The Crystal Structure of Leishmania Major N5,N10

Molecular & Biochemical Parasitology 181 (2012) 178–185

Contents lists available at SciVerse ScienceDirect

Molecular & Biochemical Parasitology

5 10

The crystal structure of Leishmania major N ,N -methylenetetrahydrofolate

ଝ

dehydrogenase/cyclohydrolase and assessment of a potential drug target

∗

Thomas C. Eadsforth, Scott Cameron, William N. Hunter

Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK

a r t i c l e i n f o a b s t r a c t

5 10

Article history: Three enzyme activities in the protozoan Leishmania major, namely N ,N -methylenetetrahydrofolate

Received 26 September 2011 5 10 10

dehydrogenase/N ,N -methenyltetrahydrofolate cyclohydrolase (DHCH) and N -formyltetrahydro-

Received in revised form 3 November 2011 10

folate ligase (FTL) produce the essential intermediate N -formyltetrahydrofolate. Although trypanoso-

Accepted 7 November 2011

matids possess at least one functional DHCH, the same is not true for FTL, which is absent in Trypanosoma

Available online 15 November 2011

brucei. Here, we present the 2.7 A˚ resolution crystal structure of the bifunctional apo-DHCH from L. major,

which is a potential drug target. Sequence alignments show that the cytosolic enzymes found in try-

Keywords:

panosomatids share a high level of identity of approximately 60%. Additionally, residues that interact

Antifolate

Cyclohydrolase and participate in catalysis in the human homologue are conserved amongst trypanosomatid sequences

Dehydrogenase and this may complicate attempts to derive potent, parasite speciﬁc DHCH inhibitors.

Trypanosoma

1. Introduction and also in the metabolism of histidine and serine (Fig. 1a) [6].

It is not surprising that enzymes involved in folate-dependent

The kinetoplastid protozoan Leishmania and Trypanosoma sp. pathways, e.g. dihydrofolate reductase (DHFR), are important

are the causal agents for a range of serious parasitic infec- antimicrobial and anticancer drug targets [7,8]. Trypanosomatids

tions [1]. Although drugs are available for the treatment of are auxotrophic for folates and pterins [9] and reliant on uptake

these diseases they are toxic, costly and with low efﬁcacy [2]. and salvage mechanisms to maintain the required level of these

Increasing levels of drug resistant parasites [3] further complicate important compounds. Inhibition of DHFR should, in principle,

this biomedical problem and the need for improved diagnos- provide a route to treat trypanosomatid infections. However, the

tic methods and treatments is as great now as ever. Modern presence of a pteridine reductase (PTR1) able to reduce dihy-

genomic-driven research is helping to unravel the molecular drofolate (DHF) to tetrahydrofolate (THF), i.e. catalyze the same

and cell biology of these primitive eukaryotes; for example a reaction as DHFR, helps to compromise the use of such inhibitors

large dataset is available on which genes might encode essen- [10]. Promising PTR1 inhibitors have been identiﬁed [11–15]

tial activities [4]. We also have an improved understanding of in support of a strategy to develop a combination treatment

what types of molecules are likely to provide drug targets or with known DHFR inhibitors to cut-off the supply of reduced

appropriate lead compounds and it is timely to identify and crit- pterins/folates. The use of drug combinations might also serve to

ically assess potential targets that might underpin future efforts alleviate the development of drug resistance [13,15]. Here, we turn

in drug discovery [5]. With this goal in mind we identiﬁed our attention onto enzymes that maintain the required levels of

5 10 10

the bifunctional N ,N -methylenetetrahydrofolate dehydroge- N -formyltetrahydrofolate, a key intermediate supporting protein

nase/cyclohydrolase, an enzyme involved in folate metabolism, as synthesis.

5 10

a potential drug target in Leishmania. THF is converted to N ,N -methylenetetrahydrofolate by ser-

Folate and derivatives are essential cofactors in the biosynthe- ine hydroxymethyl transferase or the glycine cleavage system [6].

+ +

sis of thymidine, purines, glycine, methionine, initiator fMet-tRNA A two-step reaction follows, initially an NADP or NAD dependant

5 10

oxidization of N ,N -methylenetetrahydrofolate to the inter-

5 10 5 10

mediate N ,N -methenyltetrahydrofolate catalyzed by N ,N -

ଝ methylenetetrahydrofolate dehydrogenase (DH) and the methenyl

Note: Coordinates and structure factor data have been deposited with the PDB, 10

derivative subsequently hydrolyzed to N -formyltetrahydrofolate code 4A26.

∗ 5 10

by N ,N -methenyltetrahydrofolate cyclohydrolase (CH, Fig. 1b)

Corresponding author. Tel.: +44 1382 385745; fax: +44 1382 385764.

E-mail address: [email protected] (W.N. Hunter). [16]. N -formyltetrahydrofolate also results from addition of

T.C. Eadsforth et al. / Molecular & Biochemical Parasitology 181 (2012) 178–185 179

Fig. 1. An overview of the folate metabolism pathway in formation of essential intermediates in Leishmania. (a) Dihydrofolate (DHF) is converted to tetrahydrofolate (THF)

by the actions of dihydrofolate reductase (DHFR) and/or pteridine reductase (PTR1). THF is modiﬁed by serine hydroxymethyl transferase (SHMT) or formyltetrahydrofolate

ligase (FTL) and the interplay between three substituted intermediates of THF is controlled by the bifunctional dehydrogenase cyclohydrolase (DHCH). Intermediates

5 10

generated at each stage are utilized by other pathways, for example N ,N -methylenetetrahydrofolate is used by thymidylate synthase (TS). (b) The action of DHCH

+ 5 10

maintains the required levels of methenyl-, methylene-, formyl-tetrahydrofolate molecules, ﬁrst by an NADP dependent oxidation from N ,N -methylenetetrahydrofolate

5 10 10

to form N ,N -methenyltetrahydrofolate, then to N -formyltetrahydrofolate by the cyclohydrolase activity. The inhibitor LY354899 is also shown. Figure produced using ChemDraw.

180 T.C. Eadsforth et al. / Molecular & Biochemical Parasitology 181 (2012) 178–185

formate onto THF in a reaction catalyzed by formyltetrahydro- 2.2. Puriﬁcation

folate ligase (FTL) [17]. Humans possess a cytosolic trifunctional

◦

C-1-tetrahydrofolate synthase with DHCH and FTL activities, in Cells were cultured in 1 L ﬂasks at 37 C with shaking (200 rpm)

−1

addition to a mitochondrial bifunctional DHCH and monofunc- in auto induction media [26] supplemented with 50 mg L car-

tional FTL [18]. Structures of cytosolic human DHCH (HsDHCH) benicillin until an OD600 of 0.6 was reached. The temperature

◦

[19,20] have formed the basis of some mechanistic understanding was subsequently reduced to 21 C overnight. Cells were collected

◦

[21]. by centrifugation (4 C at 4000 × g for 30 min). Cells were resus-

All trypanosomatids possess this dehydrogenase and cyclo- pended in 20 mL of buffer A (50 mM Tris–HCl, 250 mM NaCl, 20 mM

hydrolase activity with a cytosolic bifunctional enzyme (DHCH). imidazole, pH 7.5) with the addition of DNAse (200 ␮g) and an

In Leishmania major and Trypanosoma cruzi a separate enzyme EDTA-free protease inhibitor tablet (Roche) prior to two rounds of

catalyzes the FTL reaction but this enzyme is absent in Try- lysis in a French press pressure cell under 16,000 psi. The resulting

◦

panosoma brucei [22,23]. The cytosolic DHCH activity appears homogenate was centrifuged (4 C at 37,500 × g for 30 min) and

essential in L. major since knockouts of the disomic copies were the supernatant loaded onto a pre-equilibrated and Ni charged

not possible without ectopic expression of FTL to provide an HisTrap HP 5 mL column (GE Healthcare) with the subsequent

alternative route to N -formyltetrahydrofolate [24]. Since try- application of a linear gradient of 20 mM to 1 M imidazole in buffer

panosomatids are purine auxotrophs [25] then the essential A. Samples containing LmDHCH were pooled and His-tagged TEV

requirement for a supply of N -formyltetrahydrofolate is likely protease was added at 1 mg TEV per 20 mg protein. The mixture was

due to its contribution to production of fMet-tRNA and ulti- dialysed using snakeskin tubing (10 kDa MWCO) against buffer A

mately for protein synthesis. Several Leishmania species, including for 4 h at room temperature. The sample was again passed over a

Leishmania donovani, possess an additional putative mitochon- HisTrap HP 5 mL column to remove the TEV protease, uncleaved

drial enzyme (DHCH2) that shares between 27 and 31% sequence LmDHCH, the cleaved tag and histidine-rich proteins from E. coli.

identity with DHCH. Although DHCH2 is larger that the cytoso- The LmDHCH was then concentrated and applied to a Superdex 200

lic enzyme, the residues predicted to interact with the substrate 26/60 column (GE Healthcare) pre-equilibrated with buffer A. Anal-

are conserved across all species [23]. In L. major a pseudogene, ysis of the elution proﬁle showed that the main peak eluted with an

dhch2, is noted [Genedb, http://www.genedb.org]. This implies approximate molecular mass of 65 kDa, indicating LmDHCH forms

that N -formyltetrahydrofolate is synthesized in the cytosol a dimer. The buffer was exchanged to 50 mM Tris–HCl, 100 mM

−1

and then transported into the mitochondria for use in fmet- NaCl, pH 7.5 and the enzyme concentrated to 9 mg mL for crys-

tRNA [23]. The presence of DHCH activity in the mitochondria tallization trials. The ﬁnal LmDHCH preparation was estimated to

of some trypanosomatids is a complicating factor. Its presence be greater than 95% pure by SDS-PAGE and MALDI-TOF mass spec-

10 −1

could compromise inhibition of the cytosolic enzyme if N - trometry, with a yield of protein of approximately 5 mg L of cell

formyltetrahydrofolate can be transported out of the organelle culture. Protein concentration was calculated using the theoretical

−1 −1

to the cytosol. Inhibition of both DHCH and DHCH2 might then extinction coefﬁcient of 6320 M cm using ProtPram [27].

be necessary to affect the parasite. In L. major, lacking a func-

tional DHCH2, FTL activity might provide a by-pass of DHCH 2.3. Crystallization and data collection

inhibition and that would have to be taken into considera-

tion. Crystallization trials were carried out using a Phoenix Liquid

5 10

We report the crystal structure of L. major N ,N - Handling System (Art Robins Instruments/Rigaku) with several

methylenetetrahydrofolate dehydrogenase/cyclohydrolase commercial screens, including MPD, PEG, Classics and ammonium

(LmDHCH), detailed structure–sequence comparisons with try- sulfate (Hampton Research) using a 1:1 ratio of 100 nL of protein

panosomatid orthologues, with the human orthologue and explore solution and an equivalent volume of reservoir solution equili-

◦

the potential for the development of potent trypanosomatid brated against 70 ␮L reservoir at 20 C. Crystals were observed after

speciﬁc inhibitors against this essential enzyme. three days with a reservoir of 20% PEG 3350 and 0.2 M ammonium

acetate. Optimization in 2 ␮L drops using the hanging drop vapor

diffusion method gave orthogonal blocks with approximate dimen-

2. Methods

sions 0.15 mm × 0.2 mm × 0.1 mm. Single crystals were transferred

to a cryo-solution containing the original reservoir solution sup- ◦

2.1. Cloning

plemented with 40% glycerol prior to ﬂash freezing at −173 C.

Crystals were characterized in-house with a Micromax-007 rotat-

The dhch1 gene, encoding LmDHCH, was identiﬁed in ++

ing anode generator and R-AXIS IV dual image plate detector

Genedb (http://www.genedb.org, accession number LmjF26.0320).

(Rigaku), prior to storage in liquid nitrogen. X-ray diffraction data

Genomic DNA from L. major (Friedlin strain) was the tem-

were then collected at beam line I04 at the Diamond Light Source.

plate for PCR with the following primers designed to amplify

Integration and scaling of data were carried out using MOSFLM [28]

the open reading frame with NdeI and BamHI restriction sites

and SCALA [29]. The crystal displayed space group C2221 with unit

(bold), respectively: 5 -CAT-ATG-CCG-TCT-GCT-CAG-ATC-AT-3 ,

cell lengths a = 117.2 A,˚ b = 220.08 A˚ and c = 56.31 A.˚ The molecular

5 -GGA-TCC-CTA-TGA-TAC-GCC-GAA-GCG-A-3 . The PCR product

mass of a subunit is 31.9 kDa, and the asymmetric unit consists

−

was ligated into pCR-BluntII-TOPO vector using the Zero Blunt 3 1

of two subunits giving a VM of 2.8 A˚ Da and solvent content of

TOPO PCR cloning kit (Invitrogen). The gene was then excised

approximately 55%.

from TOPO with NdeI/BamHI and ligated into a modiﬁed pET15b

(Novagen) containing a Tobacco Etch Virus (TEV) protease recog-

2.4. Structure determination

nition sequence in place of a thrombin recognition sequence

(pET15bTEV). This results in recombinant expression of a product

The structure was solved by molecular replacement and reﬁned

carrying an N-terminal hexa-histidine tag (His-tag), which is cleav-

to 2.7 A˚ resolution. The search model was a monomer of the

able with TEV protease. The recombinant plasmid was ampliﬁed in

E. coli enzyme (EcDHCH) which shares 46% sequence identity,

XL-1 blue Escherichia coli, and the gene sequence veriﬁed, before

(Protein Data Bank (PDB) code 1B0A) [30] with all side chains

being transformed into E. coli BL21 (DE3) (Stratagene) for protein

removed using CHAINSAW [31]. Rotation and translation functions

production.

were solved with PHASER [32] in the Collaborative Computational

T.C. Eadsforth et al. / Molecular & Biochemical Parasitology 181 (2012) 178–185 181

Fig. 2. A cartoon representation of the LmDHCH homodimer. Subunit A is yellow, B is green. The elements of secondary structure of subunit B are labeled in addition to the

N- and C-termini. A small section of a loop (residues 248–250, highlighted with black asterisks) is absent in subunit B due to disorder.

Suite 4 [33]. The positions of two molecules, forming a dimer between the two domains and at one end an NADP binding site,

␤␣␤␣␤

were identiﬁed with a log likelihood gain of 364. Following rigid- typical of a Rossmann fold ( ), is formed. The substrate-

body reﬁnement in REFMAC5 [34], the side chains were built binding site is at the other end of the cleft. Analysis of the domains of

into electron and difference density maps and iterative rounds LmDHCH from the crystallographically independent chains shows

of restrained reﬁnement carried out with Babinet scaling, elec- a signiﬁcant pivot around a hinge (residues 150–152 and 274–280)

◦

tron and difference density map inspection, model manipulation with a rotation of almost 14 evident (Fig. 3). The hinge involves

␣

and the addition of solvent molecules using COOT [35] and REF- 6 and the loop leading into ␣11. A similar observation has been

MAC5. Tight non-crystallographic symmetry (NCS) restraints were made in HsDHCH structures [19,20]. The subunits appear in distinct

initially applied and subsequently released. In addition, Transla-

tion/Libration/Screw analysis (TLS) [36] was applied. Model quality

was checked using MolProbity [37] and crystallographic statistics

are summarized in Table 1. Structural superpositions were calcu- Table 1

Crystallographic statistics.

lated using LSQKAB [38], domain motion calculated using DynDom

[39] and active site volumes calculated using the software program Space group C2221

ICM PocketFinder (MolSoft Limited). Figures were prepared using Unit cell lengths a, b, c (Å) 117.22, 220.08, 56.31,

Resolution range (Å) 40.0–2.7 (2.85–2.7)

PyMOL (DeLano Scientiﬁc). Sequence alignments were calculated

Wavelength (Å) 0.972

using ClustalW [40] and visualized using Aline [41].

Number of measurements 77501 (11497)

Number of unique reﬂections 17725 (2631)

Multiplicity 4.4 (4.4)

3. Results and discussion Completeness (%) 88.4 (90.7)

Mean I/I 6.5 (2.8)

Wilson B (Å ) 60.5

3.1. Overall structure and comparisons b Rmerge 0.134 (0.488) c Rwork 0.231

The crystal structure of LmDHCH has been determined at 2.7 A Rfree 0.290

resolution. The asymmetric unit is a homodimer and subunits are RMSD bonds (Å) 0.0054

◦

RMSD angles ( ) 0.871

labeled A and B (Fig. 2). There was no evidence of a monomer in

solution and the presence of a stable dimer is consistent with gel Ramachandran analysis (%)

ﬁltration data and in common with all other DHCH orthologues Favoured 97.8

Allowed 2.0

[19,42]. The ﬁrst and last few residues (1–2 and 1–3, 297–298 in

Outliers 0.2

chains A and B, respectively) are disordered and an additional three

Protein residues 586

residues are disordered in chain B (248–250). Superposition of sub-

Protein atoms total 4398

unit A onto subunit B gives an RMSD of 1.7 A˚ over 286 main chain Overall B (Å ) subunit A, B 38.9, 58.4

␣ Waters 22

C atoms. This high value reﬂects differences in domain positions,

Overall B (Å ) 29.3

which will be discussed, and in a loop adjacent to the active site

Cl− 1

␣

(residues 240–255; RMSD 4.1 A˚ over 13 C ). This loop is disordered 2

Overall B (Å ) 47.2

and absent in other DHCH structures and in LmDHCH, although

Values inparentheses refer tothe highest resolution bin of 2.85–2.7 A.˚

modelled satisfactorily, elevated thermal parameters are noted. b

|| −

Rmerge = h i (h,i) I(h) h i I(h,i).

The LmDHCH subunit consists of 298 residues displaying a high c

Rwork = h k l||Fo| − |Fc||/ |Fo|, where Fo is the observed structure factor and Fc

level of secondary structure; 11 ␣-helices and 11 ␤-strands (Fig. 2).

is the calculated structure factor.

The polypeptide folds into N- and C-terminal domains compris- Rfree is the same as Rwork except calculated using 5% of the data that are not

ing residues 5–147 and 148–298, respectively. A cleft is formed included in any reﬁnement calculations.

182 T.C. Eadsforth et al. / Molecular & Biochemical Parasitology 181 (2012) 178–185

steric effect of changing Ile238 to Thr238 is small, but in LmDHCH

the hydroxyl group of Thr238 might form a hydrogen bond to the

nicotinamide ribose (data not shown). In addition, Lys156 from

the opposing subunit appears primed to interact with the NADP

phosphate in LmDHCH. This is not the case in HsDHCH, as Gly138

occupies the corresponding position. In LmDHCH subunit B a chlo-

ride ion, derived from the crystallization conditions, is assigned to a

large feature in the electron density maps at a position that mimics

the 2 phosphate binding site (data not shown).

The N-terminal domain primarily forms the catalytic centre. It

contains a highly conserved YXXXK motif, starting at Tyr52, found

in the small chain dehydrogenase/reductase family, indicative of

the substrate-binding site [21]. Seven key residues with respect

to substrate binding and catalysis in HsDHCH are Tyr52, Lys56,

Gln100, Leu101, Asp125, Gly273 and Gly276 [20,21]. Lys56 acts

with Gln100 to provide the right environment to activate substrate

and support hydride abstraction by NADP for the dehydrogenase

activity, whilst during the cyclohydrolase reaction it may transfer a

proton during formation of N -formyltetrahydrofolate. Hydropho-

bic residues surrounding the catalytic Lys56 (Leu38, Ile40, Ile53,

Val55, Leu57, Leu98, Val280 and Leu283), create an environment

Fig. 3. An overlay of the LmDHCH subunits based on superposition of N-terminal

␧

implicated in maintaining the -amino group in a non-protonated

domains. The N-terminal domains (chain A – blue, chain B – cyan) agree closely

state, essential for a role in dehydrogenase and cyclohydrolase

however a pivot (shown in green for chain A and forest for chain B) causes the C-

◦ activity. Tyr52 and Asp125 are important for binding and orient-

terminal domains (chain A – red, chain B – magenta) to differ by approximately 14 .

Such a shift appears accentuated particularly around the loop leading to ␤10. ing the substrate for catalysis. The aromatic residue stacks against

the p-amino benzoic acid moiety of the substrate, the acidic residue

binding the pterin head group (Fig. 6b).

states, with the active site slightly more compressed in one than the

In common with the cofactor-binding site there is a high level

other and this may represent a feature of DHCH activity.

of conservation at the catalytic centre with all seven key residues

The human and L. major enzymes share 44% sequence identity

strictly conserved. In addition the catalytic Lys56 of LmDHCH is

(Fig. 4) and the structures are similar. Superposition of LmDHCH

surrounded by aliphatic, hydrophobic side chains (Leu38, Val53,

subunits onto binary- or tertiary complexes of HsDHCH subunits

Leu55, Ile98, Ile280 and Leu283) as seen in HsDHCH. Two residues in

in the same open or closed forms reveals RMSD values of approx-

the vicinity of the catalytic lysine differ between host and pathogen.

imately 1.0 A˚ over 270 C␣ positions (Fig. 5). Superposition of the

The positions occupied by Ile40 and Leu57 in HsDHCH are Ser40 and

+ +

HsDHCH NADP binary and an NADP -inhibitor tertiary complexes

His57 in LmDHCH. These residues are over 6 A˚ distant from the ␧-

gives an RMSD of 0.1 A˚ over 285 C␣ atoms, suggesting no gross

amino group on the other side of the lysine side chain from where

structural change occurs following inhibitor binding.

substrate will bind (data not shown).

3.2. The active site 3.3. Assessing the potential for structure-based drug design

Attempts to obtain structures of LmDHCH ligand complexes Several questions should be addressed in assessing a target

proved fruitless and efforts to crystallize T. brucei and T. for potential in antimicrobial drug discovery [5]. Does the target

cruzi enzymes were also unsuccessful. However the similarities provide an essential function in the pathogen? Is the structure com-

described allow comparisons with HsDHCH complexes (Fig. 5) patible with tight binding of small organic molecules? Is it present

to inform on aspects of molecular recognition in the active site only in the pathogen? If an orthologue occurs in the human host, are

(Fig. 6) and the potential for developing inhibitors speciﬁc for there sufﬁcient differences to ensure that selective inhibition of the

the parasite enzymes over that of the host. Of particular value is pathogen target can be achieved? We now consider these issues. On

the ternary complex of HsDHCH with cofactor and the inhibitor the basis of biological and metabolic considerations DHCH appears

5 10

5,6,7,8-tetrahydro N ,N -caronylfolic acid (LY354899, Fig. 1b, PDB a promising drug target in L. major. Genetic approaches suggest

code 1DIB) developed at Lilly Research Laboratories [43]. LY354899 essentiality in L. major [24] and the absence of FTL in T. brucei [23]

mimics the substrate and inhibits both HsDHCH and LmDHCH makes DHCH an interesting target in that parasite also as there

with Ki values of 18 nM and 105 nM, respectively [20,43]. It also is no other obvious means by which that organism can produce

inhibits other enzymes involved in folate metabolism, DHFR, thymi N -formyltetrahydrofolate.

dylate synthase and glycinamide ribonucleotide formyl transferase The concept of a druggable protein concerns the properties

[43]. of a site able to bind molecules with the right physicochemi-

In HsDHCH, 13 residues, mainly using side chains are key in cal attributes to make them bioavailable, with high afﬁnity for

binding NADP or in creating the cofactor-binding site (Thr148, the target and low toxicity to humans [44]. The optimal target

Arg173, Ser174, Ile176, His196, Ser197, Ala215, Thr216, Gln218, would be a small, well-deﬁned and ordered cavity in the protein,

Met221, Cys236, Gly237 and Ile238 [20]). Nine of these residues with pronounced hydrophobic components [44]. The active sites of

are strictly conserved in LmDHCH and we note the arginine, serine, enzymes such as PTR1 and DHFR would be described as ‘druggable’.

histidine combination (Arg171, His194, Ser195) that binds the 2 The volume of their active sites [45] is estimated as 350 A˚ and

+ 3

phosphate of NADP is spatially conserved between the two species 380 A˚ , respectively from PDB codes 2X9G and 3CL9. The substrate

(Fig. 6a). The four differences involve Thr216 to Met216, Met221 to binding cavity of LmDHCH has a volume of approximately 430 A˚

Tyr221, Cys236 to Val236 and Ile238 to Thr238. The ﬁrst two pro- which is a modest increase compared to DHFR and of suitable size

vide a hydrophobic surface to bind the adenine moiety. At position to bind drug-like molecules [45]. Indeed drug-like inhibitors of

236 it is the main chain that is involved in cofactor binding. The HsDHCH are known supporting this conclusion. These inhibitors

T.C. Eadsforth et al. / Molecular & Biochemical Parasitology 181 (2012) 178–185 183

Fig. 4. Structure-based sequence alignment of L. major and H. sapiens cytosolic DHCH enzymes. Helices and strands are red and blue, respectively. Residues that are strictly

or highly conserved in H. sapiens, L. major, T. brucei, T. cruzi and L. donovani enzymes are highlighted in black and grey, respectively. Residues that directly bind ligands (as

shown in the HsDHCH structures) are marked with a yellow star and are highly conserved across all species. Residues that are within 5 A˚ of the bound ligand in the HsDHCH

structure are enclosed in green boxes.

␣ +

Fig. 5. Stereo view C trace showing the superposition of a subunit of LmDHCH (green) with an equivalent from HsDHCH (marine) ternary complex with NADP (yellow)

and LY354899 (black) (PDB code 1DIB). In both cases a loop that remains disordered is marked with two asterisks.

were investigated as anticancer agents since the folate pathway LmDHCH and an EC50 of approximately 1 ␮M against the parasite

produces essential co-factors for cell division [43]. The observation itself [24] is supportive of a mode of action being DHCH inhibition

that an HsDHCH inhibitor, LY354899, displays nM inhibition against with the caveat that there are other potential targets present.

184 T.C. Eadsforth et al. / Molecular & Biochemical Parasitology 181 (2012) 178–185

presents a signiﬁcant challenge due to the high level of speciﬁcity

involved and the relatively large areas involved in oligomeriza-

tion [46]. Residues on ␣5, ␣7, ␣8 and ␤6 form the dimer interface

of LmDHCH. In particular the ␤6 strands associate in anti-parallel

fashion to create a 12-stranded ␤-sheet (Fig. 2). The catalytic site is

remote from the dimer interface, however, the adenine-end of the

cofactor binds nearby indeed Lys136 from one subunit is placed to

interact with the 2 -phosphate of the cofactor bound by the partner

subunit. However, comparisons indicate a high level of conser-

vation between LmDHCH and HsDHCH in ␣5, ␣7 and ␤6 (Fig. 4)

extending to similarities in hydrogen bonding patterns (data not shown).

4. Conclusion

The crystal structure of apo-LmDHCH has been determined to

2.7 A˚ resolution allowing for detailed comparisons with HsDHCH.

Our data provide details of the active site and a template for the

rational development of selective inhibitors against the enzyme

from trypanosomatid parasites. However, two major factors may

compromise the search for selective inhibitors. Firstly, the inabil-

ity to co-crystallize a binary or tertiary complex of any protozoan

DHCH has limited efforts to exploit structure-based methods for

inhibitor development. Most HsDHCH ligand complexes in the PDB

display poorly ordered ligands [20] and DHCH itself may present

signiﬁcant challenges for structure-based approaches to inhibitor

development. Secondly, and perhaps most important is the close

structural relationship between the host and parasite enzymes not

only at the catalytic centre but also at the dimer interface. This prob-

ably represents the biggest challenge to early stage drug discovery

seeking to develop potent and parasite speciﬁc inhibitors of DHCH.

Acknowledgments

This work was funded by The Wellcome Trust grants 082596

and 083481. We thank Diamond Light Source for beam time and

+ technical support.

Fig. 6. Comparison of the (a) NADP binding and (b) catalytic sites of LmDHCH

(green) with HsDHCH (marine) containing the competitive inhibitor NADP (yellow)

and LY354899 (black), respectively. Residues shown as sticks are within hydrogen References

bonding distance of the ligands and with labels colored according to species. The

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