The activity of Plasmodium falciparum arginase is mediated by a novel inter-monomer salt-bridge between Glu295–Arg404 Gordon A Wells1, Ingrid B Muller2, Carsten Wrenger2 and Abraham I Louw1 ă Department of Biochemistry, University of Pretoria, South Africa Department of Biochemical Parasitology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany Keywords arginase; malaria; metal; modelling; trimer Correspondence A I Louw, Department of Biochemistry, University of Pretoria, Lynwood Road, Pretoria 0002, South Africa Fax: +27 (0)12 362 5302 Tel: +27 (0)12 420 2480 E-mail: braam.louw@up.ac.za (Received 27 January 2009, revised 26 March 2009, accepted 23 April 2009) doi:10.1111/j.1742-4658.2009.07073.x A recent study implicated a role for Plasmodium falciparum arginase in the systemic depletion of arginine levels, which in turn has been associated with human cerebral malaria pathogenesis Arginase (EC 3.5.3.1) is a multimeric metallo-protein that catalyses the hydrolysis of arginine to ornithine and urea by means of a binuclear spin-coupled Mn2+ cluster in the active site A previous report indicated that P falciparum arginase has a strong dependency between trimer formation, enzyme activity and metal co-ordination Mutations that abolished Mn2+ binding also caused dissociation of the trimer; conversely, mutations that abolished trimer formation resulted in inactive monomers By contrast, the monomers of mammalian (and therefore host) arginase are also active P falciparum arginase thus appears to be an obligate trimer and interfering with trimer formation may therefore serve as an alternative route to enzyme inhibition In the present study, the mechanism of the metal dependency was explored by means of homology modelling and molecular dynamics When the active site metals are removed, loss of structural integrity is observed This is reflected by a larger equilibration rmsd for the protein when the active site metal is removed and some loss of secondary structure Furthermore, modelling revealed the existence of a novel inter-monomer salt-bridge between Glu295 and Arg404, which was shown to be associated with the metal dependency Mutational studies not only confirmed the importance of this salt-bridge in trimer formation, but also provided evidence for the independence of P falciparum arginase activity on trimer formation The polyamines putrescine, spermine and spermidine are near ubiquitous polycationic aliphatic amines required for a number of essential cellular processes, particularly in organisms undergoing rapid proliferation [1–3] These processes involve the stabilization of macromolecules [4–6] and progression through the cell cycle [7] Additionally, certain secondary metabolites, such as the post-translationally modified amino acid, hypusine [1,8], and the glutathione analogue, trypano- thione, in Trypanosoma [9], require polyamines for their biosynthesis Polyamine biosynthesis has been identified as a possible therapeutic target for various parasitic diseases [10,11], cancers [12] and even HIV via the requirement for hypusine [13] Putrescine is synthesized by the decarboxylation of ornithine (ornithine decarboxylase) and serves as substrate for the addition of aminopropyl groups to form spermidine and spermine The aminopropyl groups are donated Abbreviations NP, constant number of atoms and constant pressure; NPT, constant number of atoms, constant pressure and temperature; PfArg, Plasmodium falciparum arginase FEBS Journal 276 (2009) 3517–3530 ª 2009 The Authors Journal compilation ª 2009 FEBS 3517 Novel interaction in plasmodial arginase G A Wells et al from decarboxylated S-adenosylmethionine, the product of S-adenosylmethionine decarboxylase [2,14] Arginase (EC 3.5.3.1) catalyses the hydrolysis of l-arginine to l-ornithine and urea The arginase fold is part of the ureohydrolase superfamily, which also includes agmatinase [15,16], histone de-acetylase and acetylpolyamine amidohydrolase [17] Agmatine is formed by decarboxylation of arginine (arginine decarboxylase) and is converted by agmatinase to putrescine and urea Arginase is thus part of one of two alternative biosynthetic routes to putrescine Polyamine biosynthesis enzymes characterized in the malaria parasite, Plasmodium falciparum, include the bifunctional S-adenosylmethionine decarboxylase ⁄ ornithine decarboxylase [18–22], spermidine synthase [23] and arginase [24] In P falciparum, the agmatinase route to putrescine has not been identified, thus making arginase the sole biosynthetic route to putrescine in the malaria parasite [24] In mammals, two isoforms of arginase have been identified: arginase I is cytosolic and largely hepatic where it catalyses the final step of the urea cycle [25,26]; arginase II is nonhepatic and occurs in the mitochondrial matrix [27–29] and is involved in homeostasis of ornithine for the further production of proline and glutamate [30] Both isoforms have been implicated in regulating NO biosynthesis as a result of competition for the common substrate arginine with inducible NO synthase [31] In bacteria, there is a single arginase isoform, whereas more than one exists in yeast [32] The yeast isoforms have been linked to glutamate accumulation during germination and asexual spore development [33,34] Arginase is a multimeric metallo-enzyme with a binuclear spin-coupled Mn2+ cluster in each active site that is restricted to a single a ⁄ b monomer The metal ˚ cluster is located in a 15 A deep cleft with the Mn2+ ˚ apart and bridged by a solvent molecule atoms 3.3 A [35] Structures from the bacteria Bacillus caldovelox [36,37], human arginase I [38] and II [39], rat arginase I [35,40] and Thermus thermophilus [Protein Data Bank (PDB) codes: 2EF4, 2EF5 and 2EIV] have been determined In the mechanism proposed by Kanyo et al [35], the metal bridging solvent is an activated hydroxyl, which attacks the guanidium carbon of arginine, followed by collapse of the tetrahedral intermediate to release ornithine and urea However, ab initio modelling of the active site suggests that the bridging solvent may be a neutral water molecule instead [41] Eukaryotic arginases are trimeric, whereas bacterial arginases are hexameric [42] However, the trimeric arginase from Saccharomyces cerevisiae forms a 3518 regulatory complex with trimeric ornithine transcarbamoylase, thus forming a hexameric complex [43] In mammals, monomers retain substantial activity if trimer formation is disrupted [44,45] In recent studies, disrupting metal binding has no reported effect on the quaternary structure [40,46,47] However, previously, it was reported that oligomerization of human arginase I could be disturbed by chelating out Mn2+ but substantially similar kinetics could be restored to nylonimmobilized monomers after reincubation with Mn2+ [48,49] Removal of the active site metals in yeast not only abolishes enzyme activity, but also affects the maintenance of the quaternary structure and sensitivity to temperature [43,50] To date, the strongest metal dependency has been reported for the arginase from P falciparum, where mutations that abolish metal binding or removal of the metal ions cause dissociation of the trimer into inactive monomers Conversely, a mutation that abolishes a conserved inter-monomer interaction located away from the active site results in inactive monomers [24] These host–parasite differences may thus provide a novel non-active site based strategy for inhibiting P falciparum arginase (PfArg) Essentially, disturbing trimer formation may serve as a novel means of inhibiting PfArg Thus, the mechanism of this structural dependency was investigated by homology modelling and molecular dynamics, aiming to establish an in silico system for exploiting this dependency Results Sequence alignment and homology modelling Searching online Plasmodium genome resource, PlasmoDB [51], revealed the arginase sequences for Plasmodium vivax, Plasmodium yoelii, Plasmodium knowlesi and Plasmodium berghei, in addition to the previously characterized PfArg From the automated alignments, two parasite-specific inserts were revealed in the various Plasmodium arginases (Fig 1) In both reference alignments, the positions of the inserts not differ markedly In the final model of the study, insert is predicted to run from residues 77–151 (75 residues), and insert from residues 377–388 (12 residues) The exact positions vary slightly depending on the alignment used for modelling Insert varies considerably in sequence and length between different Plasmodium species, ranging from approximately 100 residues (P vivax) to only 15 residues (P berghei) It is predicted to lie between the second b-strand and second a-helix of the model on the outer edge of the trimer By contrast, insert is highly conserved in length and FEBS Journal 276 (2009) 3517–3530 ª 2009 The Authors Journal compilation ª 2009 FEBS G A Wells et al Novel interaction in plasmodial arginase Fig Alignment used for modelling P falciparum (pfam), H sapiens (human), R norvegicus (rat) and B caldovelox (bacc) The positions of the Plasmodium-specific inserts are indicated Identical residues are shaded dark grey, and similar residues indicated by lighter shades Helices are indicated by rods, and b-strands by arrows sequence in all Plasmodium species Insert is located between the last b-strand and the last a-helix The sequence identity between the P falciparum and templates was approximately 35%, 30% and 27%, respectively, for the bacterial, rat and human arginases Even a slight difference in the alignment used for insert was found to have a significant effect on its conformation Modelling with insert shifted one residue upstream (fugue derived alignment; see Experimental procedures) caused the insert to fold away from the trimer interface, interacting with its respective monomer (Fig 2) The model preserves standard active site residues observed in other arginase structures All Mn2+ coordinating residues (discussed below) previously identified are conserved in the model The only substitutions are in second shell ligands when compared with the bacterial template, where Ser176 and Glu268 (B caldovelox) are replaced by Asp272 and Asp365 (P falciparum), respectively Residues implicated in substrate binding are also highly conserved There is only one conservative substitution compared to the mammalian templates, and none compared to the bacterial template Thr135 (rat) is replaced by Ser227 in the model In the model, Mg2+ was modelled instead of Mn2+ as a result of limitations of the forcefield, which was not parameterized for Mn2+ (see Experimental procedures) Visual inspection of the model suggested that a novel inter-monomer salt-bridge forms between Glu295x and Arg404y (where subscripts designate different monomers) (Fig 3) In multiple sequence alignments, Glu295 aligns with conserved acidic resi- Fig Effect of alignment on conformation of insert When moving the insert one residue upstream, the insert folds away from the trimer interface (yellow) compared to making contact (red) The active site Mg2+ atoms are indicated in green Monomers are distinguished by different shades of blue The image was generated using PYMOL dues in the bacterial and mammalian templates P falciparum Glu295 aligns with an Asp in mammals (human arginase II: Asp223; rat arginase I: Asp204), fungi and bacteria (B caldovelox arginase: Asp199) In the other Plasmodium species, Glu295 FEBS Journal 276 (2009) 3517–3530 ª 2009 The Authors Journal compilation ª 2009 FEBS 3519 Novel interaction in plasmodial arginase G A Wells et al Arg308/327 Asp204/223 To determine other possible interactions, the saltbridge analysis tool of vmd [52] was employed to search for all possible salt-bridges in the protein, using co-ordinates prior to sampling All salt-bridges with a hydrogen bond donor ⁄ acceptor distance less than ˚ 3.2 A were identified Only one other interaction between adjacent monomers was found between Glu400x and Lys340y However, this interaction was not stable during molecular dynamics This instability was observed both with and without the active-site metal, Mg2+ Thus, this interaction is likely to be only of secondary importance in maintaining quaternary structure, and attention was focused on the Glu295x– Arg404y interaction instead Protein stability Fig Salt-bridges in PfArg Template residue numbers are shown in italics (rat ⁄ human) The conserved interaction between Arg346x and Glu347y (P falciparum) is indicated Glu295x aligns with acidic residues in the template structures (Asp204 ⁄ Asp223) but forms a novel interaction with Arg404y compared to the template residues that interact with Arg308 ⁄ Arg327 in the C-terminus Monomers are shown in green, light blue and mauve Template backbones are transparent, and template salt-bridge residues are depicted in lighter shades of red (acidic) and blue (basic) The image was generated using VMD aligns either with Asp (P yoelii and P berghei) or Glu (P knowlesi and P vivax) The only exception is in plants, where Glu295 aligns with Ser instead (Arabidopsis thaliana arginase I: Ser247) In the model, Glu295 forms an interaction with the adjacent monomer via partner residues that not align in sequence in the mammalian and bacterial templates In mammalian arginases, the Asp223 ⁄ 204x (rat arginase I ⁄ human arginase II) cognate forms an inter-monomer salt-bridge with Arg308 ⁄ 327y This salt-bridge nucleates considerable inter-monomer interactions, characterized by an S-shaped C-terminus [35,39], which is absent in the Plasmodium sequences Arg308 from rat arginase I aligns with Ile in Plasmodium (408: P falciparum; 368: P knowlesi; 436: P vivax; 353: P berghei; 376: P yoelii) The P falciparum Arg404y salt-bridge partner to Glu295x aligns with small and ⁄ or hydrophilic residues in other organisms (e.g Ser, Thr, Cys, Ala and Glu) In the bacterial structure, the Asp199x cognate forms an inter-monomer bridge with Glu256y that is mediated either by urea or by free arginine, depending on the crystallization conditions [36] 3520 Before proceeding with detailed analysis, it was necessary to ensure that gross changes to the protein necessitated by the modelling dificulties would not compromise the interpretation of the results The omission of parts of the protein is potentially problematic in that it introduces an unnatural chain break and therefore potential instability The deletion of insert creates a protein fragment on the outer edge of the trimer complex that does not interact extensively with any neighbouring monomers, and largely makes intramonomer contacts This fragment was stable for at least 50 ns of simulation and, apart from the loss of some secondary structures in this region (described below), remained in contact with the rest of the protein The protein stability of PfArg was monitored by the change in Ca rmsd during equilibration and sampling compared to the starting co-ordinates In both cases, with and without Mg2+, there is an increase in rmsd, which typically equilibrates after approximately 20 ns (Fig 4) However, without Mg2+, the Ca rmsd equili˚ brates at approximately A more than in the presence 2+ of Mg , which was usually observed by 20 ns in both the constant number of atoms and constant pressure (NP) and constant number of atoms, constant pressure and temperature (NPT) ensembles and persisted up to 50 ns in the NPT simulations The effect of removing the metal on conservation of secondary structure during sampling was also monitored In general, a greater loss of secondary structural integrity was observed for nonmetal systems in both the NP and NPT ensembles In the absence of Mg2+, a complete loss of secondary structure is observed for certain elements Combining both NP and NPT ensembles gives a total of six simulations of the monomer, which can be used to observe any general loss of FEBS Journal 276 (2009) 3517–3530 ª 2009 The Authors Journal compilation ª 2009 FEBS G A Wells et al Novel interaction in plasmodial arginase Cα deviation (NP ensemble) RMSD (Å) 0 10 Time (ns) 12 14 16 18 20 Cα deviation (NPT ensemble) RMSD (Å) 0 10 20 30 40 possibility of another conformation in the P falciparum structure During sampling, insert moves considerably and does not retain its interaction as predicted by the original homology model prior to molecular dynamics Furthermore, there are some noticeable differences between the metal and nonmetal simulations In both the NP and NPT ensembles, insert partially occupies the interface between two adjacent monomers, which is more pronounced, however, when Mg2+ is included (results not shown) Similar effects on protein stability were observed for simulations of five mutants (Glu295 Ala, Glu295 Arg, Arg404 Ala, Glu295x Ala ⁄ Arg404y Ala, Glu347 Gln) of the PfArg model For all mutations, there is also a greater increase in rmsd (described above) compared to the wild-type model with Mg2+ The largest increase is observed for Glu347 Gln, which is approximately double that of wild-type enzyme without Mg2+ The mutations Glu295 Ala, Glu295 Arg, Glu295x Ala ⁄ Arg404y Ala show a similar increase to wild-type enzyme without Mg2+ The smallest effect is observed for Arg404 Ala, which is similar to the wildtype enzyme without Mg2+ for part of the 50 ns run (results not shown) 50 Time (ns) Fig Effect of removing Mg2+ on backbone Ca rmsd A running average was calculated using a window of 500 frames (250 fs per frame) With Mg2+, removed ( ), a greater increase is observed than with Mg2+ (+) included, implicating Mg2+ in the structural stabilization of the enzyme secondary structure However, these data are not sufficient to determine possible co-operative effects between monomers The monomers ⁄ chains are arbitrarily designated A, B and C In chain B of the NP simulation, the first and second b-strands are both lost, whereas the second half of the first a-helix is lost in chain C of the NPT simulation The first half of the third a-helix is also lost in chains B and A of the NP and NPT simulations, respectively All of these secondary structural elements align with cognate elements in all of the templates The sixth helix (310) of the model is lost in some chains of both the metal and metal-free simulations Whether this element is a helix is uncertain because it only aligns with a 310-helix in the bacterial template In both P falciparum and the bacteria, the N-terminal residue is a proline, which often forms the N-terminal cap of both a-helices and 310-helices, and is also over-represented as the helix capping residue when followed by a b-strand [53] The absence of helical structure in the mammalian templates indicates the Stability of inter-monomer salt-bridges In all arginases studied to date, there is a conserved inter-monomer salt-bridge represented in P falciparum by Arg346x–Glu347y (Fig 3) The cognate salt-bridge in the templates used is between Arg255 ⁄ 274 ⁄ 249x– Glu256 ⁄ 275 ⁄ 250y (rat, human and bacterial templates, respectively) These residues align unambiguously and the salt-bridge forms reliably during modelling Considering the established importance of this interaction, its integrity was monitored during modelling and simulation In the sampling runs, the Arg346x–Glu347y interaction was generally stable and intact for both the Mg2+ and Mg2+ -absent cases One inter-monomer bridge did break in the presence of Mg2+ in the NP ensemble In the NPT ensembles, the interactions remain intact with and without Mg2+ but there is an increase in the average standard deviation of the salt-bridge distance in the absence of Mg2+ (Fig 5) This suggests that the Arg346x–Glu347y interaction is susceptible to removal of Mg2+, even though the interaction remained intact As described above, visual inspection of the homology models suggested a further interaction between Glu295x and Arg404y Although not fully formed in the homology models, the salt-bridge distance did FEBS Journal 276 (2009) 3517–3530 ª 2009 The Authors Journal compilation ª 2009 FEBS 3521 Novel interaction in plasmodial arginase G A Wells et al Glu295–Arg404 salt bridge Arg346–Glu347 salt bridge 20 0.2 15 Distance (Å) Distance (Å) Standard deviation (Å) 10 0.1 10 20 30 40 50 Time (ns) Fig Effect of removing Mg2+ on backbone Arg346x–Glu347y salt-bridge A running average was calculated using a window of 500 frames (250 fs per frame) Distances for all interactions from the NP and NPT ensembles are plotted against the right y-axis In the NP ensemble, including Mg2+, one of the salt-bridges is broken between chains A and B (d) All other salt-bridges remain intact for both the NP and NPT ensembles with (black) and without (purple) Mg2+ The average standard deviation for the NPT ensemble is plotted against the left y-axis with (black) and without (red) Mg2+ Solid lines indicate the average sum for all three chains, and the mean over the entire run is indicated by the dashed lines With Mg2+ removed, there is an increase in the standard deviation ˚ adopt standard values (± A) during minimization and heating of the systems The integrity of this interaction was found to be more susceptible to removal of Mg2+ than the Arg346x–Glu347y interaction Between the NP and NPT ensembles, there are six Glu295x– Arg404y salt-bridges In the Mg2+ -free systems, the salt-bridge was broken in half of these (Fig 6) In the NP ensemble, two interactions are broken between chain A and B, and chain B and C by the end of 20 ns The third interaction was transiently broken (chain C and A) In the NPT ensemble, only one of these interactions (chain A and B) was broken by the end of 50 ns The alignment used to model insert also affected the Arg346x–Glu347y interaction In models where insert was predicted to interact at the trimer interface, the interaction was broken during in vacuo simulations in charmm [54] It was also observed during simulation with charmm that models built without the imposition of symmetry on the internal co-ordinates tended to disturb the Arg346x–Glu347y interaction The stability of this interaction was improved by using models built with symmetry imposed on internal co-ordinates (i.e with perfectly super-imposable monomers) (results not shown) 3522 0 10 20 30 40 50 Time (ns) Fig Effect of removing Mg2+ on backbone Glu295x–Arg404y saltbridge A running average for the interaction between each chain-pair combination was calculated using a sliding window of 500 frames (250 fs per frame) The NP simulation was terminated after 20 ns In the absence of Mg2+ (purple) in the NP ensemble, all three interactions were broken (chains: d, AB; r, BC; , CA), albeit that between chains CA only transiently In the NPT ensemble, only the bridge between chains A and B ( ) was broken in the absence of Mg2+ The interactions remained intact with Mg2+ present (black) During the simulation of the mutant enzymes, there was no effect on the integrity of Arg346x–Glu347y interaction for mutations directed at Glu295x and ⁄ or Arg404y For the simulation of Glu347 Gln, however, an interesting result was obtained: two out of three of the Glu295x–Arg404y interactions were broken during the sampling run This indicates that Glu347 Gln may also effect trimer destabilization through disturbing Glu295x–Arg404y as well by the loss of the Arg346x– Glu347y interaction Conversely, the results not indicate that disturbing the Glu295x–Arg404y interaction affects the Arg346x–Glu347y salt-bridge Coordination geometry of Mn2+/Mg2+ Highly conserved residues are involved in a specific co-ordination pattern by donation of free electron pairs for the binuclear Mn2+ cluster in existing crystal structures In rat I arginase, the more deeply buried ion (Mn2+A) is co-ordinated by His101, Asp124, Asp128, Asp232 and the bridging solvent in a square pyramidal geometry The respective residues in P falciparum are His193, Asp216, Asp220 and Asp323 The second metal, Mn2+B is co-ordinated by His126, Asp124, Asp232, Asp234 and the bridging solvent in a distorted octahedral geometry in rat I arginase (His218, Asp216, Asp323, Asp325 in P falciparum) FEBS Journal 276 (2009) 3517–3530 ª 2009 The Authors Journal compilation ª 2009 FEBS G A Wells et al Novel interaction in plasmodial arginase During modelling, the conformations adopted by the co-ordinating residues did not entirely conform to known crystal structures from homologues The most notable difference is Asp323, which is expected to form a monodentate bridging interaction between the two ions During the simulations, it formed a bidentate bridge instead All other expected co-ordinating atoms were oriented close enough to interact with the ions The only other missing interaction was that of the bridging OH– because no attempt was made to introduce the bridging solvent molecule The Mg2+–Mg2+ ˚ distance was also approximately 0.6 A greater than the 2+ 2+ distance The Mg2+ ions known Mn –Mn remained in the active site during the simulations and restricted the movement of the interacting ligands When Mg2+ is removed, considerable movement is observed in the co-ordinating residues in both the NPT and NP simulation Site-directed mutagenesis of Glu295x and Arg404y Modelling predicts that Glu295x–Arg404y is necessary for trimer formation The existence of a structuralmetal dependency between trimer formation and activity in P falciparum arginase supports the involvement of the Glu295x–Arg404y interaction The effects of mutating Glu295x and Arg404y were therefore determined in the recombinantly expressed enzyme and are summarized in Table PfArg was found to be more susceptible to mutations introduced at Glu295x than at Arg404y Mutating Glu295 to Ala or Arg considerably reduces enzyme activity (by 96% and 73%, respecTable Comparison of kinetic parameters for wild-type (WT) and mutant arginases The results are derived from at least three independent assays with standard deviations ND, not detectable Vmax Km (lmolỈmin)1Ỉmg)1) (mM) c WT 31 Glu295 Ala 1.3 ± 0.3 Glu295 Arg 8.4 ± 0.9 Arg404 Ala 14.3 ± 0.9 Glu295 Ala ⁄ Arg404 Ala 1.6 ± 0.1 a 13 d d c kcata,b (s)1) 24.8 (100%) ND 1.0 ± 0.2 d (4%) 146 ± 6.7 ± 0.7 (27%) 45 ± 11.4 ± 0.7 (46%) ND 1.3 ± 0.1 (5%) kcat ⁄ Kma,b (mM)1Ỉs)1) 1.9 (100%) ND 0.03 (1.6%) 0.25 (13%) ND Percentage of WT value is shown in parentheses b Calculated from 48 kDa per monomer c Values, without standard deviation, are taken from Muller et al [24] d £ 5% of WT activity in standard ă assay Fig Effect of Glu295xArg404y salt-bridge mutations on trimer formation Recombinant proteins were separated on a Superdex S-200 gel sizing column (1 · 30 cm) using a buffer containing 50 mM Tris–HCl, pH 8, mM dithiothreitol and mM MnCl2 Aliquots of 100 lL of the elution fractions (0.5 mL) were analysed by western dot-blotting using monoclonal anti-Strep-tag serum (Institut fur Bioanalytik) at a dilution of : 5000 The corresponding ă molecular masses are given above the dots tively) under standard assay conditions, which is also the case for the double mutant Glu295 Ala ⁄ Arg404 Ala (95%) However, single mutations of Glu295 to Arg and Arg404 to Ala leads to altered Km values of 146 mm and 45 mm for l-Arg, respectively, which is up to 11-fold higher compared to the wild-type arginase The catalytic activity (as kcat) of these mutants were and 11 s)1, respectively, and were thus 27% and 46% of that for the wild-type enzyme The resulting efficiencies expressed as kcat ⁄ km values are reduced in both mutants to 1.6% and 46%, respectively, compared to the wild-type The elution profile of all mutants analysed by gel filtration revealed monomeric forms, except for Glu295 Ala, which is partially trimeric (Fig 7) By contrast, trimer formation is more susceptible to mutation of Arg404 The partial activity of Arg404 Ala is the first clear evidence of active monomers for PfArg Discussion From the multiple sequence alignment used for modelling, two parasite-specific inserts were identified in the P falciparum sequence Proteins from Plasmodium frequently have large inserts relative to sequences from homologues in other organisms [55] These inserts are often characterized by low complexity [56,57] and ⁄ or have a strong amino acid bias towards small and hydrophilic residues Apart from possible global functions [55–57], it has been demonstrated that inserts may have local functions relative to their enzymes [22,58,59] Plasmodium-specific inserts can be difficult to delineate in sequences of low homology Thus, FEBS Journal 276 (2009) 3517–3530 ª 2009 The Authors Journal compilation ª 2009 FEBS 3523 Novel interaction in plasmodial arginase G A Wells et al where possible, other Plasmodium sequences were included to assist with the insert delineation Because of its length, most of insert was left unmodelled (residues 81–147 removed; Fig 1) Insert is considerably shorter and more conserved and was therefore retained for ab initio modelling The choice of alignment was found to have a considerable effect on the conformation of insert Because a small change in alignment had a substantial effect on insert 2, it is important to justify the choice of reference alignment used In the fugue [60] derived alignment (see Experimental procedures), insert was predicted to fold away from the trimer interface, compared to the clustalw derived alignment The fugue alignment was favoured, however, because the fugue software makes use of environment-specific substitution tables and structuredependent gap penalties, and is thus generally expected to give a more accurate starting alignment for modelling purposes The function of the inserts in PfArg has yet to be established Comparing the active site of the model with the templates revealed only a small number of substitutions The high conservation of the active site suggests that inhibitors specific to the P falciparum active site will be difficult to find Thus, an alternative means of inhibition may be necessary if PfArg is to be of potential therapeutic value Therefore, attention was directed at the inter-monomer interactions A novel intermonomer salt-bridge forms between Glu295x and Arg404y Although Glu295 aligns with conserved acidic residues in the templates, its interacting partner does not align In mammalian structures, the acidic equivalent nucleates considerable inter-monomer interactions by means of an S-shaped C-terminus by forming a salt-bridge with Arg308 The importance of the S-shaped tail is still in doubt because products truncated after Arg308 can still form active trimers [61] In bacterial structures, an interaction is formed with another acidic residue (Glu256) that is mediated by either urea or free arginine Finally, in the P falciparum model, Glu295 is predicted to interact with Arg404, which does not align with mammalian Arg308 or bacterial Glu256 Thus, there appears to have been evolutionary pressure to establish a strong inter-monomer interaction in this region of the monomer-monomer interface The differences between the P falciparum model and templates suggest this saltbridge as a possibly unique interaction and was therefore subjected to scrutiny using molecular dynamics and site-directed mutagenesis The deletion of insert for modelling did not adversely affect the stability of the model Although potential problems with respect to introducing a chain 3524 break could have been avoided by ligating the ends of the gap, this would also be unnatural Because the fragment was apparently stable and closing the gap unligated is less parsimonious, the break was left in The equilibration of Ca rmsd during molecular dynamics at a larger distance for the Mg2+ -free systems suggests that removing the active site metals has a detrimental effect on protein stability It was previously reported that removing Mn2+, either by dialysis and chelation with EDTA, or by mutagenesis of Mn2+ co-ordinating residues in the active site, of PfArg not only abolished enzyme activity, but also promoted dissociation of the trimer, which could be reversed by addition of Mn2+ [24] The general loss of secondary structure further mirrors the increase in rmsd upon removing the metal and confirms the necessity of the active site metals for protein stability Removing the active site metals also affected the conformation of insert during molecular dynamics, which generally remained more solvent exposed and made less inter-monomer contacts This suggests that insert may also be involved in maintaining the trimer and thus part of the structural metal dependency The temperature of the NP ensemble was allowed to increase (310 to 332 °K) by not coupling it to a temperature bath Although it is usual to apply some means of keeping temperature constant (isothermal ensemble), sampling at higher temperatures allows the system to overcome energy barriers faster In the present study, the increase in temperature accelerates the effects of removing Mg2+ In the NPT ensemble, only one Glu295x–Arg404y interaction is broken after 20 ns, whereas, in the NP ensemble with increasing temperature, all three have been broken before 20 ns The effect of the increasing temperature is also reflected in the rmsd, which is more pronounced and more rapid in the NP ensemble The increasing temperature may be detrimental, however, as reflected by breaking an Arg346x–Glu347y interaction in the NP ensemble with Mg2+ For this reason, subsequent simulations were carried out in the NPT ensemble Because most simulations based on classical mechanics not model metal co-ordination, the conformations adopted by the co-ordinating residues did not entirely conform to known crystal structures from homologues The larger distance between the Mg2+ atoms compared to Mn2+ in known structures is partly a result of the inability of the software to recognize co-ordination chemistry natively as well as the larger van der Waals radius of Mg2+ compared to Mn2+ Because of the stability of the Mg2+ cluster, it was considered unnecessary to introduce artificial FEBS Journal 276 (2009) 3517–3530 ª 2009 The Authors Journal compilation ª 2009 FEBS G A Wells et al restraints to replicate metal-co-ordination Because the presence of Mg2+ was able to stabilize the co-ordinating residues by electrostatic interactions alone, this approach appears to be viable for investigating the structural metal dependency These results also suggest that structural metal dependency involves free movement of the metal co-ordinating residues The existence of the inter-monomer salt-bridge between Glu295x and Arg404y was corroborated by site-directed mutagenesis of the recombinant enzyme All mutants tested promoted trimer dissociation, with incomplete dissociation for Glu295 Ala but contrasts with Glu295 Arg, which led to complete dissociation Mutating Glu295 to Arg is expected to be more drastic compared to Ala because this would introduce a positive charge and thus an electrostatic repulsion in the vicinity of the Glu295x–Arg404y interaction Interestingly, this mutation leads to active but less efficient monomers with a 11-fold increased Km value of 146 mm for l-arginine, indicating altered substrate binding By contrast, mutating Glu295 to Ala reduced the activity to 4% of the wild-type enzyme but with its trimeric conformation partially retained The Km value for the Glu295 Ala mutant was not measurable because it was not saturated up to 200 mm arginine Mutating Arg404 to Ala abolished trimer formation However, this mutant enzyme shows 46% activity (as kcat) and 13% efficiency (as kcat ⁄ Km) and its Km value is approximately three-fold increased compared to the wild-type enzyme This result is similar to the previously reported behaviour of the rat liver arginase Arg308 mutants, which, as monomers, still had a residual activity of 41% and an efficiency in the range 13–17% [44] Size-exclusion chromatography therefore suggests that certain mutations abolish trimerization, although the enzymatic data suggests that trimerization is not absolutely necessary for activity However, the possibility that a weakened trimer can form under enzyme assay conditions cannot be excluded Such a possibility is suggested by rat arginase, where the Arg308 Lys mutant is apparently active as a monomer, but nonetheless crystallizes as a trimer [44] Although it has been demonstrated that disturbing the oligomer via the conserved interaction between Arg346x and Glu347y largely inactivates the enzyme, it still has 10% residual activity [24] The results of the Arg404 Ala mutation indicates that it is possible to produce active monomers and, furthermore, that certain mutations can partially compensate for induced structural instability of monomerization by long range allosteric effects Although there is a dependency between trimer formation and enzyme activity, these results indicate that it is not complete This incompleteness was sug- Novel interaction in plasmodial arginase gested by previous results where mutating His193 in the active site also results in an inactive trimer [24] as was also found for the Glu295 Ala mutation in the present study Mutations that disturb the Arg346x– Glu347y and Glu295x–Arg404y interactions both result in decreased activity Furthermore, during simulations of the Glu347 Gln mutant, the Glu295x–Arg404y interaction is also disturbed These observations suggest that disruption of both interactions may provide a novel means of inhibiting PfArg These results suggest that formation of the Glu295x–Arg404y salt-bridge is necessary for trimer formation, and that the hypothesis that the enzyme can be inhibited via disturbing the trimer warrants further investigation It is expected that disturbing the interactions involved in trimer formation mediate their effects via the co-ordination of Mn2+ in the active site, which is required for the arginase chemistry This is reflected by the increased equilibrium rmsd during molecular dynamics, which should ultimately translate into lost co-ordination of Mn2+ in the active site The loss of Mn2+ under such conditions, however, has yet to be observed directly Nonetheless, the decreased activity of monomeric mutants and the increased equilibrium rmsd of modelled mutants suggests that this may be the case It has been demonstrated that rat arginase I loses some activity (33–41% of kcat) when the trimerization is disturbed by mutagenesis [44] This has not been observed for human arginase I, where fully functional monomers have been obtained [45,48,62] Despite the high sequence similarity (87%) between arginase I from rat and human [63], they differ in their kinetic properties Human arginase I has a substantially lower Km for arginine compared to rat arginase I Furthermore, the Kd values for the inhibitors S-(2-boronoethyl)-l-cysteine and 2-amino-6-boronohexanoic acid are one order of magnitude less than for the rat counter part [38,46,64] This suggests that it may be possible to inhibit PfArg via disturbing oligomerization without affecting the human counterpart Arginine levels in in vitro cultures of P falciparum are depleted by PfArg, although the relevance of arginase as a malaria drug target remains to be demonstrated [65] Hypoargininaemia has been linked to the progression of severe malaria and may be related to the requirement of arginine for NO biosynthesis [66] It has been speculated that low host NO benefits the parasite by causing increased expression of host intracellular adhesion molecule-1, which is used by parasatized red blood cells to adhere to the vascular endothelium and thus avoid spleen clearance Arginase knockouts of the rodent malaria parasite, FEBS Journal 276 (2009) 3517–3530 ª 2009 The Authors Journal compilation ª 2009 FEBS 3525 Novel interaction in plasmodial arginase G A Wells et al P berghei (ANKA strain), are viable and show similar growth behaviour ex vivo and in infected mice [65], although this has yet to be established for the human parasite Experimental procedures Sequence alignments Reference multiple alignments were generated using clustalw 1.82 and the fugue server The clustalw alignment included eukaryotic arginases types I and II, and bacterial arginases The sequences used for the clustalw alignment (Entrez accession number are given in brackets for non-Plasmodium species, PlasmoDB reference numbers are used for Plasmodium sequences) were: A thaliana (P46637, Q9ZPF5), Schizosaccharomyces pombe (P37818, Q10066), Xenopus laevis (Q91553, Q91554, Q91555, P30759), Homo sapiens (P78540, P05089), Mus musculus (O08691, Q61176), Rattus norvegicus (O08701, P07824), Agrobacterium tumefaciens (P14012), B caldovelox (P53608), Bacillus subtilis (P39138), Brucella melitensis (Q59174), Coccidioides immitis (P40906), Emericella nidulans (Q12611), Neurospora crassa (P33280), Rana catesbeiana (P49900), Glycine max (O49046), Staphylococcus aureus (P60086), S cerevisiae (P00812), P knowlesi (PKH_ 070380), P vivax (Pv098770), P falciparum (PFI0320w), P yoelii (PY03443) and P berghei (PB000787.03.0) Sequences for P knowlesi, P vivax, P yoelii and P berghei were obtained using blast (available at: http://plasmodb org) [51] with the P falciparum sequence as query Although the reference alignments were often highly redundant, all sequences were retained to offset the bias of including five Plasmodium sequences Homology modelling modeller 8v0 [67,68] was used to build the homology models Trimeric models were constructed on the rat arginase I (PDB code: 1RLA[abc]), human arginase II (PDB code: 1PQ3[abc]) and B caldovelox (PDB code: 1CEV[abc]) templates Superimposable monomers were constructed by imposing symmetry restraints on the internal coordinates of all atoms during the model building process The effect of various sequence alignments was determined by generating multiple models with different random number seeds and monitoring the effect on the number of residues in disallowed regions of the Ramachandran plot and on the overall G-factor score from procheck [69] Problem areas were identified as residues that frequently fell in disallowed regions Models that minimized residues with poor phi ⁄ psi values and maximized the G-factor were used for molecular dynamics In the final run, one model was chosen from a total of 33 for molecular dynamics 3526 Molecular dynamics Hydrogen atoms were added automatically using charmm 32b1 [54,70] All residue positions are given relative to their own sequence The histidine protonation scheme adopted was based on the requirements for co-ordination of metal atoms in the active site in known structures Thus, His193 and His218 (which align with His101 and His126 in rat arginase I, respectively) were protonated on Ne, and His233 was protonated on both nitrogen atoms His233 aligns with His141 in human arginase I The double protonation of ˚ His233 was based on the high resolution (1.29 A) crystal structure of human arginase I [38], for which hydrogen positions were also determined, as well as previous speculation concerning activity [35] All remaining histidines were protonated on the Nd atom Glutamate, aspartic acid and lysine residues were charged Because the current common protein forcefields (charmm, amber, gromos) were not parameterized for Mn2+, the Mg2+ ion was used instead It was thus assumed that any effects of the metal on trimer formation were largely electrostatic in nature Although charmm was initially used for molecular dynamics, it was found to perform too slowly on the available hardware (Pentium IV Beowulf cluster with GigaBit Ethernet) namd 2.6 [71] was used instead, which provided better scaling pymol [72] was used for visualization, as well as vmd, which was also used for analysis of molecular dynamics trajectories [52] stride software, as provided with vmd, was used to assign secondary structure The multiseq plugin [73] was used to visualize secondary structure alignments Salt-bridges between arginine and glutamate residues were measured between the Cf and Cd atoms, respectively A distance between ˚ these atoms of A corresponds to the typical distance of ˚ between the hydrogen bond donor (PDB naming: NHx) 2.8 A and acceptor atoms (PDB naming: OEx) respectively The system was explicitly solvated (transferable intermolecular potential water model) using the solvate plugin ˚ of vmd The protein was padded with solvent for 12 A in the x- and y-axis (xy being coplanar with the trimer) and ˚ 10 A in the z-axis NaCl counter ions were added with the autoionize plugin from vmd to an ionic strength of 50 mm with charge balancing to create a net system charge of zero The system with metals (Mg2+) included 14 Na+ ions and 11 Cl– ions, whereas the system without metal finally contained 20 Na+ ions and Cl) ions ˚ Nonbonded interactions were shifted to zero from 10 A ˚ to a cut-off of 12 A All nonbonded interactions connected by more than four covalent bonds were included Prior to solvation, the trimer complex was minimized in namd in vacuo for 450 steps All steps including the complete system with solvent and ions were simulated with periodic boundary conditions using particle mesh Ewald Sums for the electrostatic calculations A typical cell size was approx˚ imately 118x · 113y · 79z A A particle mesh Ewald ˚ ngstrom grid size of 120 · 120 · 90 was used sub-A FEBS Journal 276 (2009) 3517–3530 ª 2009 The Authors Journal compilation ª 2009 FEBS G A Wells et al Multiple protocols were used to verify the general robustness of the system Two different heating protocols were used In the first protocol, the solvent of the entire system was minimized for 2000 steps, followed by the solvent and nonbackbone atoms (backbone = C,N,O,Ca) for another 2000 steps The entire system was then heated over 20 000 steps (20 ps) from 60 °K at 10 °K increments every 500 steps using velocity reassignment All atoms were then minimized (2000 steps), followed by another heating step as described, but for 200 ps In the second protocol, all steps were the same except that, during the first heating, only solvent atoms were allowed to move During heating, timesteps were fs and a Langevin piston (piston period 100 fs, piston decay 50 fs) was used to maintain pressure at one atmosphere Two different protocols were used during the production runs In the first protocol, a Langevin piston was used to maintain pressure at one atmosphere (NP ensemble) with a piston period of 200 fs and a piston decay of 100 fs, whereas temperature was left to fluctuate In the second protocol, Langevin dynamics was used to also maintain temperature at 310 °K (NPT ensemble) with a damping constant of ps)1 The first protocol was used in conjunction with the first heating protocol and the second sampling protocol was used with the second heating protocol described above Total run lengths for this stage were in the range 20–50 ns In accordance with the results of the initial simulations and site-directed mutagenesis, various mutants of the model were also simulated Mutations were introduced using scwrl [74] into the homology model followed by addition of hydrogens and molecular dynamics according to the second sampling protocol described above with Mg2+ included A number of Linux clusters were used for molecular dynamics simulations These clusters include a 64 processor Gentoo Linux cluster of Pentium IV processors (University of Pretoria), Clusters of Intel XeonÒ, AMD OpteronÒ or Intel Itanium2Ò processors (Bio-Medical Informatics Centre, Meraka Institute, Council for Scientific and Industrial Research) running Scientific Linux In both cases, the interconnect comprises GigaBit Ethernet During the set-up phases of a new national supercomputer at The Centre for High Performance Computing (Cape Town, South Africa), temporary access was granted to the iQudu cluster The iQudu hardware comprises Multicore AMD OpteronÒ processors with InfiniBand interconnect Activity and oligomeric status of mutant Glu295x and Arg404y arginase Simulations with the arginase model suggested further experiments to be performed on the recombinant enzyme The mutations Glu295 Ala, Glu295 Arg, Arg404 Ala, as well as the double mutation Glu259x Ala ⁄ Arg404y Ala, were introduced into the recombinant PfArg by Novel interaction in plasmodial arginase site-directed mutagenesis; activity and oligomeric status were detected as described previously [24] Briefly, PfArg was cloned into the pASK-IBA3 expression vector with a C-terminal Strep-tag (Institut fur Bioanalytik, Gottingen, ă ă Germany) for affinity purification The construct was transformed and expressed in Escherichia coli BL21-CodonPlusÔ (DE3)-RIL (Stratagene, Amsterdam, The Netherlands) A single colony was picked and grown overnight in LB medium The bacterial culture was diluted : 50 and grown at 37 °C until A600 of 0.5 was reached The expression was initiated with 200 ngỈmL)1 of anhydrotetracycline and the cells were grown for h at 37 °C before being harvested The cell pellet was resuspended in 100 mm Tris–HCl, 150 mm NaCl, pH 8.0, containing 0.1 mm phenylmethanesulfonyl fluoride, sonicated, and centrifuged at 100 000 g for h at 48 °C Strep-tag fusion protein was purified according to the manufacturers recommendations (Institut fur Bioanalytik) ă Site-directed mutagenesis was carried out according to the manufacturer’s recommendations (QuikChange protocol; Stratagene, Amsterndam, The Netherlands) All mutations were verified by nucleotide sequencing using the Sanger dideoxy chain termination reaction for doublestranded DNA [75] The purification and expression of the protein variants were carried out as described above The arginase activity was assayed by measuring the formation of urea in a colorimetric method with a-isonitroso propiophenone at a wavelength of 540 nm as described previously [76] The standard assay was carried out in 50 mm Tris–HCl, pH 8.0, mm dithiothreitol, mm MnCl2 and 30 mm arginine in a total volume of 750 mL l-arginine levels up to 200 mm were used to determine the Km values The molecular size and oligomeric state of PfArg were assessed by subjecting the affinity-purified protein to FPLC on a calibrated Superdex S-200 column (GE Healthcare, Munich, Germany) equilibrated with 50 mm Tris–HCl, pH 8.0, 150 mm NaCl, mm dithiothreitol, mm MnCl2 PfArg was detected in aliquots of each fraction using Western dotblotting, and a monoclonal antibody against Strep-tag (Institut fur Bioanalytik) at a dilution of : 5000 Antiă mouse-horseradish peroxidase-coupled secondary serum (Invitrogen, Karlsruhe, Germany) was used for detection at a dilution of : 5000 Acknowledgements Temporary access was granted to the iQudu cluster at the Centre for High Performance Computing, Meraka Institute, Council for Scientific and Industrial Research, Cape Town, South Africa Access was also granted to the computer clusters at the BioMedical Informatics Centre, Meraka Institute, Council 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Cold Spring Harbor, NY 76 Archibald RM (1945) Colorimetric determination of urea J Biol Chem 157, 507–518 FEBS Journal 276 (2009) 3517–3530 ª 2009 The Authors Journal compilation ª 2009 FEBS ... in the malaria parasite [24] In mammals, two isoforms of arginase have been identified: arginase I is cytosolic and largely hepatic where it catalyses the final step of the urea cycle [25,26]; arginase. .. the mammalian and bacterial templates In mammalian arginases, the Asp223 ⁄ 204x (rat arginase I ⁄ human arginase II) cognate forms an inter-monomer salt-bridge with Arg308 ⁄ 327y This salt-bridge. .. enzymes: a promising approach to therapy of African sleeping sickness, Chagas’ disease, and leishmaniasis Amino Acids 33, 359–366 12 Wallace HM (2007) Targeting polyamine metabolism: a viable therapeutic