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The Crystal Structure of Trypanosoma Cruzi Dutpase Reveals a Novel Dutp/Dudp Binding Fold

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The Crystal Structure of Trypanosoma Cruzi Dutpase Reveals a Novel Dutp/Dudp Binding Fold Structure, Vol. 12, 41–53, January, 2004, 2004 Elsevier Science Ltd. All rights reserved. DOI 10.1016/j.str.2003.11.016 The Crystal Structure of Trypanosoma cruzi dUTPase Reveals a Novel dUTP/dUDP Binding Fold Maria Harkiolaki,1,3 Eleanor J. Dodson,1 dUTPase whose subunits are composed primarily of Victor Bernier-Villamor,2 Johan P. Turkenburg,1 ␤-pleated sheets (Persson et al., 2001). Each active site Dolores Gonza´ lez-Pacanowska,2 and Keith S. Wilson1,* in the trimeric dUTPases lies at the interface of adjacent 1Structural Biology Laboratory subunits and is made up from five highly conserved Department of Chemistry motifs, contributed from all three subunits within a single University of York molecular unit. The mammalian herpesviral dUTPases Heslington, York YO10 5YW function as monomers (McGeoch, 1990), and possess United Kingdom the same motifs as the trimeric enzymes but in a different 2 Instituto de Parasitologı´a y Biomedicina order. No structures are available for these enzymes. In “Lo´ pez-Neyra” contrast, the dUTPases from Leishmania major, Trypa- C/Ventanilla, 11. 18001-Granada nosoma cruzi, Campylobacter jejuni, and T4 bacterio- Spain phage form a totally different sequence family and function as physiological dimers (Hidalgo-Zarco and Gonzalez-Pacanowska, 2001) which contain none of Summary the five conserved sequence motifs that form the active site of the trimeric or monomeric dUTPases (Persson et dUTPase is an essential enzyme involved with nucleo- al., 2001). More recently, sequences encoding dimeric- tide metabolism and replication. We report here the dUTPases have also been identified in the genomes X-ray structure of Trypanosoma cruzi dUTPase in its of Trypanosoma brucei (AC105378) and Microbulbifer native conformation and as a complex with dUDP. degradans (ZP_00066466). These reveal a novel protein fold that displays no dUTPase expression appears to be essential in L. structural similarities to previously described dUT- major (Hidalgo-Zarco and Gonzalez-Pacanowska, 2001) Pases. The molecular unit is a dimer with two active and given the high degree of similarity of this enzyme sites. Nucleotide binding promotes extensive struc- to the T. cruzi homolog this assumption can be projected tural rearrangements, secondary structure remodel- to other members of the Trypanosomatidae family. The ing, and rigid body displacements of 20 A˚ or more, protozoan T. cruzi is the causative agent of Chagas’ which effectively bury the substrate within the enzyme disease and is one of three species of the genus Trypa- core for the purpose of hydrolysis. The molecular com- nosoma that are pathogenic to humans. According to plex is a trapped enzyme-substrate arrangement which the Center for Disease Control, an estimated 16–18 mil- clearly demonstrates structure-induced specificity and lion people are currently suffering from Chagas’ disease catalytic potential. This enzyme is a novel dUTPase and and 50,000 people die each year as a result (see http:// therefore a potential drug target in the treatment of www.cdc.gov/ncidod/dpd/parasites/chagasdisease). Chagas’ disease. There is as yet no cure for Chagas’ (World Health Organi- zation reports at http://www.who.int/ctd/chagas), al- Introduction though advances in the treatment of experimental T. cruzi infections have been reported (Engel et al., 1998). dUTPase, also known as dUTP nucleotidohydrolase or Hence the identification of further protein drug targets dUTP pyrophosphatase (EC 3.6.1.23), is a ubiquitous against trypanosomes such as dUTPase are of para- enzyme that catalyses the hydrolysis of 2-deoxyuridine mount importance. triphosphate (dUTP) to 2-deoxyuridine monophosphate We report here the X-ray structure of T. cruzi dUTPase (dUMP) (Bertani et al., 1961). Hydrolysis of 2-deoxyuri- in its native state and as a complex with dUDP. In the dine diphosphate (dUDP) to dUMP has also been re- complex there are two crystallographically independent ported and is a unique feature of a subset of dUTPases dimers, the first with dUDP in both subunits, and the (Hidalgo-Zarco and Gonzalez-Pacanowska, 2001). The second (the hybrid) with only one active site occupied. action of dUTPases ensures a balanced ratio of dTTP/ The structure has a novel protein fold suggestive of a dUTP within the cellular nucleotide pool and thus safe- mechanism of action quite dissimilar to that of the tri- guards DNA integrity during replication (Pearl and meric dUTPases. Savva, 1996). The lack of dUTPase activity has been shown to be detrimental to cell survival (el-Hajj et al., 1988; Gadsden et al., 1993) Subunit Structure Currently dUTPases are classified according to their The T. cruzi dUTPase subunit is a predominantly helical associative state in the molecular unit as either homotri- arrangement (Figure 1A). It is comprised of 12 helices, meric, homodimeric, or monomeric, corresponding to h1–h12 (residues 12–29, 33–36, 39–55, 71–93, 102–111, members of three families of homologous proteins. The 153–168, 172–186, 190–205, 207–210, 223–231, 245– majority of organisms studied to date possess a trimeric 259, and 264–274) (Figure 1B), as assessed by DSSP (Kabsch and Sander, 1983). These are arranged in two *Correspondence: [email protected] distinct domains (Figure 1C), termed “rigid” and “mo- 3Present address: Weatherall Institute of Molecular Medicine, John bile,” angled at about 60Њ to one another. The molecular Radcliffe Hospital, Oxford OX39 DS, United Kingdom. unit is a homodimer formed primarily through associa- Structure 42 Figure 1. Structure of the T. cruzi dUTPase Subunit (A) Stereo backbone trace with every tenth residue numbered. (B) Stereo view of the native subunit with helices represented as rods numbered from the amino terminus. (C) Schematic of the subunit domains. (D) Stereo view of a subunit in complex with dUDP. (E) Electrostatic surface representation of a single subunit with characteristics highlighted. tion of the two rigid domains that constitute its core. regions lie at the opposite side of the molecule from These remain static during substrate binding, in contrast the active sites and probably have no significance with to the mobile domains which undergo substantial struc- regards to catalysis, although such an assumption tural changes to accommodate incoming nucleotides. would need to be substantiated by mutation studies. The rigid domain extends through residues 30–187 and When an active site becomes occupied the mobile the mobile through residues 9–30 and 188–278. The fold domain moves to engulf the bound nucleotide through is complemented by several loops connecting individual a series of structural rearrangements that include re- helices and domains and two helical 3/4 turns (residues modeling of secondary structural features as well as 142–145 and 236–240). Both native and complex struc- rigid body displacements of 20 A˚ or more (Figure 1D). tures contain regions of poor electron density that could Prominent structural rearrangements occur at h9, which not be modeled and are therefore presumed to be flexi- becomes a helical 3/4 turn (residues 208–212), and h10, ble (residues 1–9, 97–100, 120–139, and 279–283). These which is segmented into a 310 part (residues 223–226) Novel Fold T. cruzi dUTPase in Complex with dUDP 43 and a ␣-helical part (residues 227–231). In addition, h12 site in a dimer lies at the depression formed by elements splits into two smaller helices, a 310 segment (h12a, resi- of the mobile and rigid domains of one subunit and is dues 264–266) and a ␣ helix (h12b, residues 268–275). complemented by the latch of the other subunit. Two newly formed ␤ strands “zip” the loop covering the Ligand features are recognized by specific structural active site into an apparently stable and tighter confor- arrangements that confer a highly selective binding po- mation. The two strands (␤1 and ␤2, residues 217–218 tential on the active sites (Figure 4). These are effectively and 221–220 respectively) are arranged in an antiparallel base-pairing mimics that bind uracil through selective fashion. In addition to secondary structure remodeling, hydrogen bonding with side chain atoms (Asn26 and elements in the mobile domain adopt new relative posi- Gln22 from h1 and Trp61 from the latch of the neigh- tions in the dimer as helices “slide” relative to one an- boring subunit). Once bound, the nucleotide is capped other to cover the active site. The rigid domains of partic- by the side chain of Met26 (h1) which stacks on the uracil ipating subunits retain the native conformation upon ring effectively locking it in position. The deoxyribose is nucleotide binding. anchored through two hydrogen bonds to the O␦1 and N␦2 atoms of Asn201 (h8). Additionally, two aromatic residues, Phe84 and His83, both components of h4, Overall Fold of the Molecular Unit flank the sugar moiety preventing any movement once The functional dimer in the native crystal is formed by it is bound. The phosphates in turn are tightly positioned two identical subunits related through 2-fold symmetry. within an intricate network of hydrogen bonds formed The dimer associates chiefly through helix-helix interac- with a number of other residues, including Trp62, Tyr209 tions of the two rigid domains which form the stable core (h9), Arg204 and Lys197 (h8), Asn224 (3 section of h10), of the enzyme (Figure 1E), complemented by insertion of 10 Lys216 (␤1), Glu49 and Glu52 (h3), and Glu77 (h4). the latch from one subunit into the groove of its partner The model thus reveals how structure affects specific- (Figure 1C). There are two active sites per dimer, each ity. The uracil moiety of dUDP is held in place by three residing in the groove formed at the interface of the hydrogen bonds to protein side chain atoms. dCTP and two domains within individual subunits (Figure 1E). The dCDP are excluded from this site because the radial amine active site is formed primarily by residues of a single of cytosine would be repelled by Trp61 due to the respec- subunit but is complemented by residues brought into tive positive charge potentials.
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