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1.  Structure-Based Design and Synthesis of Antiparasitic Pyrrolopyrimidines Targeting Pteridine Reductase 1 
Journal of Medicinal Chemistry  2014;57(15):6479-6494.
The treatment of Human African trypanosomiasis remains a major unmet health need in sub-Saharan Africa. Approaches involving new molecular targets are important; pteridine reductase 1 (PTR1), an enzyme that reduces dihydrobiopterin in Trypanosoma spp., has been identified as a candidate target, and it has been shown previously that substituted pyrrolo[2,3-d]pyrimidines are inhibitors of PTR1 from Trypanosoma brucei (J. Med. Chem.2010, 53, 221–229). In this study, 61 new pyrrolo[2,3-d]pyrimidines have been prepared, designed with input from new crystal structures of 23 of these compounds complexed with PTR1, and evaluated in screens for enzyme inhibitory activity against PTR1 and in vitro antitrypanosomal activity. Eight compounds were sufficiently active in both screens to take forward to in vivo evaluation. Thus, although evidence for trypanocidal activity in a stage I disease model in mice was obtained, the compounds were too toxic to mice for further development.
doi:10.1021/jm500483b
PMCID: PMC4136963  PMID: 25007262
2.  High-resolution structure of the M14-type cytosolic carboxypeptidase from Burkholderia cenocepacia refined exploiting PDB_REDO strategies 
The structure of a bacterial M14-family carboxypeptidase determined exploiting microfocus synchrotron radiation and highly automated refinement protocols reveals its potential to act as a polyglutamylase.
A potential cytosolic metallocarboxypeptidase from Burk­holderia cenocepacia has been crystallized and a synchrotron-radiation microfocus beamline allowed the acquisition of diffraction data to 1.9 Å resolution. The asymmetric unit comprises a tetramer containing over 1500 amino acids, and the high-throughput automated protocols embedded in PDB_REDO were coupled with model–map inspections in refinement. This approach has highlighted the value of such protocols for efficient analyses. The subunit is constructed from two domains. The N-terminal domain has previously only been observed in cytosolic carboxypeptidase (CCP) proteins. The C-terminal domain, which carries the Zn2+-containing active site, serves to classify this protein as a member of the M14D subfamily of carboxypeptidases. Although eukaryotic CCPs possess deglutamylase activity and are implicated in processing modified tubulin, the function and substrates of the bacterial family members remain unknown. The B. cenocepacia protein did not display deglutamylase activity towards a furylacryloyl glutamate derivative, a potential substrate. Residues previously shown to coordinate the divalent cation and that contribute to peptide-bond cleavage in related enzymes such as bovine carboxypeptidase are conserved. The location of a conserved basic patch in the active site adjacent to the catalytic Zn2+, where an acetate ion is identified, suggests recognition of the carboxy-terminus in a similar fashion to other carboxypeptidases. However, there are significant differences that indicate the recognition of substrates with different properties. Of note is the presence of a lysine in the S1′ recognition subsite that suggests specificity towards an acidic substrate.
doi:10.1107/S1399004713026801
PMCID: PMC3940198  PMID: 24531462
carboxypeptidases; metalloproteins; refinement; specificity; zinc enzymes
3.  Two high-resolution structures of the human E3 ubiquitin ligase Siah1 
Two structures of human Siah1 at 1.95 and 1.58 Å resolution provide more complete models for this protein and identify conformational variability in the subdomain organization.
Siah1 is an E3 ubiquitin ligase that contributes to proteasome-mediated degradation of multiple targets in key cellular processes and which shows promise as a therapeutic target in oncology. Structures of a truncated Siah1 bound to peptide-based inhibitors have been reported. Here, new crystallization conditions have allowed the determination of a construct encompassing dual zinc-finger subdomains and substrate-binding domains at significantly higher resolution. Although the crystals appear isomorphous, two structures present distinct states in which the spatial orientation of one zinc-finger subdomain differs with respect to the rest of the dimeric protein. Such a difference, which is indicative of conformational freedom, infers potential biological relevance related to recognition of binding partners. The crystallization conditions and improved models of Siah1 may aid future studies investigating Siah1–ligand complexes.
doi:10.1107/S1744309113031448
PMCID: PMC3855715  PMID: 24316825
E3 ubiquitin ligase; seven-in-absentia homologue 1 (Siah1); zinc finger
4.  Structural basis for type VI secreted peptidoglycan dl-endopeptidase function, specificity and neutralization in Serratia marcescens  
Crystal structures of type VI secretion system-associated immunity proteins, a peptidoglycan endopeptidase and a complex of the endopeptidase and its cognate immunity protein are reported together with assays of endopeptidase activity and functional assessment.
Some Gram-negative bacteria target their competitors by exploiting the type VI secretion system to extrude toxic effector proteins. To prevent self-harm, these bacteria also produce highly specific immunity proteins that neutralize these antagonistic effectors. Here, the peptidoglycan endopeptidase specificity of two type VI secretion-system-associated effectors from Serratia marcescens is characterized. These small secreted proteins, Ssp1 and Ssp2, cleave between γ-d-glutamic acid and l-meso-diaminopimelic acid with different specificities. Ssp2 degrades the acceptor part of cross-linked tetratetrapeptides. Ssp1 displays greater promiscuity and cleaves monomeric tripeptides, tetrapeptides and pentapeptides and dimeric tetratetra and tetrapenta muropeptides on both the acceptor and donor strands. Functional assays confirm the identity of a catalytic cysteine in these endopeptidases and crystal structures provide information on the structure–activity relationships of Ssp1 and, by comparison, of related effectors. Functional assays also reveal that neutralization of these effectors by their cognate immunity proteins, which are called resistance-associated proteins (Raps), contributes an essential role to cell fitness. The structures of two immunity proteins, Rap1a and Rap2a, responsible for the neutralization of Ssp1 and Ssp2-like endopeptidases, respectively, revealed two distinct folds, with that of Rap1a not having previously been observed. The structure of the Ssp1–Rap1a complex revealed a tightly bound heteromeric assembly with two effector molecules flanking a Rap1a dimer. A highly effective steric block of the Ssp1 active site forms the basis of effector neutralization. Comparisons with Ssp2–Rap2a orthologues suggest that the specificity of these immunity proteins for neutralizing effectors is fold-dependent and that in cases where the fold is conserved sequence differences contribute to the specificity of effector–immunity protein interactions.
doi:10.1107/S0907444913022725
PMCID: PMC3852654  PMID: 24311588
amidases; cysteine proteases; disulfide linkage; effector; endopeptidases; Gram-negative; immunity protein; peptidoglycan; Serratia marcescens; type VI secretion system
5.  Structure of the SCAN Domain of Human Paternally Expressed Gene 3 Protein 
PLoS ONE  2013;8(7):e69538.
Human paternally expressed gene 3 protein (PEG3) is a large multi-domain entity with diverse biological functions, including acting as a transcription factor. PEG3 contains twelve Cys2-His2 type zinc finger domains, extended regions of predicted disorder and at the N-terminus a SCAN domain. PEG3 has been identified as partner of the E3 ubiquitin-protein ligase Siah1, an association we sought to investigate. An efficient bacterial recombinant expression system of the human PEG3-SCAN domain was prepared and crystals appeared spontaneously when the protein was being concentrated after purification. The structure was determined at 1.95 Å resolution and reveals a polypeptide fold of five helices in an extended configuration. An extensive dimerization interface, using almost a quarter of the solvent accessible surface, and key salt bridge interactions explain the stability of the dimer. Comparison with other SCAN domains reveals a high degree of conservation involving residues that contribute to the dimer interface. The PEG3-SCAN domain appears to constitute an assembly block, enabling PEG3 homo- or heterodimerization to control gene expression in a combinatorial fashion.
doi:10.1371/journal.pone.0069538
PMCID: PMC3720700  PMID: 23936039
6.  Structure of diaminohydroxyphosphoribosylaminopyrimidine deaminase/5-amino-6-(5-phospho­ribosylamino)uracil reductase from Acinetobacter baumannii  
The structure of a bifunctional deaminase/reductase involved in riboflavin biosynthesis in the pathogen A. baumannii has been determined in two crystal forms.
The bifunctional diaminohydroxyphosphoribosylaminopyrimidine deaminase/5-­amino-6-(5-phosphoribosylamino)uracil reductase (RibD) represents a potential antibacterial drug target. The structure of recombinant Acinetobacter baumannii RibD is reported in orthorhombic and tetragonal crystal forms at 2.2 and 2.0 Å resolution, respectively. Comparisons with orthologous structures in the Protein Data Bank indicated close similarities. The tetragonal crystal form was obtained in the presence of guanosine monophosphate, which surprisingly was observed to occupy the adenine-binding site of the reductase domain.
doi:10.1107/S174430911301292X
PMCID: PMC3668577  PMID: 23722836
bifunctional deaminase/reductase; Acinetobacter baumannii; RibD; riboflavin biosynthesis
7.  Specificity and Reactivity in Menaquinone Biosynthesis: The Structure of Escherichia coli MenD (2-Succinyl-5-Enolpyruvyl-6-Hydroxy-3-Cyclohexadiene-1-Carboxylate Synthase) 
Journal of molecular biology  2008;384(5):1353-1368.
The thiamine diphosphate (ThDP) and metal-ion-dependent enzyme 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase, or MenD, catalyze the Stetter-like conjugate addition of α-ketoglutarate with isochorismate to release 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate and carbon dioxide. This reaction represents the first committed step for biosynthesis of menaquinone, or vitamin K2, a key cofactor for electron transport in bacteria and a metabolite for posttranslational modification of proteins in mammals. The medium-resolution structure of MenD from Escherichia coli (EcMenD) in complex with its cofactor and Mn2+ has been determined in two related hexagonal crystal forms. The subunit displays the typical three-domain structure observed for ThDP-dependent enzymes in which two of the domains bind and force the cofactor into a configuration that supports formation of a reactive ylide. The structures reveal a stable dimer-of-dimers association in agreement with gel filtration and analytical ultracentrifugation studies and confirm the classification of MenD in the pyruvate oxidase family of ThDP-dependent enzymes. The active site, created by contributions from a pair of subunits, is highly basic with a pronounced hydrophobic patch. These features, formed by highly conserved amino acids, match well to the chemical properties of the substrates. A model of the covalent intermediate formed after reaction with the first substrate α-ketoglutarate and with the second substrate isochorismate positioned to accept nucleophilic attack has been prepared. This, in addition to structural and sequence comparisons with putative MenD orthologues, provides insight into the specificity and reactivity of MenD and allows a two-stage reaction mechanism to be proposed.
doi:10.1016/j.jmb.2008.10.048
PMCID: PMC3656419  PMID: 18983854
crystal structure; enzyme mechanism; menaquinone biosynthesis; thiamine diphosphate cofactor
8.  High-resolution structures of Trypanosoma brucei pteridine reductase ligand complexes inform on the placement of new molecular entities in the active site of a potential drug target 
Pteridine reductase (PTR1) is a potential target for drug development against parasitic Trypanosoma and Leishmania species. These protozoa cause serious diseases for which current therapies are inadequate. High-resolution structures have been determined, using data between 1.6 and 1.1 Å resolution, of T. brucei PTR1 in complex with pemetrexed, trimetrexate, cyromazine and a 2,4-diaminopyrimidine derivative. The structures provide insight into the interactions formed by new molecular entities in the enzyme active site with ligands that represent lead compounds for structure-based inhibitor development and to support early-stage drug discovery.
doi:10.1107/S0907444910040886
PMCID: PMC3655514  PMID: 21123874
9.  Acinetobacter baumannii FolD ligand complexes – potent inhibitors of folate metabolism and a re-evaluation of the structure of LY374571 
The FEBS journal  2012;279(23):4350-4360.
The bifunctional N5,N10-methylenetetrahydrofolate dehydrogenase/cyclohydrolase (DHCH or FolD), which is widely distributed in prokaryotes and eukaryotes, is involved in the biosynthesis of folate cofactors that are essential for growth and cellular development. The enzyme activities represent a potential antimicrobial drug target. We have characterized the kinetic properties of FolD from the Gram-negative pathogen Acinetobacter baumanni and determined high-resolution crystal structures of complexes with a cofactor and two potent inhibitors. The data reveal new details with respect to the molecular basis of catalysis and potent inhibition. A unexpected finding was that our crystallographic data revealed a different structure for LY374571 (an inhibitor studied as an antifolate) than that previously published. The implications of this observation are discussed.
doi:10.1111/febs.12025
PMCID: PMC3655515  PMID: 23050773
antifolate; cyclohydrolase; dehydrogenase; enzyme inhibition; X-ray structure
10.  The role of Co2+ in the crystallization of human SENP1 and comments on the limitations of automated refinement protocols 
Crystallographic analysis of the human SENP1 catalytic domain identified a well ordered Co2+ ion that contributes to intermolecular interactions relevant to crystallization of the enzyme. The presence of this ion was overlooked in previous studies.
Metal ions often stabilize intermolecular contacts between macromolecules, thereby promoting crystallization. When interpreting a medium-resolution electron-density map of the catalytic domain of human sentrin-specific protease 1 (SENP1), a strong feature indicative of an ordered divalent cation was noted. This was assigned as Co2+, an essential component of the crystallization mixture. The ion displays tetrahedral coordination by Glu430 and His640 from one molecule and the corresponding residues from a symmetry-related molecule. Analysis of the data derived from a previous structure of SENP1 suggested that Co2+ had been overlooked and re-refinement supported this conclusion. High-throughput automated re-refinement protocols also failed to mark the Co2+ position, supporting the requirement for the incorporation of as much information as possible to enhance the value of such protocols.
doi:10.1107/S1744309111005835
PMCID: PMC3080145  PMID: 21505236
cobalt; sentrin-specific protease 1; SUMO
11.  Structure of Pseudomonas aeruginosa inosine 5′-monophosphate dehydrogenase 
The crystal structure of inosine 5′-monophosphate dehydrogenase from P. aeruginosa has been determined to 2.25 Å resolution.
Inosine 5′-monophosphate dehydrogenase (IMPDH) represents a potential antimicrobial drug target. The crystal structure of recombinant Pseudomonas aeruginosa IMPDH has been determined to a resolution of 2.25 Å. The structure is a homotetramer of subunits dominated by a (β/α)8-barrel fold, consistent with other known structures of IMPDH. Also in common with previous work, the cystathionine β-synthase domains, residues 92–204, are not present in the model owing to disorder. However, unlike the majority of available structures, clearly defined electron density exists for a loop that creates part of the active site. This loop, composed of residues 297–315, links α8 and β9 and carries the catalytic Cys304. P. aeruginosa IMPDH shares a high level of sequence identity with bacterial and protozoan homologues, with residues involved in binding substrate and the NAD+ cofactor being conserved. Specific differences that have been proven to contribute to selectivity against the human enzyme in a study of Cryptosporidium parvum IMPDH are also conserved, highlighting the potential value of IMPDH as a drug target.
doi:10.1107/S1744309113002352
PMCID: PMC3606566  PMID: 23519796
inosine 5′-monophosphate dehydrogenase; Pseudomonas aeruginosa; antimicrobial drug targets
12.  The AEROPATH project targeting Pseudomonas aeruginosa: crystallographic studies for assessment of potential targets in early-stage drug discovery 
A focused strategy has been directed towards the structural characterization of selected proteins from the bacterial pathogen P. aeruginosa. The objective is to exploit the resulting structural data, in combination with ligand-binding studies, and to assess the potential of these proteins for early-stage antimicrobial drug discovery.
Bacterial infections are increasingly difficult to treat owing to the spread of antibiotic resistance. A major concern is Gram-negative bacteria, for which the discovery of new antimicrobial drugs has been particularly scarce. In an effort to accelerate early steps in drug discovery, the EU-funded AEROPATH project aims to identify novel targets in the opportunistic pathogen Pseudomonas aeruginosa by applying a multidisciplinary approach encompassing target validation, structural characterization, assay development and hit identification from small-molecule libraries. Here, the strategies used for target selection are described and progress in protein production and structure analysis is reported. Of the 102 selected targets, 84 could be produced in soluble form and the de novo structures of 39 proteins have been determined. The crystal structures of eight of these targets, ranging from hypothetical unknown proteins to metabolic enzymes from different functional classes (PA1645, PA1648, PA2169, PA3770, PA4098, PA4485, PA4992 and PA5259), are reported here. The structural information is expected to provide a firm basis for the improvement of hit compounds identified from fragment-based and high-throughput screening campaigns.
doi:10.1107/S1744309112044739
PMCID: PMC3539698  PMID: 23295481
protein structure; Gram-negative bacteria; Pseudomonas aeruginosa; infectious diseases; structure-based inhibitor design
13.  Two interacting binding sites for quinacrine derivatives in the active site of trypanothione reductase – a template for drug design 
The Journal of biological chemistry  2004;279(28):29493-29500.
SUMMARY
Trypanothione reductase is a key enzyme in the trypanothione-based redox metabolism of pathogenic trypanosomes. Since this system is absent in humans, being replaced with glutathione and glutathione reductase, it offers a target for selective inhibition. The rational design of potent inhibitors requires accurate structures of enzyme-inhibitor complexes, but this is lacking for trypanothione reductase. We therefore used quinacrine mustard, an alkylating derivative of the competitive inhibitor quinacrine, to probe the active site of this dimeric flavoprotein. Quinacrine mustard irreversibly inactivates Trypanosoma cruzi trypanothione reductase, but not human glutathione reductase, in a time-dependent manner with a stoichiometry of two inhibitors bound per monomer. The rate of inactivation is dependent upon the oxidation state of trypanothione reductase, with the NADPH-reduced form being inactivated significantly faster than the oxidised form. Inactivation is slowed by clomipramine and a melarsen oxide-trypanothione adduct (both are competitive inhibitors) but accelerated by quinacrine. The structure of the trypanothione reductase-quinacrine mustard adduct was determined to 2.7 Å, revealing two molecules of inhibitor bound in the trypanothione-binding site. The acridine moieties interact with each other through π-stacking effects, and one acridine interacts in a similar fashion with a tryptophan residue. These interactions provide a molecular explanation for the differing effects of clomipramine and quinacrine on inactivation by quinacrine mustard. Synergism with quinacrine occurs as a result of these planar acridines being able to stack together in the active site cleft, thereby gaining an increased number of binding interactions, whereas antagonism occurs with non-planar molecules, such as clomipramine, where stacking is not possible.
doi:10.1074/jbc.M403187200
PMCID: PMC3491871  PMID: 15102853
enzyme-inhibitor complex; Trypanosoma cruzi; trypanothione reductase; quinacrine mustard; X-ray diffraction
14.  Isoprenoid precursor biosynthesis offers potential targets for drug discovery against diseases caused by apicomplexan parasites 
Current topics in medicinal chemistry  2011;11(16):2048-2059.
Two, simple, C5 compounds, dimethylally diphosphate and isopentenyl diphosphate, are the universal precursors of isoprenoids, a large family of natural products involved in numerous important biological processes. Two distinct biosynthetic pathways have evolved to supply these precursors. Humans use the mevalonate route whilst many species of bacteria including important pathogens, plant chloroplasts and apicomplexan parasites exploit the non-mevalonate pathway. The absence from humans, combined with genetic and chemical validation suggests that the non-mevalonate pathway holds the potential to support new drug discovery programmes targeting Gram-negative bacteria and the apicomplexan parasites responsible for causing serious human diseases, and also infections of veterinary importance. The non-mevalonate pathway relies on eight enzyme-catalyzed stages exploiting a range of cofactors and metal ions. A wealth of structural and mechanistic data, mainly derived from studies of bacterial enzymes, now exists for most components of the pathway and these will be described. Particular attention will be paid to how these data inform on the apicomplexan orthologues concentrating on the enzymes from Plasmodium spp.; these cause malaria, one the most important parasitic diseases in the world today.
PMCID: PMC3466564  PMID: 21619509
Antimicrobial drug discovery; isoprenoid precursor biosynthesis; malaria; structure-based inhibitor discovery; toxoplasmosis
16.  Structure of Staphylococcus aureus EsxA suggests a contribution to virulence by action as a transport chaperone and/or adaptor protein 
Journal of molecular biology  2008;383(3):603-614.
Staphylococcus aureus pathogenesis depends on a specialized protein secretion system, ESX-1, that delivers a range of virulence factors to assist infectivity. We report the characterization of two such factors, EsxA and EsxB; small acidic dimeric proteins carrying a distinctive WXG motif. EsxA crystallized in triclinic and monoclinic forms and high-resolution structures were determined. The asymmetric unit of each crystal form is a dimer. The EsxA subunit forms an elongated cylindrical structure created from side-by-side α-helices linked with a hairpin bend formed by the WXG motif. Approximately 25% of the solvent accessible surface area of each subunit is involved in interactions, predominantly hydrophobic, with the partner subunit. Secondary structure predictions suggest that EsxB displays a similar structure. The WXG motif helps to create a shallow cleft at each end of the dimer, forming a short β-sheet-like feature with an N-terminal segment of the partner subunit. Structural and sequence comparisons, exploiting biological data on related proteins found in Mycobacteria tuberculosis suggest that this family of proteins may contribute to pathogenesis by transporting protein cargo through the ESX-1 system exploiting a C-terminal secretion signal and / or are capable of acting as adaptor proteins to facilitate interactions with host receptor proteins.
doi:10.1016/j.jmb.2008.08.047
PMCID: PMC3465917  PMID: 18773907
adaptor protein; chaperone; helical bundle; secretion system; virulence factor
17.  Structure of Leishmania major cysteine synthase 
A crystallographic and biochemical study of L. major cysteine synthase, which is a pyridoxyl phosphate-dependent enzyme, is reported. The structure was determined to 1.8 Å resolution and revealed that the cofactor has been lost and that a fragment of γ-poly-d-glutamic acid, a crystallization ingredient, was bound in the active site. The enzyme was inhibited by peptides.
Cysteine biosynthesis is a potential target for drug development against parasitic Leishmania species; these protozoa are responsible for a range of serious diseases. To improve understanding of this aspect of Leishmania biology, a crystallographic and biochemical study of L. major cysteine synthase has been undertaken, seeking to understand its structure, enzyme activity and modes of inhibition. Active enzyme was purified, assayed and crystallized in an orthorhombic form with a dimer in the asymmetric unit. Diffraction data extending to 1.8 Å resolution were measured and the structure was solved by molecular replacement. A fragment of γ-poly-d-glutamic acid, a constituent of the crystallization mixture, was bound in the enzyme active site. Although a d-­glutamate tetrapeptide had insignificant inhibitory activity, the enzyme was competitively inhibited (K i = 4 µM) by DYVI, a peptide based on the C-­terminus of the partner serine acetyltransferase with which the enzyme forms a complex. The structure surprisingly revealed that the cofactor pyridoxal phosphate had been lost during crystallization.
doi:10.1107/S1744309112019124
PMCID: PMC3388911  PMID: 22750854
Arabidopsis thaliana; cysteine synthase; Leishmania major
18.  Assessment of Pseudomonas aeruginosa N5,N10-Methylenetetrahydrofolate Dehydrogenase - Cyclohydrolase as a Potential Antibacterial Drug Target 
PLoS ONE  2012;7(4):e35973.
The bifunctional enzyme methylenetetrahydrofolate dehydrogenase – cyclohydrolase (FolD) is identified as a potential drug target in Gram-negative bacteria, in particular the troublesome Pseudomonas aeruginosa. In order to provide a comprehensive and realistic assessment of the potential of this target for drug discovery we generated a highly efficient recombinant protein production system and purification protocol, characterized the enzyme, carried out screening of two commercial compound libraries by differential scanning fluorimetry, developed a high-throughput enzyme assay and prosecuted a screening campaign against almost 80,000 compounds. The crystal structure of P. aeruginosa FolD was determined at 2.2 Å resolution and provided a template for an assessment of druggability and for modelling of ligand complexes as well as for comparisons with the human enzyme. New FolD inhibitors were identified and characterized but the weak levels of enzyme inhibition suggest that these compounds are not optimal starting points for future development. Furthermore, the close similarity of the bacterial and human enzyme structures suggest that selective inhibition might be difficult to attain. In conclusion, although the preliminary biological data indicates that FolD represents a valuable target for the development of new antibacterial drugs, indeed spurred us to investigate it, our screening results and structural data suggest that this would be a difficult enzyme to target with respect to developing the appropriate lead molecules required to underpin a serious drug discovery effort.
doi:10.1371/journal.pone.0035973
PMCID: PMC3338484  PMID: 22558288
19.  IspE Inhibitors Identified by a Combination of In Silico and In Vitro High-Throughput Screening 
PLoS ONE  2012;7(4):e35792.
CDP-ME kinase (IspE) contributes to the non-mevalonate or deoxy-xylulose phosphate (DOXP) pathway for isoprenoid precursor biosynthesis found in many species of bacteria and apicomplexan parasites. IspE has been shown to be essential by genetic methods and since it is absent from humans it constitutes a promising target for antimicrobial drug development. Using in silico screening directed against the substrate binding site and in vitro high-throughput screening directed against both, the substrate and co-factor binding sites, non-substrate-like IspE inhibitors have been discovered and structure-activity relationships were derived. The best inhibitors in each series have high ligand efficiencies and favourable physico-chemical properties rendering them promising starting points for drug discovery. Putative binding modes of the ligands were suggested which are consistent with established structure-activity relationships. The applied screening methods were complementary in discovering hit compounds, and a comparison of both approaches highlights their strengths and weaknesses. It is noteworthy that compounds identified by virtual screening methods provided the controls for the biochemical screens.
doi:10.1371/journal.pone.0035792
PMCID: PMC3340893  PMID: 22563402
20.  The crystal structure of Leishmania major N5,N10-methylenetetrahydrofolate dehydrogenase/cyclohydrolase and assessment of a potential drug target☆ 
Molecular and Biochemical Parasitology  2012;181(2-6):178-185.
Graphical abstract
The crystal structure of Leishmania major N5,N10-methylenetetrahydrofolate dehydrogenase/N5,N10-methenyltetrahydrofolate cyclohydrolase is used to assess the potential of this bifunctional enzyme as a drug target.
Highlights
► We report the structure of Leishmania major methylenetetrahydrofolate dehydrogenase/cyclohydrolase. ► Sequence–structure comparisons are carried out with homologues from kinetoplastids and the human host. ► The potential of this bifunctional enzyme as a drug target is assessed. ► The similarities between parasite and human enzymes suggest a difficult target for drug discovery.
Three enzyme activities in the protozoan Leishmania major, namely N5,N10-methylenetetrahydrofolate dehydrogenase/N5,N10-methenyltetrahydrofolate cyclohydrolase (DHCH) and N10-formyltetrahydrofolate ligase (FTL) produce the essential intermediate N10-formyltetrahydrofolate. Although trypanosomatids possess at least one functional DHCH, the same is not true for FTL, which is absent in Trypanosoma brucei. Here, we present the 2.7 Å resolution crystal structure of the bifunctional apo-DHCH from L. major, which is a potential drug target. Sequence alignments show that the cytosolic enzymes found in trypanosomatids share a high level of identity of approximately 60%. Additionally, residues that interact and participate in catalysis in the human homologue are conserved amongst trypanosomatid sequences and this may complicate attempts to derive potent, parasite specific DHCH inhibitors.
doi:10.1016/j.molbiopara.2011.11.004
PMCID: PMC3368264  PMID: 22108435
Antifolate; Cyclohydrolase; Dehydrogenase; Drug target; Leishmania; Trypanosoma
21.  The structure of Serratia marcescens Lip, a membrane-bound component of the type VI secretion system 
The high-resolution crystal structure of S. marcescens Lip reveals a new member of the transthyretin family of proteins. Lip, a core component of the type VI secretion apparatus, is localized to the outer membrane and is positioned to interact with other proteins forming this complex system.
Lip is a membrane-bound lipoprotein and a core component of the type VI secretion system found in Gram-negative bacteria. The structure of a Lip construct (residues 29–176) from Serratia marcescens (SmLip) has been determined at 1.92 Å resolution. Experimental phases were derived using a single-wavelength anomalous dispersion approach on a sample cocrystallized with iodide. The membrane localization of the native protein was confirmed. The structure is that of the globular domain lacking only the lipoprotein signal peptide and the lipidated N-terminus of the mature protein. The protein fold is dominated by an eight-stranded β-sandwich and identifies SmLip as a new member of the transthyretin family of proteins. Transthyretin and the only other member of the family fold, 5-hydroxyisourate hydrolase, form homo­tetramers important for their function. The asymmetric unit of SmLip is a tetramer with 222 symmetry, but the assembly is distinct from that previously noted for the transthyretin protein family. However, structural comparisons and bacterial two-hybrid data suggest that the SmLip tetramer is not relevant to its role as a core component of the type VI secretion system, but rather reflects a propensity for SmLip to participate in protein–protein interactions. A relatively low level of sequence conservation amongst Lip homologues is noted and is restricted to parts of the structure that might be involved in interactions with physiological partners.
doi:10.1107/S0907444911046300
PMCID: PMC3225178  PMID: 22120744
β-sandwich; Gram-negative pathogens; lipoproteins; protein secretion; transthyretin; virulence
22.  Pseudomonas aeruginosa 4-Amino-4-Deoxychorismate Lyase: Spatial Conservation of an Active Site Tyrosine and Classification of Two Types of Enzyme 
PLoS ONE  2011;6(9):e24158.
4-Amino-4-deoxychorismate lyase (PabC) catalyzes the formation of 4-aminobenzoate, and release of pyruvate, during folate biosynthesis. This is an essential activity for the growth of Gram-negative bacteria, including important pathogens such as Pseudomonas aeruginosa. A high-resolution (1.75 Å) crystal structure of PabC from P. aeruginosa has been determined, and sequence-structure comparisons with orthologous structures are reported. Residues around the pyridoxal 5′-phosphate cofactor are highly conserved adding support to aspects of a mechanism generic for enzymes carrying that cofactor. However, we suggest that PabC can be classified into two groups depending upon whether an active site and structurally conserved tyrosine is provided from the polypeptide that mainly forms an active site or from the partner subunit in the dimeric assembly. We considered that the conserved tyrosine might indicate a direct role in catalysis: that of providing a proton to reduce the olefin moiety of substrate as pyruvate is released. A threonine had previously been suggested to fulfill such a role prior to our observation of the structurally conserved tyrosine. We have been unable to elucidate an experimentally determined structure of PabC in complex with ligands to inform on mechanism and substrate specificity. Therefore we constructed a computational model of the catalytic intermediate docked into the enzyme active site. The model suggests that the conserved tyrosine helps to create a hydrophobic wall on one side of the active site that provides important interactions to bind the catalytic intermediate. However, this residue does not appear to participate in interactions with the C atom that undergoes an sp2 to sp3 conversion as pyruvate is produced. The model and our comparisons rather support the hypothesis that an active site threonine hydroxyl contributes a proton used in the reduction of the substrate methylene to pyruvate methyl in the final stage of the mechanism.
doi:10.1371/journal.pone.0024158
PMCID: PMC3174152  PMID: 21935381
23.  Crystal structure of Leishmania major ADP ribosylation factor-like 1 and a classification of related GTPase family members in this Kinetoplastid 
ADP ribosylation factor-like (ARL) proteins are small GTPases that undergo conformational changes upon nucleotide binding, and which regulate the affinity of ARLs for binding other proteins, lipids or membranes. There is a paucity of structural data on this family of proteins in the Kinetoplastida, despite studies implicating them in key events related to vesicular transport and regulation of microtubule dependent processes. The crystal structure of Leishmania major ARL1 in complex with GDP has been determined to 2.1 Å resolution and reveals a high degree of structural conservation with human ADP ribosylation factor 1 (ARF1). Putative L. major and Trypanosoma brucei ARF/ARL family members have been classified based on structural considerations, amino acid sequence conservation combined with functional data on Kinetoplastid and human orthologues. This classification may guide future studies designed to elucidate the function of specific family members.
doi:10.1016/j.molbiopara.2010.08.002
PMCID: PMC3065712  PMID: 20801163
ADP ribosylation factor-like; GTPase; Leishmania; protein structure
24.  A triclinic crystal form of Escherichia coli 4-diphosphocytidyl-2C-methyl-d-erythritol kinase and reassessment of the quaternary structure 
The structure of a triclinic crystal form of 4-diphosphocytidyl-2C-methyl-d-erythritol kinase has been determined. Comparisons with a previously reported monoclinic crystal form raise questions about our knowledge of the quaternary structure of this enzyme.
4-Diphosphocytidyl-2C-methyl-d-erythritol kinase (IspE; EC 2.7.1.148) contributes to the 1-deoxy-d-xylulose 5-phosphate or mevalonate-independent biosynthetic pathway that produces the isomers isopentenyl diphosphate and dimethylallyl diphosphate. These five-carbon compounds are the fundamental building blocks for the biosynthesis of isoprenoids. The mevalonate-independent pathway does not occur in humans, but is present and has been shown to be essential in many dangerous pathogens, i.e. Plasmodium species, which cause malaria, and Gram-negative bacteria. Thus, the enzymes involved in this pathway have attracted attention as potential drug targets. IspE produces 4-­diphosphos­phocytidyl-2C-methyl-d-erythritol 2-phosphate by ATP-dependent phosphorylation of 4-diphosphocytidyl-2C-methyl-d-erythritol. A triclinic crystal structure of the Escherichia coli IspE–ADP complex with two molecules in the asymmetric unit was determined at 2 Å resolution and compared with a monoclinic crystal form of a ternary complex of E. coli IspE also with two molecules in the asymmetric unit. The molecular packing is different in the two forms. In the asymmetric unit of the triclinic crystal form the substrate-binding sites of IspE are occluded by structural elements of the partner, suggesting that the ‘triclinic dimer’ is an artefact of the crystal lattice. The surface area of interaction in the triclinic form is almost double that observed in the monoclinic form, implying that the dimeric assembly in the monoclinic form may also be an artifact of crystallization.
doi:10.1107/S1744309109054591
PMCID: PMC2833027  PMID: 20208151
mevalonate-independent pathway; isoprenoid biosynthesis; kinases
25.  Crystal Structures of Penicillin-Binding Protein 3 from Pseudomonas aeruginosa: Comparison of Native and Antibiotic-Bound Forms 
Journal of Molecular Biology  2011;405(1-3):173-184.
We report the first crystal structures of a penicillin-binding protein (PBP), PBP3, from Pseudomonas aeruginosa in native form and covalently linked to two important β-lactam antibiotics, carbenicillin and ceftazidime. Overall, the structures of apo and acyl complexes are very similar; however, variations in the orientation of the amino-terminal membrane-proximal domain relative to that of the carboxy-terminal transpeptidase domain indicate interdomain flexibility. Binding of either carbenicillin or ceftazidime to purified PBP3 increases the thermostability of the enzyme significantly and is associated with local conformational changes, which lead to a narrowing of the substrate-binding cleft. The orientations of the two β-lactams in the active site and the key interactions formed between the ligands and PBP3 are similar despite differences in the two drugs, indicating a degree of flexibility in the binding site. The conserved binding mode of β-lactam-based inhibitors appears to extend to other PBPs, as suggested by a comparison of the PBP3/ceftazidime complex and the Escherichia coli PBP1b/ceftoxamine complex. Since P. aeruginosa is an important human pathogen, the structural data reveal the mode of action of the frontline antibiotic ceftazidime at the molecular level. Improved drugs to combat infections by P. aeruginosa and related Gram-negative bacteria are sought and our study provides templates to assist that process and allows us to discuss new ways of inhibiting PBPs.
doi:10.1016/j.jmb.2010.10.024
PMCID: PMC3025346  PMID: 20974151
PBP, penicillin-binding protein; HMM, high molecular mass; LMM, low molecular mass; PDB, Protein Data Bank; ESRF, European Synchrotron Radiation Facility; anti-bacterial; Pseudomonas aeruginosa; carbenicillin; ceftazidime; enzyme structure

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