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Structural flexibility in Trypanosoma brucei enolase revealed by X-ray crystallography and molecular dynamics Marcos V de A S Navarro1,*,‡, Sandra M Gomes Dias1,*,§, Luciane V Mello2,3,*, Maria T da Silva Giotto1,†, Sabine Gavalda4,–, Casimir Blonski4, Richard C Garratt1 and Daniel J Rigden2 ´ Instituto de Fısica de Sao Carlos, Universidade de Sao Paulo, Sao Carlos SP, Brazil ˜ ˜ ˜ School of Biological Sciences, University of Liverpool, UK Northwest Institute for Bio-Health Informatics, University of Liverpool, UK ´ Groupe de Chimie Organique Biologique, Universite Paul Sabatier, Toulouse, France Keywords crystal structure; drug design; enolase; molecular dynamics; structural flexibility Correspondence D J Rigden, School of Biological Sciences, Crown Street, University of Liverpool, Liverpool L69 7ZB, UK Fax: +44 151 7954406 Tel: +44 151 7954467 E-mail: drigden@liv.ac.uk Website: http://www.liv.ac.uk/biolsci/ *These authors contributed equally to this work †Deceased Present address ´ ´ ‡Laboratorio Nacional de Luz Sıncrotron, Campinas, SP, Brazil §Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA –Department of Molecular Mechanisms of Mycobacterial Infections, Institut de Pharmacologie et de Biologie Structurale, CNRS, UPS (UMR5089), Toulouse, France Enolase is a validated drug target in Trypanosoma brucei To better characterize its properties and guide drug design efforts, we have determined six new crystal structures of the enzyme, in various ligation states and conformations, and have carried out complementary molecular dynamics simulations The results show a striking structural diversity of loops near the catalytic site, for which variation can be interpreted as distinct modes of conformational variability that are explored during the molecular dynamics simulations Our results show that sulfate may, unexpectedly, induce full closure of catalytic site loops whereas, conversely, binding of inhibitor phosphonoacetohydroxamate may leave open a tunnel from the catalytic site to protein surface offering possibilities for drug development We also present the first complex of enolase with a novel inhibitor 2-fluoro-2-phosphonoacetohydroxamate The molecular dynamics results further encourage efforts to design irreversible species-specific inhibitors: they reveal that a parasite enzyme-specific lysine may approach the catalytic site more closely than crystal structures suggest and also cast light on the issue of accessibility of parasite enzyme-specific cysteines to chemically modifying reagents One of the new sulfate structures contains a novel metal-binding site IV within the catalytic site cleft (Received June 2007, revised 25 July 2007, accepted August 2007) doi:10.1111/j.1742-4658.2007.06027.x Enolase (2-phospho-d-glycerate hydrolase, EC 4.2.1.11) catalyses the reversible dehydration of d-2-phosphoglycerate to phosphoenolpyruvate (PEP) and partici- pates in both glycolysis and gluconeogenesis In common with most glycolytic enzymes, enolases from a wide variety of organisms, including Archaea, Bacteria Abbreviations EV, eigenvector; FPAH, 2-fluoro-2-phosphonoacetohydroxamate; PAH, phosphonoacetohydroxamate; PDB, protein databank; PEP, phosphoenolpyruvate FEBS Journal 274 (2007) 5077–5089 ª 2007 The Authors Journal compilation ª 2007 FEBS 5077 Structural flexibility in T brucei enolase M V A S Navarro et al and Eukarya, are highly conserved [1] The catalytic site is particularly well conserved, leading to broadly similar kinetic parameters for enzymes of different origins [2,3] The quaternary structure of enolase is typically a homodimer, although some bacteria apparently contain octameric enzymes [4,5] Each subunit of enolase contains an eightfold b ⁄ a barrel domain preceded by an N-terminal a + b domain [6] The catalytic site is contained completely within a single subunit and lies at the interface of the two domains: monomeric enolase is catalytically active [7] Catalysis results from acid–base chemistry involving a Lys-Glu dyad [8,9] Also essential is the binding of two divalent metal ions to distinct sites: the first ‘conformational’ site being required for substrate binding and the second ‘catalytic’ site, occupied after substrate has bound, stabilizing the reaction intermediate [6] This ordered binding is accompanied by dramatic rearrangements of three protein loops lying near the catalytic site Note that, although the conventional loop nomenclature is maintained here, regular secondary structure is sometimes present in these regions Briefly, when the catalytic site is occupied by sulfate, phosphate or phosphoglycolate, all three loops typically adopt an open conformation, as seen, for example, in our previous Trypanosoma brucei enolase structure [10] When occupied by substrate, or the phosphonoacetohydroxamate (PAH) inhibitor, and two metal ions, the loops are generally all in a closed conformation, as in some yeast structures [11] Intermediate semiclosed conformations have been observed when one metal ion is absent or in some complexes with PEP [12,13] As well as its key roles in glycolysis and gluconeogenesis, enolase, in common with other glycolytic enzymes [14], has a remarkable number of ‘moonlighting’ roles in diverse organisms that are unrelated to its catalytic activity [15] These include roles in the RNA degradosome in Escherichia coli [16], as a structure lens protein (s-crystallin) in the eye [17], as a transcription factor in both animals [18] and plants [19] and, on cell surfaces, as a receptor for plasminogen [15] In this last role, the expression of enolase on the surface of streptococci is particularly interesting, where its interaction with host plasminogen is presumed to facilitate entry of the parasite into host tissues [20] Very recently, the enolase of the trypanosomatid parasite Leishmania mexicana has also been detected on the cell surface [21] A role for enolase as plasminogen receptor in this organism is highly plausible because interaction between parasite and plasminogen has been demonstrated [22] Our interest in T brucei enolase [2,10] stems from the promise of the glycolytic pathway as a target for 5078 drugs against parasitic protozoa [23] With few exceptions, homologues of the enzymes involved are present in the human host, and a premium is placed on seeking and exploiting structural differences between parasite and host proteins Irreversible inhibition is particularly desirable because it would be impervious to high substrate levels that could displace competitive inhibitors [23] Using parasite enzyme-specific residues (e.g lysines in both cases), selective inhibitors against aldolase [24] and phosphofructokinase [25] have been developed Despite bearing chemically reactive groups, by combining high affinity and low reactivity, optimized inhibitors of this kind should have minimal effects on other proteins in vivo Indeed, a prodrug version of an aldolase inhibitor kills parasite cells without detectable cytotoxicity against human MRC-5 cells [26] Like other glycolytic enzymes, T brucei enolase has been validated as a drug target: RNA interference of enolase in the bloodstream form of the parasite leads to an effect on its growth within 24 h and death commences at approximately 48 h [27] Encouragingly, the same study also demonstrated that a reduction in enolase activity to approximately 15–20% of its original level was sufficient for cell death to occur This suggests that incomplete inhibition of this enzyme in vivo might prove sufficient for effective treatment The presence of homologous enolase isoenzymes in the human host raises the complication of selectivity In this respect, enolase is not the best target because the parasite and host enzymes share 58% sequence identity Nevertheless, modelling showed that there are three particularly interesting T brucei enzyme residues, two cysteines (numbered 147 and 241) and lysine 155, near to the catalytic site, which are not conserved in the human enzymes [2] (Fig S3) The chemical characteristics of the side chains of these residues offer the potential for species-selective permanent target inactivation by appropriately designed covalent inhibitors The T brucei crystal structure suggested that the cysteines were almost entirely solvent inaccessible, yet, most surprisingly, at least Cys147 could be chemically modified by iodoacetamide with consequent enzyme inhibition [10] In that crystal structure, Lys155 is pointed away from the catalytic site, being unfavourably positioned to make additional interactions with a catalytic sitebound inhibitor In the present study, we present six new enolase structures that enhance our understanding of the structural and dynamic properties of the T brucei enolase catalytic site, which is essential for further drug design The new structures demonstrate that the enzyme can adopt three distinct catalytic site structures in the FEBS Journal 274 (2007) 5077–5089 ª 2007 The Authors Journal compilation ª 2007 FEBS M V A S Navarro et al sulfate-bound form, including one containing a novel metal binding site Furthermore, they show structural heterogeneity in their inhibitor-bound forms, highlighting the potential to extend future inhibitors out of the ligand-binding pocket We also present extensive molecular dynamics simulations aiming to address how the apparently buried cysteine residues achieve solvent accessibility and show that Lys155 may indeed offer a useful alternative possibility for covalent inhibition Results and Discussion Overview of the new structures Characteristics and statistics of data collection and refinement for the six new structures are presented in Table The crystal form is the same in each case, namely the C2221 form previously reported [10], although the precipitant used was PEG 1000 rather than the PEG monomethylether 550 In the subsequent analyses, we compare these structures with the previously published sulfate-bound structure, refined to ˚ 2.35 A, and containing Zn2+ ions bound to sites I and III [10], which we refer to here as sulfate_1 The new structures, all obtained by co-crystallization, are all of significantly better resolution than sulfate_1, in particular a complex with PAH inhibitor that diffracted well ˚ to 1.65 A In common with previous structures, from T brucei and other organisms, a single Arg residue, numbered 400 in T brucei, lies in the disallowed region of the Ramachandran plot [10] Among the three important catalytic site loops previously described (and discussed further below), there is only one that makes a crystal contact This involves Glu272 of loop 3, its last residue and the most distant from the catalytic site Thus, we can be confident that the conformations observed represent readily achieved structures of the native enzyme, rather than crystal packing artefacts In our initial sulfate_1 structure, density did not allow for chain tracing of two stretches, from Thr41-Gly42 and Thr260-Pro266, regions that are frequently poorly ordered in other enolase structures With the exception of sulfate_2, all the structures presented here could be unambiguously fully traced In sulfate_2, density did not allow for the tracing of the polypeptide chain between Asp251 and Gln273 inclusive As with sulfate_1, one or two artefactual residues preceding the N-terminal Met of the natural sequence could be traced in each new structure, and these result from thrombin cleavage of the His-tag used in purification (see Experimental procedures) In the three inhibitorbound structures, artefactual Zn2+ ions bound, with partial occupancy (0.5–0.7), at the crystal packing Structural flexibility in T brucei enolase interface to residues ‘His0’ and Glu27, and to His283 from a crystal symmetry-related chain The new structures are diverse in the contents of their catalytic sites, both in terms of substrate ⁄ inhibitor and in terms of bound metal (Table 1) The two new sulfate complexes and the previous sulfate-bound structure were all achieved at highly similar crystallization conditions (Table 1) As such, there is no clear explanation why they should differ in conformation (see below) and we view their structural diversity as being the result of ‘freezing out’ of similarly accessible catalytic site conformations Substrate (PEP) and inhibitor (phosphonoacetohydroxamate, PAH) [28] bind with their phospho and phosphono groups, respectively, occupying the same position as that occupied by sulfate in the earlier sulfate_1 structure [10] Schematic diagrams of the interactions of inhibitors and metal ions with enolase are given in Fig S4 The binding mode of PEP seen is essentially the same fully closed conformation as that seen for yeast enolase [protein databank (PDB) code 1one][29] One PAH structure is also fully closed, as in an earlier yeast complex (PDB code 1ebg) [11], whereas the second, as discussed below, represents a novel conformation for bound PAH Electron density maps for each complex are given in Fig S5 The novel compound 2-fluoro-2-phosphonoacetohydroxamate (FPAH), a derivative with a pKa value more resembling that of the phosphate of substrate PEP, was also synthesized and its complex determined It is a competitive inhibitor of enolase which, despite its lower pKa value compared to PAH, binds more poorly with a Ki at pH 7.2 of 1.4 lm compared to approximately 15 nm for PAH (see supplementary Doc S1 and Figs S1 and S2) [30] It binds in the same way as PEP and PAH with an electron density suggesting that both isomers of the R,S racemic mixture bind equally well (Fig S5) Despite the uniformity of ligand binding, protein structure varies considerably at the active site in the new set of structures Rather than try to explain their differences in the typical qualitative way (i.e open, closed, semiopen, loose, etc.), we attempt a more quantitative description As shown in Figs and 2, the principal conformational differences between the structures lie in three catalytic site loops, 1–3, corresponding to those highlighted in many other studies However, unlike the results obtained in a similar analysis for Saccharomyces cerevisiae crystal structures (data not shown), a fourth peak for the region from residues 215–220 is present This loop is a neighbour of loop and moves in a coordinated way in T brucei but not in yeast structures Because loop is distant from the catalytic site, it is not discussed further FEBS Journal 274 (2007) 5077–5089 ª 2007 The Authors Journal compilation ª 2007 FEBS 5079 5080 a Sulfate_2 Sulfate_3 PEP PAH_1 PAH_2 FPAH 1, 0.9, 0.8 111.26 109.97 26.0 1.90 2.02 99.4 (100.0) 15.2 (2.2) 4.9 (4.8) 5.7 (49.7) 111.17 108.98 26.1 1.90 2.02 C2221 73.62 20.1 16.1 21.6 29.9 0.015 1.505 89.4 10.3 0.3 308 33900 (4812) 21.4 (27.1) 25.0 (30.0) 28.0 28.1 33.1 39.0 26.9 0.019 1.672 89.8 9.0 0.9 0.3 See [10]; PDB code 1oep bValues within parentheses are for the highest resolution bin cCalculated with 382 33961 (4764) 16.4 (25.3) 20.6 (33.7) 21.2 99.0 (96.7) 17.0 (3.9) 3.2 (3.2) 5.8 (54.2) C2221 74.77 1, 1.0, 1.0 PROCHECK 91.1 8.4 0.3 0.3 1.680 37.3 30.2 33.9 36.1 0.018 [50] 179 28944 (4161) 17.3 (24.7) 22.4 (37.1) 37.2 99.8 (100.0) 12.9 (1.9) 2.9 (2.8) 6.4 (54.5) 110.72 109.3 25.0 2.00 2.11 C2221 73.99 1, 1.0, 1.0 90.0 9.5 0.3 0.3 1.668 28.5 22.7 27.9 38.5 0.017 421 47566 (6562) 16.5 (32.4) 20.3 (39.7) 29.6 95.5 (91.4) 26.4 (3.9) 8.3 (8.3) 5.5 (40.9) 109.28 107.95 28.0 1.65 1.74 C2221 73.86 1, 1.0, 1.0 90.0 9.8 0.3 1.487 23.9 28.9 29.2 29.9 0.016 257 33851 (4700) 16.5 (20.7) 20.5 (30.1) 24.3 98.8 (96.1) 16.1 (3.1) 4.6 (4.6) 7.0 (40.3) 110.76 109.22 17.8 1.90 2.02 C2221 74.95 1, 1.0, 1.0 90.5 8.9 0.3 0.3 1.611 27.2 21.0 25.5 35.5 0.018 356 40063 (5784) 16.2 (22.2) 20.6 (31.3) 27.9 99.9 (100) 23.3 (4.3) 7.6 (7.7) 5.6 (43.5) 110.64 109.01 21.0 1.80 1.99 C2221 74.81 1, 1.0, 1.0 0.1 M Mes pH 6.5, 0.1 M Mes pH 6.5, 0.1 M Mes pH 6.5, 0.1 M Mes pH 6.5, 0.1 M Mes pH 6.5, 0.1 M Mes pH 5.0, 0.1 M Mes pH 5.0, 10 mM ZnSO4, 10 mM ZnSO4, 10 mM ZnSO4, 10 mM ZnSO4, 10 mM ZnSO4, 10 mM ZnCl2, 10 mM ZnCl2, 25% PEGMME550 10% PEG1000 10% PEG1000 10% PEG1000 10% PEG1000 10% PEG1000 10% PEG1000 Metal content Metal ion sites occupied at catalytic site 1, Occupancy of catalytic site metals 1.0, 0.7 Data collection Space group C2221 Unit cell dimensions 74.02 ˚ a (A) ˚ b (A) 110.54 ˚ c (A) 109.1 ˚ Low resolution diffraction limit (A) 38.8 ˚ High resolution diffraction limit (A) 2.3 Lower resolution limit of highest 2.38 resolution bin Completeness (%) 97.2 (93.4)b I ⁄ r(I ) 14.2 (1.9) Multiplicity 4.7 (4.5) Rmerge (%) 6.52 (46.8) Refinement Number of water molecules 240 Number of reflections 17334 (1942) R (%) 21.0 (28.5) Rfree (%) 25.1 (33.7) ˚ Mean temperature factor B (A2) 43.7 All atoms Protein 42.7 Ligand 71.3 Zinc 56.2 Solvent 42.7 rmsd from ideal values 0.007 ˚ Bond lengths (A) Bond angles (°) 1.4 Ramachandran (%)c Most favoured regions 86.4 Additional allowed regions 13.4 Generously allowed regions Disallowed regions 0.3 Crystallization conditions Sulfate_1a Name of structure Table Crystallisation conditions, metal content, data collection statistics and refinement statistics for structures of T brucei enolase Structural flexibility in T brucei enolase M V A S Navarro et al FEBS Journal 274 (2007) 5077–5089 ª 2007 The Authors Journal compilation ª 2007 FEBS M V A S Navarro et al Structural flexibility in T brucei enolase Borrowing a technique more commonly associated with molecular dynamics studies, we analysed the conformational differences in the new set of structures using essential dynamics [31] This also allowed us to visualize to what extent the resulting modes of conformational variability were explored during molecular dynamics simulations (see later) The positions of the six new structures projected onto eigenvectors (EVs) and are shown in Fig 3A Visual inspection of the maximum and minimum projections of EV1 shows A Fig Multi-rms plot of the enolase structures in Table produced with LSQMAN[56] The multi-rms value is defined as the rms value of the distances between all unique pairs of Ca atoms for a given residue Loops 1–4 (see text) are labelled Note that the value for a section of loop is artificially low in a stretch, coloured grey, for which density did not allow tracing of the chain in either of the open forms, sulfate_1 or sulfate_2 B Fig Comparison of sulfate_1, sulfate_2, PEP and PAH_1 structures, coloured, respectively, in shades of green, blue, magenta and orange The FPAH ligand position closely resembles that of PAH_1 A complete cartoon representation of sulfate_2 is shown Backbone structure is shown for the other three structures only for loops 1–4, which are labelled Note the gaps in loops and of the sulfate_1 structure and the loop gap in the sulfate_2 structure Side chains of Lys155 and His156 are shown as sticks, as are the structures’ respective ligands showing the overlay of bound sulfate with phospho and phosphono groups Zinc atoms are shown as spheres occupying the labelled sites I–IV Black dashes mark the hydrogen bonding interactions of His156 with PEP or with metals in sites III or IV in the sulfate_1 and sulfate_2 structures, respectively Fig (A) Projections of the six new crystal structures and molecular dynamics trajectories on to EVs and resulting from the essential dynamics analysis Blue circles are used for sulfate structures [open for sulfate_1* (see Experimental procedures), filled for sulfate_3], green triangles for PAH complexes (open for PAH_1, filled for PAH_2), a magenta square for the PEP structure and an orange diamond for the FPAH complex Black dots mark the PEP + Mg trajectory and red dots the single Mg trajectory starting from the same PEP complex protein conformation Dots are shown at ps intervals along the trajectory (B) Path of the single Mg trajectory, indicated at 20 ps intervals, showing a structural switch at approximately ns from a closed (low values for EVs and 2) to an open structure (high EVs) The trajectory start is marked with a circle whereas the end is indicated by a square FEBS Journal 274 (2007) 5077–5089 ª 2007 The Authors Journal compilation ª 2007 FEBS 5081 Structural flexibility in T brucei enolase M V A S Navarro et al that it captures the coordinated closure of loops 1–4 over the catalytic site The structure sulfate_2 (high value of EV1 projection) has the loops in a fully open conformation whereas, in the other structures, they close over the active site EV2 splits this group of five to two sets, with positive projection values signifying structures in which His156 remains outside the catalytic site, whereas negative values mean that His156 enters the site so that the enzyme achieves a catalytically competent conformation A comparison of sulfate_1, sulfate_2, PEP and PAH_1 structures is shown in Fig Unexpected variability in inhibitor complex structures Comparison of the substrate and inhibitor complexes shows that the His156-out and His156-in structures are equally represented, the former by PAH_1 and FPAH and the latter by PEP and PAH_2 This appears to be the first time that an enolase-PAH complex has crystallised in a nonfully closed conformation The PAH_1 and PAH_2 structures were crystallised at different pH values We therefore considered whether varying charge on the phosphono groups of the substrate, with which His156 interacts on entering the catalytic site, could be responsible However, the negative charge on the PAH phosphono group would be greater at pH 6.5, at which the His-out structure was obtained, compared to crystallization at pH 5.0 of the His-in PAH structure Furthermore, a greater negative charge would be expected on the phosphono group of FPAH than of PAH due to the lower pKa value of the fluoro derivative, yet the FPAH structure was also His-out The pKa of His165 is not known experimentally, although an observed value of 5.9 has been ascribed to it in the yeast enzyme [32] If this is true, then its ionization state will also differ at pH values of 5.0 and 6.5 A greater attraction for bound PAH of the more positively charged His165 is consistent with the His-in structure observed at pH 5.0 (PAH_2) and the His-out PAH_1 structure observed at pH 6.5 However, consideration of the ionization state of His165 does not explain why the FPAH structure at pH 5.0 should be His-out There is no obvious explanation for this structural difference, leading to the conclusion that the His-in and His-out conformations may be similarly energetically favourable and perhaps only chance leads to the freezing of one or the other in a given crystal The previously unsuspected existence of His-out inhibitor-bound conformations has important implications for further ligand design In the fully closed, His-in conformation, the ligand is fully enclosed in a substrate-sized cavity with little potential for the design of a larger inhibitor of better affinity or selectivity By contrast, as shown in Fig 4, the outward pointing His156 conformation leaves a tunnel open leading from the protein surface down to the bound ligand This allows ‘growing room’ for a catalytic site-bound inhibitor, enabling access to a larger number of target residues and hence increasing the chance of achieving selectivity for the parasite enzyme over the human counterpart EV3 from the essential dynamics analysis splits the two His-out structures, PAH_1 and FPAH (data not shown) The difference between these can be described as a localized twist of loop containing His156 In this case, an explanation is forthcoming The fluorine atom of one of the isomers of the racemic FPAH makes a ˚ close nonbonded contact (3.0 A) with Gln164, inducing a small displacement of the entire loop Although evidently non-natural, the existence of this loop conformation emphasizes just how conformationally plastic the catalytic sites loops are Fig A tunnel leading to the catalytic site is present in PAH_1 (left) but not in PAH_2 (right) A semitransparent surface is shown, uniformly coloured with the exception of the surface contributions from bound PAH (coloured magenta), Lys155 (dark grey) and His156 (light grey) These residues and the ligand are shown as sticks 5082 FEBS Journal 274 (2007) 5077–5089 ª 2007 The Authors Journal compilation ª 2007 FEBS M V A S Navarro et al Sulfate complexes and a novel metal binding site IV The protein conformation most similar to the sulfate_1 structure [10] is sulfate_2 (Table 1) to which 405 Ca ˚ atoms could be fit with an rmsd of 0.4 A and a maxi˚ at position 276, a surface mum displacement of 2.0 A residue distant from the catalytic site Remarkably, however, the sulfate_2 structure binds its two zinc ions differently to sulfate_1 Both have fully occupied I sites, the so-called conformational site [6], but although sulfate_1 showed the position of the inhibitory metal site III, the sulfate_2 structure reveals a further novel metal-binding site IV at the enolase catalytic site As with site III, the metal in site IV is ligated by His156 but, whereas metal in site III is also bound by Gln164, Glu165 and Glu208, His156 is the only protein ligand of the metal in site IV (Fig 5) Zinc ions bound by single protein ligands are comparatively rare in the Metalloprotein Database [33] but there are several other examples The Zn2+ ion in site IV is fully occupied and there appears to be no doubt regarding the identity of this feature in the electron density map: there are no other components of the crystallization solution that could be responsible Additionally, anomalous scattering maps reveal clear, although somewhat noisy, density for both metal sites (Fig S6A) The density is similar to that observed for the sulfur atoms of cysteine and methionine residues, which have similar scattering power to zinc at the ˚ wavelength used (1.54 A) (Fig S6B) The zinc ion in site IV is further ligated by five solvent molecules with Structural flexibility in T brucei enolase ˚ interatomic separations of 1.75–2.20 A (Fig 5) The B-factor of the metal ion in site IV of 41.3 is close to that of the ligating nitrogen atom of His156 (39.2) The His156 conformations in the sulfate_1 and sulfate_2 structures differ by only 21° at the v1 rotation, but by a 180° flip of the imidazole ring because the Ne2 atom is involved in both cases (Fig 2) The occupation of site IV is unexpected because site III, with additional, negatively charged ligands, would be expected to have a higher affinity for the metal We can be confident that site III, and not site IV, corresponds to the inhibitory metal site characterized kinetically because the H156A mutant of the S cerevisiae enzyme retains an inhibitory site with one third of the native enzyme’s affinity [34] Such a mutant would simply lack a site IV because the His side chain contributes its only protein coordination Nevertheless, it remains possible that binding to site IV contributes to the inhibition of enolase at elevated metal concentrations The sulfate_3 structure closely resembles the PEP and PAH_2 structures, with loops 1–4 fully closed Its Ca atoms can be fit to those of the PEP complex to ˚ produce an rmsd of 0.23 A Additionally, its two zinc ions in sites I and II superimpose on those of the PEP complex, as does the sulfate on the phospho group of PEP (Fig 2) It is unusual for occupation of the catalytic site by a small sulfate or phosphate to be sufficient to support full closure This situation was seen in one subunit of the E coli structure, but the influence of crystal packing was suspected [35] More recently, one subunit of the asymmetric human neuron enolase Fig Coordination of zinc ions occupying metal site I and novel site IV (labelled) in the sulfate_2 structure Side chains and sulfate are shown as sticks, water molecules and Zn2+ ions as spheres, coloured cyan and grey, respectively Electron density from a metal-deleted Fo–Fc omit map contoured at 10 r is shown in magenta Density in a 2Fo–Fc map (shades of blue) is contoured at r in the vicinity of site IV, and at r around the sulfate and metal site I FEBS Journal 274 (2007) 5077–5089 ª 2007 The Authors Journal compilation ª 2007 FEBS 5083 Structural flexibility in T brucei enolase M V A S Navarro et al was demonstrated to adopt the closed conformation while containing only phosphate or sulfate [36] Molecular dynamics simulations The static description of crystal structures is incomplete for many proteins but particularly so in the case of enolase Not only multiple structures from several species demonstrate large conformational changes at the catalytic site but also, in the case of the T brucei enzyme, crystal structures show Cys147 and Cys241 to be entirely buried in the second layer of protein residues below the base of the catalytic site [10] whereas experimental data show that at least Cys147 can be modified by iodoacetamide with resulting protein inactivation [10] To explore this and other issues, we carried out 10 ns duration molecular dynamics simulations on two fully solvated dimeric enolase structures, the PEP complex and a PEP structure derivative with active site contents removed to leave a single divalent metal ion, the ‘conformational’ ion in site I In each case, Zn2+ was replaced by the more physiologically relevant Mg2+ Initial modeling also highlighted Lys155 as a residue near the catalytic site, present only in enolases from T brucei and Leishmania major, Euglena gracilis and Treponema pallidum, which could be a target for irreversible modification by a suitable inhibitor Such an inhibitor would likely occupy the catalytic site; thus, we assessed how closely the Lys155 side chain approached ligands in that site In the previous sulfate_1 structure [10], the Nf atom of Lys155 was far, ˚ around 12 A, from the catalytic site-bound sulfate In the new PEP, PAH and FPAH structures, the Nf atom is separated from the phospho(no) group by ˚ approximately 7.5 A Remarkably, although the position of its neighbour, His156 varies dramatically among these structures (Fig 2), the position of the Nf atom is constant (Figs and 4), making a hydrogen bond with the backbone carbonyl of Ala39 Encouragingly, Lys155 lies at the mouth of the tunnel leading from the protein surface to the bound ligand in the His-out structures (Fig 4) As such, it would lie near to an expanded inhibitor occupying that tunnel The molecular dynamics results show that it approaches the catalytic site even more closely The separations of its Nf atom and the oxygen atoms of the PEP phospho group were monitored throughout the PEP trajec˚ tory and reached values as low as 6.5 A Clearly, the prospects for the exploitation of this parasite-specific residue are much better than first supposed To address the issue of Cys solvent accessibility, the solvent-accessible surface area of Cys147 and Cys241 5084 was monitored in both subunits throughout the molecular dynamics simulations It is already known that the presence of PEP or PAH does not affect the chemical modification of the cysteines, suggesting that iodoacetamide and other reagents not access the cysteines via the catalytic site Examination of the structures shows that the modifiable cysteine(s), and the adjacent conserved buried water molecules [10], lie quite close to the opposite surface of the protein Only the side chain of the penultimate residue, Trp428 separates them from bulk solvent The Trp side chain remains firmly in place throughout the course of our simulations, and neither buried water molecule exchanges with bulk solvent, but nevertheless transient displacement of the Trp side chain remains the most likely means of access to the cysteines by modifying reagents Given that modification is a slow process [10], it may be that the timescale of our simulations is simply too short for it to be observed Also, the actual presence of the rather hydrophobic reagents, rather than pure bulk solvent, may be necessary to induce the necessary structural alterations that allow access, as seen in other systems [37] The trajectories were mapped onto the EVs obtained by analysis of the crystal structures (Fig 3) to determine to what extent these modes of structural variability are explored The PEP complex simulation remains in the vicinity of the starting point There is little tendency toward the His156-out conformation (high values of EV2) and no evidence at all of coordinated loop opening (high values of EV1) Because we have so far only observed the His156-out conformation with inhibitors, and not with substrate, it may be that the His156-out is favoured only for the former ligands for reasons that remain unclear (see above) The results for the single Mg trajectory (obtained by removing PEP and the site II metal from the PEP complex structure) are intriguingly different After exploring the neighbourhood of the starting conformation for approximately ns, there is a transition (Fig 3B) and the protein explores an area of much higher values for both EV1 (centred around 0.6) and EV2 (centred around 0.2) This implies that, in the absence of PEP and site II metal, there is a shift towards a more open structure along both the coordinated loop dimension (EV1) and the His156-out dimension (EV2) The trajectory reaches the EV1 value of the open sulfate_1* structure (maximum 1.73) and exceeds the EV2 values of the His156-out inhibitor complexes (maximum 0.45) These results are fully consistent with the prevailing notion of an ordered mechanism for enolase [38] With only metal site I occupied, the enzyme adopts an open conformation (high values of EVs FEBS Journal 274 (2007) 5077–5089 ª 2007 The Authors Journal compilation ª 2007 FEBS M V A S Navarro et al and 2) but with substrate present, and site II occupied is stable in a closed conformation (lower values of EVs and 2) in which catalytic residues align precisely for reaction to occur The mapping of the trajectories onto EV3 (not shown), confirms that the twisted His156-out loop induced by the fluorine atoms in the FPAH is a conformation not explored naturally and therefore an unfavourable one This suggests that the lower pKa value of that inhibitor, compared to parent PAH, comes at an energetic cost, consistent with the higher experimental Ki of FPAH (Doc S1, Figs S1–S2) Conclusions Although previous work has painted a picture of flexible loops near the enolase catalytic site, the diversity of structures observed for the T brucei enzyme, in a single crystal form and at broadly similar pH values, is impressive Particularly notable are the findings that sulfate occupation of the catalytic site alone can lead to full closure of all loops whereas, for reasons unknown, other sulfate-bound structures are open and exhibit unexpected diversity of metal binding [10] Similarly, occupation with the inhibitor PAH (or our novel fluorinated PAH derivative) need not lead to full closure of catalytic site loops, leaving open a tunnel allowing for the design of enlarged inhibitors occupying more than the immediate vicinity of the small, enclosed catalytic site Equally encouraging for future drug design is the discovery that a potentially modifiable Lys155 side chain lies near to this tunnel, not far from the catalytic site as previously supposed [10] Our molecular dynamics results fail to demonstrate the appearance of a channel exposing the modifiable cysteine residue(s) to solvent, consistent with modification being a slow process In summary, our results emphasize the importance of a full understanding of the dynamic properties of a drug target for the effective design of tight-binding and specific ligands Experimental procedures Chemical synthesis Phosphonoacetohydroxamate, lithium salt (PAH) and the corresponding fluoro analog (FPAH) were obtained from the diethylphosphonoacetic acid and the diethyl-2-fluorophosphonoacetic acid, respectively, using an improved reaction sequence distinct from that previously described [28] Diethyl-2-fluoro-phosphonoacetic was obtained by saponification of the triethyl-2-fluoro-phosphonoacetate Diethylphosphonoacetic acid and the diethyl-2-fluoro-phosphonoacetic acid were linked to O-benzylhydroylamine in Structural flexibility in T brucei enolase the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 4-di(methylamino)pyridine [39] to obtain the protected form of PAH and FPAH The next step consisted of the deprotection of these compounds by a catalytic hydrogenation on Pd ⁄ BaSO4 [40] followed by the deprotection of the phosphonate group by bromotrimethylsilane [41] Neutralization of the resulting acid derivatives with LiOH provided the expected products PAH and FPAH as lithium salts in 28% and 15% overall yield, respectively X-ray crystallography Recombinant T brucei enolase was expressed and purified as previously described [2] Briefly, bacterial cells (E coli BL21(DE3)pLysS strain) harbouring the recombinant pET28a plasmid were grown at 37 °C until an attenuance (D) at 600 nm of approximately 0.5 was reached and protein expression was induced with mm isopropyl thio-b-d-galactoside for 20 h at 30 °C After centrifugation at 5000 g in a Sorvall RC26 plus centrifuge with Sorvall GS-3 rotor, the harvested cells were resuspended in TEA buffer, lysed through alternating cycles of freezing–thawing and then centrifuged The resulting clarified supernatant was directly subjected to nickel–nitrilotriacetic acid (Qiagen, Valencia, CA, USA) affinity chromatography and the purified fusion protein was treated with thrombin to remove the His-tag Crystallization was carried out based on the reported conditions for T brucei enolase [2], but using PEG1000 as precipitant rather than PEG monomethylether 550 Orthorhombic C2221 crystals were obtained by the hanging drop method using a reservoir solution of 10% (w ⁄ v) PEG1000, 0.01 m ZnSO4 or ZnCl2, and 0.1 m Mes, pH 5.0–6.5, with or without ligand at a concentration of 20 mm Before data collection, crystals of native T brucei enolase were immersed in the cryo-solution (mother liquor, 20% ethylene glycol) with or without 20 mm of ligand (PEP, PAH or FPAH) for and flash-cooled X-ray diffraction data were collected from two native crystals (referred to as sulfate_2 and sulfate_3 in Table 1) and four ligand-cocrystallised crystals (referred to as PEP, PAH_1, PAH_2 and FPAH in Table 1) using a Mar345 image plate detector (X-Ray Research GmbH, Norderstedt, Germany) mounted on a Rigaku UltraX 18 generator (Rigaku Corporation, ´ Tokyo, Japan) or at the MX1 beam line at the Laboratorio Nacional de Luz Sı´ ncrotron (Campinas, Brazil) using Mar˚ CCD 125 mm detector and radiation at 1.431 A (PAH_1) All data sets were processed and scaled with the software mosflm [42] and scala [43] from the ccp4 suite [44] The structures were straightforwardly solved by molecular replacement with the software molrep [45], using the previously determined T brucei enolase structure (PDB code 1oep) as the search model The resulting molecular replacement solutions were subjected to interactive rounds of manual rebuilding into 2Fo–Fc and Fo–Fc electron density maps using coot [46] and restrained refinement FEBS Journal 274 (2007) 5077–5089 ª 2007 The Authors Journal compilation ª 2007 FEBS 5085 Structural flexibility in T brucei enolase M V A S Navarro et al implemented in refmac [47] The ligands were built into difference electron density maps using coot As with the previously reported T brucei structure, intense positive peaks in the difference maps were observed near the active site and were modelled as metal ions Their identities and occupancy were determined based on the resulting maps and B-factors Water molecules were located automatically with the program warp [48] An additional, partially occupied (occupancy 0.5–0.7) and artefactual metal site involving a His residue at position ‘0’ (i.e immediately preceding the natural initiator Met within the tail downstream of the thrombin cleavage site in the N-terminal extension containing the His tag) was observed at a crystal lattice interface between in the PAH and FPAH complexes In all cases, final rounds of refinement were carried out with the entire subunit defined as a translation ⁄ libration ⁄ screw group in the modelling of anisotropy within refmac Isotropic B-factors were calculated from the refined translation ⁄ libration ⁄ screw parameters and residual isotropic B-factors with tlsanl [49] Stereochemical parameters were analysed with procheck [50] Details of the data collection and refinement statistics are shown in Table The PDB [51] codes for the new structures are: sulfate_2 (2ptw), sulfate_3 (2ptx), PEP (2pty), PAH_1 (1ptz), PAH_2 (2pu0) and FPAH (2pu1) Molecular dynamics Molecular dynamics simulations of 10 ns duration each were performed on the PEP complex structure, and also a structure in which the PEP and site II metal had been removed leaving a single metal ion in site I The molecular dynamics calculations employed the gromacs simulation suite [52] using the force field appropriate for proteins in water Sodium ions were added to the simulation system to compensate for the net negative charge of the protein The simulation was carried out in a cubic box with a minimal distance between solute and box edge of 0.7 nm Periodic boundary conditions were used The topology file for PEP was built using the small-molecule topology generator prodrg [53], followed by manual examination The crystal structures were relaxed by the default protocol of energy minimization and 100 ps of positionrestrained molecular dynamics, in which the protein is restrained to its starting conformation, prior to the start of the simulations proper After approximately 2000 ps of the simulation proper, both trajectories were stable, fluctuating at Ca rms deviations from the starting structure of approximately 0.18 nm (PEP + Mg trajectory) and 0.22 nm (single Mg trajectory) Monitoring of interatomic distances was performed using other gromacs programs whereas solvent accessible surface areas were calculated using dssp [54] Other methods Essential dynamics analysis [31] of a set of crystal structures (Table 1) was performed with programs from the 5086 gromacs package [52] Because the sulfate_2 structure lacked a large number of loop residues, it was omitted from the set However, the well-defined loop of sulfate_2 was used to fill the gap of two residues (Thr41 and Gly42) in the sulfate_1 structure, leaving only the gap in loop from Thr260-Pro266 The sulfate_1 and sulfate_2 structures are similar overall and in the vicinity enabling a simple splicing of these two residues Limited energy minimization of residues 40–43 of the result with modeller [55] was carried out to regularize bond lengths and angles The spliced version of sulfate_1, called sulfate_1*, was used in the essential dynamics analysis The essential dynamics method is based on the diagonalization of the covariance matrix of atomic fluctuations, which yields a set of eigenvalues and EVs The EVs indicate directions in a 3n-dimensional space (where n ¼ the number of atoms in the protein) and describe concerted fluctuations of the atoms The eigenvalues reflect the magnitude of the fluctuation along the respective EVs Structural superpositions and other conformational analyses were performed using lsqman [56] and mustang [57] Structural figures were made with pymol [58] Acknowledgements We are grateful to Paul Michels for useful discussions regarding this manuscript An early part of this work was supported by the European Commission through its INCO-DEV programme (contract ICA4-CT-200110075) References Fothergill-Gilmore LA & Michels PA (1993) Evolution of glycolysis Prog Biophys Mol Biol 59, 105–235 Hannaert V, Albert MA, Rigden DJ, da Silva Giotto MT, Thiemann O, Garratt RC, Van Roy J, Opperdoes FR & Michels PA (2003) Kinetic characterization, structure modelling 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and trypanosomatids Fig S4 LIGPLOT [59] figures of the interactions with enzyme of ligands in the new complexes Fig S5 Stereo figures generated with PYMOL showing electron density for ligands in the new complexes Structural flexibility in T brucei enolase Fig S6 Anomalous scattering maps for the sulfate_3 ˚ structure at 1.54 A This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 274 (2007) 5077–5089 ª 2007 The Authors Journal compilation ª 2007 FEBS 5089 ... barrel domain preceded by an N-terminal a + b domain [6] The catalytic site is contained completely within a single subunit and lies at the interface of the two domains: monomeric enolase is... (e.g lysines in both cases), selective inhibitors against aldolase [24] and phosphofructokinase [25] have been developed Despite bearing chemically reactive groups, by combining high affinity and. .. crystal packing Structural flexibility in T brucei enolase interface to residues ‘His0’ and Glu27, and to His283 from a crystal symmetry-related chain The new structures are diverse in the contents