysis could be calculated using atomic coordinates of the isomor- Parameter Values phous wild-type NDP kinase–ADP–AlF3 structure (9). Rela- tive to this structure, the electron density map clearly showed Diffraction data the absence of the Asn-119 side chain and the replacement of Space group P3121 ADP–AlF3 with AZT-DP. The density for the nucleotide Cell parameters a ϭ b, c, Å 71.59, 152.7 analog was easily interpretable at two of three active sites in the Resolution, Å 2.3 asymmetric unit (subunits A and C). At the third site (subunit Measured intensities 134,673 B), the phosphate positions were obvious, but the sugar and Unique reflections 19,887 base density was weaker. An occupancy of 1 was therefore Completeness, % 86 assumed for AZT-DP in subunit A and C, and 0.7 only at Rmerge,% 6.3 subunit B. The AZT moiety was taken from the x-ray structure Refinement (11) deposited in the Cambridge Structural Data Bank. Re- † Rcryst (Rfree), % 21.6 (28.0) finement was done with X-PLOR (12). The final model has an Reflections 16,845 R factor of 21.6% and correct geometry (Table 1). It contains ‡ Protein atoms 3515 three NDP kinase subunits (residues 6–155), three bound Solvent atoms 140 AZT-DP–Mg2ϩ complexes, and 140 water molecules. As in 2 Average B, Å 23.5 other x-ray structures of Dictyostelium NDP kinase (13), § Geometry N-terminal residues are disordered. The electron density is Bond length, Å 0.013 absent up to residue 5 and weak for residues 6–7. The three Bond angle, deg. 1.9 subunits in the asymmetric unit are identical to within exper- ␻ torsion angle, deg. 1.5 imental error over most of the polypeptide chain, the rms C␣ *Rmerge ϭ •hi ͉I(h)i Ϫ͗I(h)͉͘͞•hi I(h)i. deviation between pairs being 0.4 Å for residues 8–155. Main † Rcryst ϭ •h ʈF0͉ Ϫ ͉Fcʈ͞•h ͉F0͉ calculated on reflections with F Ͼ 2␴. chain discrepancies up to 2 Å are nevertheless observed in the Rfree was estimated by omitting 5% of the reflexions and running a extended C-terminal segment at residues 145–147, which are final 100 steps of refinement. poorly ordered and have above average temperature factors. ‡Includes Mg2ϩ and nucleotide atoms. §Root-mean-square deviation from ideal values. RESULTS AND DISCUSSION withdrawn at time intervals, heated for 2 min at 86°C, and then cooled on ice. An excess of cold nucleotides was added and the NDP kinase transfers the ␥-phosphate of a nucleoside triphos- mixture was analyzed by thin layer chromatography using 0.4 phate donor to a diphosphate acceptor via a phospho-histidine M ammonium carbonate as solvent. After drying, the plates intermediate (2, 14). It has a ‘‘ping-pong’’ type of mechanism were exposed to a PhosphorImager screen (Molecular Dy- and binds both donor and acceptor substrates at the same site namics) and the radioactivity in each spot was quantified as located in a cleft on the surface of the hexamer. The cleft has described (4). a ␣-helix hairpin (helices ␣A–␣2) on one side, a large loop Crystallographic Analysis. X-ray diffraction data were col- called the Kpn loop on the other; the name refers to the lected at the W32 station of the Laboratoire pour l’Utilisation killer-of-prune mutation of Drosophila (15), which is a point du Rayonnement Electromagnetique (LURE-DCI) synchro- substitution in the equivalent loop of Drosophila NDP kinase. tron radiation center (Orsay, France) using a MAResearch The bound nucleotide is oriented with the nucleobase pointing imaging plate system. A single N119A–AZT-DP crystal was outside the protein, the phosphate groups pointing inside and used. The wavelength was ␭ ϭ 0.994 Å and the temperature toward the active site histidine, His-122 in Dictyostelium NDP was 4°C. Data reduction used the CCP4 package (10). The data kinase. Fig. 1 shows that AZT-DP binds at that site and in the had good statistics to 2.3-Å resolution (Table 1) and phases same orientation as the natural substrate dTDP (5). The

FIG. 1. Stereoview of the superimposed AZT-DP and TDP structures at the NDP kinase active site. The N119A–AZT-DP complex is colored by atom type; the wild-type–dTDP complex (5) has white bonds. The electron density for AZT-DP is contoured at 3 ␴ in a 2.3 Å Fo Ϫ Fc map. The thymine base points down toward outside the protein. The phosphates carry a Mg2ϩ ion (orange ball) and point toward the active site His-122 on top. Though the N119A mutation locally affects the main chain conformation, the protein structure is essentially the same in the two complexes and crowding by the 3Ј-azido group of AZT-DP is chiefly responsible for the observed movement of the Lys 16 side chain. Drawn with TURBO (A. Roussel and C. Cambillau, Marseille, France, personal communication). Downloaded by guest on September 28, 2021 7164 Biochemistry: Xu et al. Proc. Natl. Acad. Sci. USA 94 (1997)

Table 2. Nucleotide–protein contacts Table 3. Conformation of bound nucleotides Nucleotide Protein Distance, Å* Dihedral angle (deg.) AZT AZT-DP dTDP Thymine ring Phe-64 cycle 3.6 (0.25) ␹ (glycosidic bond) Ϫ125 Ϫ128 (2) Ϫ156 Val-116 C␥1 3.7 (0.25) ␥ (C5Ј-C4Ј bond) 52 63 (2) 57 ␣-phosphate O11 His-59 N␧ 3.2 (0.15) P (sugar pucker) 173 152 (6) 31 2ϩ Mg 2.7 (0.25) Dihedral angles and the pseudo-rotation angle P are defined as in † 2ϩ ␤-phosphate O21 Mg 2.6 (0.1) ref. 18. AZT is from the crystal structure of the free compound (11). Arg-92 N␩1 2.7 (0.2) AZT-DP are average values, with the standard deviation in paren- O22 Arg-109 N␩2 3.0 (0.15) theses, observed in the three independent subunits of the NDP kinase O7 Arg-109 N␩1 3.1 (0.25) complex (this work). dTDP is from the complex with NDP kinase (5). Azido N4 Lys-16 N␨ 3.6 (0.15) the nucleoside moiety of AZT-DP is C -endo with the base N6 Ala-119 C␤ 3.7 (0.4) 2Ј Ile-121 O 3.0 (0.4) anti, essentially the same as in the crystal structure of AZT alone (11). In contrast, the deoxyribose is C -endo in bound His-122 C␤ 3.5 (0.1) 3Ј dTDP (Table 3). As a result of the change in sugar pucker, the Arg-109 N␩1 3.0 (0.15) 3Ј-azido group moves away from the site normally occupied by *Average of three values observed in the crystallographic unit; the the hydroxyl, where it would collide with protein atoms. standard deviation is in parentheses. Instead, it overlaps with the mobile ␧-amino group of Lys-16. †In subunit B, the Mg2ϩ site is poorly occupied and appears to coordinate only the ␣-phosphate. This is displaced by as much as 2.6 Å, whereas other active site residues stay essentially unperturbed. The azido group is also protein fold is essentially unchanged. The rms C␣ deviation is in van der Waals contact with the side chains of Ala-119 and 0.41–0.46 Å between subunits of the dTDP and AZT-DP His-122, and with the carbonyl group of Ile-121. In addition, complexes, comparable to the deviation between the three it may receive a hydrogen bond from Arg-109 (Table 2). independent subunits of the latter. The Asn to Ala substitution In NDP kinase, the 3ЈOH of the sugar in the bound substrate at residue 119 has a small local effect on the main chain, which normally receives a hydrogen bond from the amide group of is displaced by 0.7 Å over two residues. Asn-119 and donates one to the oxygen bridging the ␤- and Interactions of AZT-DP with protein groups and Mg2ϩ are ␥-phosphates (O7 in Table 2) (5–7, 17). The azido group of listed in Table 2. The thymine base lies in between the side AZT cannot make these interactions. Moreover, it is much bulkier than a hydroxyl, and the N119A mutant was designed chains of Phe-64 at the tip of the ␣ –␣ helix hairpin, and A 2 to make room for it. The lack of a carboxamide group in Val-116 of the Kpn loop (Fig. 2). Its polar groups make no alanine has only a local effect on the conformation of the direct interaction with the protein. Compared with dTDP, the polypeptide chain, but it does affect the catalytic activity, base shifts in its plane by about 1 Å, and the Phe-64 side chain which in N119A NDP kinase is of the order of 10% of that of accompanies its movement. A very similar shift is observed the Dictyostelium wild type (Table 4). With AZT-DP as the with GDP or ADP (7, 17), the mobility of Phe-64 contributing phosphate acceptor and GTP as the phosphate donor, the to the lack of discrimination between purine and pyrimidine kinetic parameters of the Dictyostelium and human enzymes substrates which is a peculiar feature of NDP kinase. The 3 4 are similar (4). There is a ratio of 10 in kcat and 10 in kcat͞KM phosphate moieties have identical positions in the AZT-DP in the relative efficiency toward dTDP and AZT-DP. In the and dTDP complexes. They bind arginine side chains and mutant, the relative efficiency is 2 ϫ 103 as measured by 2ϩ coordinate a Mg ion in the same way, though the metal– kcat͞KM, the most significant parameter in an enzyme with a oxygen bonds are long with AZT-DP suggesting that the metal ‘‘ping-pong’’ mechanism, since its value for one substrate is site is partly occupied by solvent. independent of the other substrate concentration. The most noticeable difference between the natural sub- The present x-ray structure excludes the possibility that the strate and the analog is the sugar pucker. The conformation of low activity on AZT-DP reflects binding of the analog in a

FIG. 2. NDP kinase interactions with AZT-DP. The N3 azido group in the center is in contact with the side chains of Lys-16, of Ala-119 replacing an asparagine in wild-type NDP kinase, and of His-122 on top. It may also hydrogen bond to Arg 109. The grey ball is a Mg2ϩ ion interacting with both phosphate groups. The stereopair was drawn with MOLSCRIPT (16). Downloaded by guest on September 28, 2021 Biochemistry: Xu et al. Proc. Natl. Acad. Sci. USA 94 (1997) 7165

Table 4. Phosphorylation of AZT-DP by wild-type and N119A Yvette) for protein purification and crystallization, Dr. L. Tchernova mutant NDP kinase (ICSN, Centre National de la Recherche Scientifique, Gif-sur-Yvette) for searching the Cambridge Structural Data Bank, and the staff of WT N119A Laboratoire pour l’Utilisation du Rayonnement Electromagnetique Enzyme acceptor AZT- AZT- (Orsay, France) for access to the W32 station and help in data substrate dTDP DP dTDP DP collection. This work was supported by Universite´Paris-Sud, Orsay, Agence Nationale de Recherche contre le SIDA, Association pour la k ,sϪ1 1,220 1.0 160 3.2 cat Recherche contre le Cancer, and the PRA B95-03 cooperative pro- K , ␮M 55 500 75 3,000 M gram between Universite´Paris-Sud and University of Science and k K ,MϪ1⅐sϪ1 2.3 107 2 103 2 106 1 103 cat͞ M ϫ ϫ ϫ ϫ Technology of China, Hefei, China. Enzyme assays were performed on wild-type (wt) and N119A Dictyostelium NDP kinases at 37°C as described in Materials and 1. Balzarini, J., Herdewijn, P. & De Clercq, E. (1989) J. Biol. Chem. Methods.[␥-32P]GTP (1 mM) was the donor substrate, and either 264, 9062–9069. dTDP or AZT-DP was the acceptor substrate. 2. Parks, R. E., Jr., & Agarwal, R. P. (1973) Enzymes 8, 307–334. 3. Gilles, A. M., Presecan, E., Vonica, A. & Lascu, I. (1991) J. Biol. nonproductive way due to steric hindrance by the azido group. Chem. 266, 8784–8789. Alternative explanations are the induced movement of the 4. Bourdais, J., Biondi, R., Sarfati, S., Guerreiro, C., Lascu, I., Lys-16 side chain and the missing hydrogen bonds. The Janin, J. & Ve´ron,M. (1996) J. Biol. Chem. 271, 7887–7890. movement of Lys-16 apparently suffices to release crowding by 5. Cherfils, J., More´ra,S., Lascu, I., Ve´ron,M. & Janin, J. (1994) the azido group. It has functional consequences, for the Biochemistry 33, 9062–9069. ␧-amino group can no longer interact with the ␥-phosphate of 6. Webb, P. A., Perisic, O., Mendola, C. E., Backer, J. M. & Wil- the donor nucleotide as it does in a normal substrate (7, 14). liams, R. L. (1995) J. Mol. Biol. 251, 574–587. Substitution of Lys-16 with an alanine reduces activity to 0.2% 7. More´ra,S., Lacombe, M. L., Xu, Y., LeBras, G. & Janin, J. (8). The role of the Asn-119 bond to the 3ЈOH is illustrated in (1995) Structure 3, 1307–13014. Table 4 by kinetic data obtained on the N119A mutant with 8. Tepper, A., Dammann, H., Bominaar, A. A. & Ve´ronM. (1994) J. Biol. Chem. 269, 32175–32180. dTDP as a substrate: kcat͞KM drops by a factor of 10 when the carboxamide group is deleted. The loss of the internal hydro- 9. Xu, Y., More´ra,S., Janin, J. & Cherfils, J. (1997) Proc. Natl. gen bond of the 3ЈOH to the bridging phosphate oxygen in Acad. Sci. USA 94, 3579–3583. AZT-DP has much more effect and it must be a major reason 10. Collaborative Computational Project, Number 4 (1994) Acta for the low activity of NDP kinase on that substrate. Dideoxy- Crystallogr. A 50, 157–163. nucleotides that, like AZT-DP, are unable to make this bond 11. Gurskaya, G. V., Tsapkina, E. N., Skaptsova, N. V., Kraevskii, A. A., Lindeman, S. V. & Struchko, Yu. T. (1986) Dokl. Akad. are equally poor substrates of the human enzyme (4). The Nauk SSSR 291, 854. bridging oxygen is the leaving group when phosphorylating, 12. Bru¨nger, A. T., Kuriyan, J. & Karplus, M. (1987) Science 235, and the attacking group when dephosphorylating the active 458–460. site histidine. Therefore, to be efficiently activated by NDP 13. More´ra,S., LeBras, G., Lascu, I., Lacombe, M.-L., Ve´ron,M. & kinase, a nucleoside analog must carry a hydrogen donor group Janin, J. (1994) J. Mol. Biol. 243, 873–890. comparable to the 3ЈOH and also occupy approximately the 14. More´raS., Chiadmi M., LeBras G., Lascu I. & Janin J. (1995) same location relative to the phosphates. The challenge is that Biochemistry 34, 11062–11070. it may not be a hydroxyl, nor any other group on which reverse 15. Timmons, L. & Shearn, A. (1997) Adv. Genet. 35, 207–252. transcriptase can add a phosphate to elongate the DNA chain. 16. Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 949–950. Our structural data should help in its design. 17. More´ra,S., Lascu, I., Dumas, C., LeBras, G., Briozzo, P., Ve´ron, M. & Janin, J. (1994) Biochemistry 33, 459–67. We thank G. LeBras (Laboratoire d’Enzymologie et de Biochimie 18. Saenger, W. (1984) Principles of Nucleic Acid Structure (Springer, Structurales, Centre National de la Recherche Scientifique, Gif-sur- New York). Downloaded by guest on September 28, 2021