ABSTRACT
Electron-driven process helps the living organism in the generations of energy, biomass production and detoxification of synthetic compounds. Soluble quinone oxidoreductases (QORs) mediate the transfer of an electron from NADPH to various quinone and other compounds, helping in the detoxification of quinones. QORs play a crucial role in cellular metabolism and are thus potential targets for drug development. Here we report the crystal structure of the NADPH-dependent QOR from Leishmania donovani (LdQOR) at 2.05 Å. The enzyme exists as a homo-dimer, with each protomer consisting of two domains, responsible for binding NADPH cofactor and the substrate. Interestingly, the human QOR exists as a tetramer. Comparative analysis of the oligomeric interfaces of LdQOR with HsQOR shows no significant differences in the protomer/dimer assembly. The tetrameric interface of HsQOR is stabilized by salt bridges formed between Arg 169 and Glu 271 which is non-existent in LdQOR, with an Alanine replacing the glutamate. This distinct feature is conserved across other dimeric QORs, indicating the importance of this interaction for tetramer association. Among the homologs, the sequences of the loop region involved in the stabilization and binding of the adenine ring of the NADPH shows significant differences except for an Arginine & glycine residues. In dimer QORs, this Arginine acts as a gate to the co-factor, while the NADPH binding mode in the human homolog is distinct, stabilized by His 200 and Asn 229, which are not conserved in LdQOR. These distinct features have the potential to be utilized for therapeutic interventions.
Subject(s)
NAD(P)H Dehydrogenase (Quinone) , Quinone Reductases , Humans , NADP/metabolism , Protein Subunits , NAD(P)H Dehydrogenase (Quinone)/metabolism , Quinone Reductases/chemistry , Quinone Reductases/metabolism , Quinones , Arginine , Binding Sites , Crystallography, X-RayABSTRACT
Leishmaniasis is caused by â¼20 species of Leishmania that affects millions in endemic areas. Available therapies are not sufficient to effectively control the disease, cause severe side effects and eventually lead to drug resistance, making the discovery of novel therapeutic molecules an immediate need. Molecular target-based drug discovery, where the target is a defined molecular gene, protein or a mechanism, is a rationale driven approach for novel therapeutics. Humans obtain the essential amino acid such as threonine from dietary sources, while Leishmania synthesize it de-novo. Enzymes of the threonine biosynthesis pathway, including the rate limiting Homoserine kinase (HSK) which converts L-homoserine into ortho-phospho homoserine are thus attractive targets for rationale driven therapy. The absence of HSK in humans and its presence in Leishmania donovani enhances the opportunity to exploit HSK as a molecular target for anti-leishmanials therapeutic development. In this study, we utilize structure-based high throughput drug discovery (SBDD), followed by biochemical validation and identified two potential inhibitors (RH00038 and S02587) from Maybridge chemical library that targets L. donovani HSK. These two inhibitors effectively induced the mortality of Leishmania donovani in both amastigote and promastigote stages, with one of them being specific to parasite and twice as effective as the standard therapeutic molecule.
ABSTRACT
The neglected tropical disease (NTD) leishmaniasis is the collective name given to a diverse group of illnesses caused by ~20 species belonging to the genus Leishmania, a majority of which are vector borne and associated with complex life cycles that cause immense health, social, and economic burdens locally, but individually are not a major global health priority. Therapeutic approaches against leishmaniasis have various inadequacies including drug resistance and a lack of effective control and eradication of the disease spread. Therefore, the development of a rationale-driven, target based approaches towards novel therapeutics against leishmaniasis is an emergent need. The utilization of Artificial Intelligence/Machine Learning methods, which have made significant advances in drug discovery applications, would benefit the discovery process. In this review, following a summary of the disease epidemiology and available therapies, we consider three important leishmanial metabolic pathways that can be attractive targets for a structure-based drug discovery approach towards the development of novel anti-leishmanials. The folate biosynthesis pathway is critical, as Leishmania is auxotrophic for folates that are essential in many metabolic pathways. Leishmania can not synthesize purines de novo, and salvage them from the host, making the purine salvage pathway an attractive target for novel therapeutics. Leishmania also possesses an organelle glycosome, evolutionarily related to peroxisomes of higher eukaryotes, which is essential for the survival of the parasite. Research towards therapeutics is underway against enzymes from the first two pathways, while the third is as yet unexplored.
ABSTRACT
Cadherins play an essential role in the attachment of the blastocyst to the endometrium, a process known as endometrial receptivity. Loss of E-cadherin expression is essential during the process, while the expression level of the other cadherin, N-cadherin, has been reported to be altered in cases of infertility. Both E-cadherin and N-cadherin can be regulated by members of the PARP family. Specifically, PARP-2, which is under the epigenetic control of miR-149, has been observed to promote E-cadherin expression in other human cells. We investigated the roles of E-cadherin and N-cadherin in endometrial receptivity using mouse models for normal endometrial receptivity, pseudopregnancy, and LPS-induced endometrial receptivity failure. E-cadherin and phosphorylated E-cadherin were predominantly expressed during pre-receptive stages as well as in the implantation site of the receptive stage, which were observed reduced during the later stages of implantation in both implantation and non-implantation regions, while N-cadherin was detected only at pre-receptive stages. E-cadherin and N-cadherin were also seen in the uterus during pseudopregnancy, showing a downregulation trend during receptive and post-receptive stages. LPS-induced failed endometrial receptivity showed upregulation of E-cadherin and downregulation of N-cadherin. The E-cadherin expression promoter, GSK-3, was lost and its suppressor, SLUG was upregulated during normal course of endometrial receptivity in mouse model, while GSK-3 was increased during LPS-induced failed embryo implantation. In an in vitro model of embryo implantation, E-cadherin expression is promoted by PARP-2 and regulated by miR-149 epigenetically in human endometrium epithelial cells. In conclusion, E-cadherin is predominantly expressed during pre-receptive stage and promoted by PARP-2, which is regulated by miR-149 in the endometrial epithelial cells.
Subject(s)
Cadherins/metabolism , Endometrium/metabolism , MicroRNAs/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Animals , Embryo Implantation/physiology , Female , Mice , Pregnancy , Signal TransductionABSTRACT
The two-domain actin associated protein coronin interacts with filamentous (F-) actin, facilitating diverse biological processes including cell proliferation, motility, phagocytosis, host-parasite interaction and cargo binding. The conserved N-terminal ß-propeller domain is involved in protein: protein interactions, while the C-terminal coiled-coil domain mediates oligomerization, transducing conformational changes. The L. donovani coronin coiled-coil (LdCoroCC) domain exhibited a novel topology and oligomer association with an inherent asymmetry, caused primarily by three a residues of successive heptads. In the T.brucei homolog (TbrCoro), two of these 'a' residues are different (Val 493 & 507 replacing LdCoroCC Ile 486 and Met 500 respectively). The elucidated structure possesses a similar topology and assembly while comparative structural analysis shows that the T.brucei coronin coiled-coil domain (TbrCoroCC) too possesses the asymmetry though its magnitude is smaller. Analysis identifies that the asymmetric state is stabilized via cyclic salt bridges formed by Arg 497 and Glu 504. Co-localization studies (LdCoro, TbrCoro and corresponding mutant coiled coil constructs) with actin show that there are subtle differences in their binding patterns, with the double mutant V493I-V507M showing maximal effect. None of the constructs have an effect on F-actin length. Taken together with LdCoroCC, we therefore conclude that the inherent asymmetric structures are essential for kinetoplastids, and are of interest in understanding and exploiting actin dynamics.
ABSTRACT
Nucleoside diphosphate kinases (NDK) are ubiquitous enzymes that catalyse the transfer of the γ phosphate from nucleoside triphosphates (NTPs) to nucleoside diphosphate (NDPs), to maintain appropriate NTP levels in cells. NDKs are associated with signal transduction, cell development, proliferation, differentiation, tumor metastasis, apoptosis and motility. The critical role of NDK in bacterial virulence renders it a potential drug target. The present manuscript reports crystal structure and functional characterization of Vibrio cholerae NDK (VNDK). The 16 kDa VNDK was crystallized in a solution containing 30% PEG 4000, 100 mM Tris-HCl pH 8.5 and 200 mM sodium acetate in orthorhombic space group P212121 with unit cell parameters a = 48.37, b = 71.21, c = 89.14 Å, α = ß = γ = 90° with 2 molecules in asymmetric unit. The crystal structure was solved by molecular replacement and refined to crystallographic Rfactor and Rfree values of 22.8% and 25.8% respectively. VNDK exists as both dimer and tetramer in solution as confirmed by size exclusion chromatography, glutaraldehyde crosslinking and small angle X-ray scattering while the crystal structure appears to be a dimer. The biophysical characterization states that VNDK has kinase and DNase activity with maximum stability at pH 8-9 and temperature up to 40 °C. VNDK shows elevated thermolability as compared to other NDK and shows preferential binding with GTP rationalized using computational studies.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Nucleoside-Diphosphate Kinase/chemistry , Nucleoside-Diphosphate Kinase/metabolism , Vibrio cholerae/enzymology , Bacterial Proteins/isolation & purification , Crystallography, X-Ray , Deoxyribonucleases/metabolism , Enzyme Stability , Guanosine Triphosphate/metabolism , Molecular Docking Simulation , Molecular Dynamics Simulation , Nucleoside-Diphosphate Kinase/isolation & purification , Protein Multimerization , Protein Structure, Quaternary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Scattering, Small Angle , Sequence Alignment , Sequence Analysis, Protein , Spectrometry, Fluorescence , Temperature , Vibrio cholerae/geneticsABSTRACT
The glycosomal membrane-associated Leishmania donovani protein PEX14, which plays a crucial role in protein import from the cytosol to the glycosomal matrix, consists of three domains: an N-terminal domain where the signalling molecule binds, a transmembrane domain and an 84-residue coiled-coil domain (CC) that is responsible for oligomerization. CCs are versatile domains that participate in a variety of functions including supramolecular assembly, cellular signalling and transport. Recombinant PEX14 CC was cloned, overexpressed, affinity-purified with in-column thrombin cleavage and further purified by size-exclusion chromatography. Crystals that diffracted to 1.98â Å resolution were obtained from a condition consisting of 1.4â M sodium citrate tribasic dihydrate, 0.1â M HEPES buffer pH 7.5. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 143.98, b = 32.62, c = 95.62â Å, ß = 94.68°. Structure determination and characterization are in progress.
Subject(s)
Crystallography, X-Ray/methods , Leishmania donovani/metabolism , Protozoan Proteins/chemistry , Protozoan Proteins/metabolism , Cloning, Molecular , Protein Binding , Protein Conformation , Protein Domains , Protozoan Proteins/genetics , Protozoan Proteins/isolation & purificationABSTRACT
Purine bases, synthesized de novo or recycled through the salvage pathway, are precursors of nucleotide synthesis and are essential in a variety of physiological processes including cell division, growth, signaling, energy metabolism and synthesis of vitamins/co-factor. The protozoan kinetoplastid parasites including Leishmania cannot synthesize de novo and rely solely on the purine salvage pathway, recycling the degraded products of nucleic acid metabolism. Enzymes of this pathway are thus of therapeutic importance. The enzyme Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) (EC 2.4.2.8) plays a central role in this pathway, converting the purine base to its monophosphate product. Towards the elucidation of its role, we have cloned, expressed, purified and determined the crystal structure of L. donovani HGPRT at 2.76 Å. Comparative structural analysis with the human homolog indicates differences in oligomer association. Comparative analyses identify insertions in the human homolog sequence in the tetramer interface. The results suggest that this difference can be exploited for therapeutic approaches.
Subject(s)
Hypoxanthine Phosphoribosyltransferase/chemistry , Leishmania donovani/enzymology , Protozoan Proteins/chemistry , Cloning, Molecular , Humans , Hypoxanthine Phosphoribosyltransferase/genetics , Hypoxanthine Phosphoribosyltransferase/isolation & purification , Models, Molecular , Protozoan Proteins/genetics , Protozoan Proteins/isolation & purification , Structural Homology, ProteinABSTRACT
Protein-protein interactions of cellular importance are mediated by coiled coils (CCs), the ubiquitous structural motif formed by the association of two or more α-helices in a knobs into holes manner. Coronins, actin-associated multi-functional proteins that possess distinct cytoskeleton-dependent and independent functions, oligomerize through their C-terminal CC domain. The structure of the L. donovani coronin CC domain (LdCoroCC; PDB ID 5CX2) revealed, in addition to a novel topology and architecture, an inherent asymmetry, with one of the helices of the 4-helix bundle axially shifted (~2 turns). The structural analysis identified that steric hindrance by Ile 486, Leu 493 and Met 500 as the cause for this asymmetry. To experimentally validate this hypothesis and to better understand the sequence-structure relationship in CCs, these amino acids have been mutated (I486A, L493A, M500V and the double mutant I486A-L493A) and characterized. Thermal CD studies suggest that the I486A and M500V mutants have comparable Tm values to LdCoroCC, while the other mutants have lower melting temperatures. The mutant crystal structures (I486A, M500V and the double mutant) retain the 'ade' core packing as LdcoroCC. While the M500V structure is similar to LdCoroCC, the I486A and the I486A-L493A structures show an asymmetry to symmetry transition. This study reveals crucial role of residues at position 'a' in coiled-coil domain play an important role in stabilizing the asymmetry in LdCoroCC, which might be necessary pursue specific biological function(s) inside the Leishmania.
Subject(s)
Leishmania/metabolism , Microfilament Proteins/chemistry , Microfilament Proteins/metabolism , Amino Acid Sequence , Animals , Cattle , Models, Molecular , Mutant Proteins/chemistry , Protein Domains , Protein Multimerization , Protein Structure, SecondaryABSTRACT
The availability of complete genome sequence of Mycobacterium tuberculosis has provided an important tool to understand the mycobacterial biology with respect to host-pathogen interaction, which is an unmet need of the hour owing to continuous increasing drug resistance. Hypothetical proteins are often an overlooked pool though half the genome encodes for such proteins of unknown function that could potentially play vital roles in mycobacterial biology. In this context, we report the structural and functional characterization of the hypothetical protein Rv3272. Sequence analysis classifies Rv3272 as a Family III CoA transferase with the classical two domain structure and conserved Aspartate residue (D175). The crystal structure of the wild type protein (2.2â¯Å) demonstrated the associated inter-locked dimer while that of the D175A mutant co-crystallized with octanoyl-CoA demonstrated relative movement between the two domains. Isothermal titration calorimetry studies indicate that Rv3272 binds to fatty acyl-CoAs of varying carbon chain lengths, with palmitoyl-CoA (C16:0) exhibiting maximum affinity. To determine the functional relevance of Rv3272 in mycobacterial biology, we ectopically expressed Rv3272 in M. smegmatis and assessed that its expression encodes significant alteration in cell surface with marked differences in triacylglycerol accumulation. Additionally, Rv3272 expression protects mycobacteria from acidic, oxidative and antibiotic stress under in vitro conditions. Taken together, these studies indicate a significant role for Rv3272 in host-pathogen interaction.
Subject(s)
Bacterial Proteins/physiology , Coenzyme A-Transferases/physiology , Mycobacterium tuberculosis/physiology , Stress, Physiological/physiology , Acyl Coenzyme A/chemistry , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/chemistry , Cell Wall/chemistry , Coenzyme A-Transferases/chemistry , Hydrogen-Ion Concentration , Ligands , Lipid Metabolism , Molecular Docking Simulation , Mycobacterium tuberculosis/drug effects , Oxidative StressABSTRACT
Nucleoside diphosphate kinases (NDKs) are ubiquitous enzymes that catalyze the transfer of the γ-phosphate moiety from an NTP donor to an NDP acceptor, crucial for maintaining the cellular level of nucleoside triphosphates (NTPs). The inability of trypanosomatids to synthesize purines de novo and their dependence on the salvage pathway makes NDK an attractive target to develop drugs for the diseases they cause. Here we report the discovery of novel inhibitors for Leishmania NDK based on the structural and functional characterization of purified recombinant NDK from Leishmania amazonensis. Recombinant LaNDK possesses auto-phosphorylation, phosphotransferase and kinase activities with Histidine 117 playing an essential role. LaNDK crystals were grown by hanging drop vapour diffusion method in a solution containing 18% PEG-MME 500, 100 mM Bis-Tris propane pH 6.0 and 50 mM MgCl2. It belongs to the hexagonal space group P6322 with unit cell parameters a = b = 115.18, c = 62.18 Å and α = ß = 90°, γ = 120°. The structure solved by molecular replacement methods was refined to crystallographic R-factor and Rfree values of 22.54 and 26.52%, respectively. Molecular docking and dynamics simulation-based virtual screening identified putative binding compounds. Protein inhibition studies of selected hits identified five inhibitors effective at micromolar concentrations. One of the compounds showed ~45% inhibition of Leishmania promastigotes proliferation. Analysis of inhibitor-NDK complexes reveals the mode of their binding, facilitating design of new compounds for optimization of activities as drugs against leishmaniasis.
Subject(s)
Antiprotozoal Agents/chemistry , Leishmania/enzymology , Nucleoside-Diphosphate Kinase/antagonists & inhibitors , Enzyme Activation , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Molecular Structure , Nucleoside-Diphosphate Kinase/chemistry , Protein Binding , Protein Conformation , Structure-Activity RelationshipABSTRACT
Trypansomatids maintain their redox balance by the trypanothione-based redox system, enzymes of which exhibit differences from mammalian homologues. γ-Glutamylcysteine synthetase (Gcs) is an essential enzyme in this pathway that performs the first and rate-limiting step. l-Buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor of Gcs, induces toxicity in hosts infected with Trypanosoma brucei, underlining the need for novel Gcs inhibitors. The present study reports identification of Leishmania donovani Gcs (LdGcs) inhibitors using computational approaches and their experimental validation. Analysis of inhibitor-LdGcs complexes shows modifications that could result in increased efficacy of these compounds.
Subject(s)
Dipeptides/antagonists & inhibitors , Dipeptides/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Leishmania donovani/enzymology , Molecular Dynamics Simulation , Amino Acid Sequence , Antiprotozoal Agents/chemical synthesis , Antiprotozoal Agents/chemistry , Antiprotozoal Agents/metabolism , Antiprotozoal Agents/pharmacology , Dipeptides/chemistry , Drug Design , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/metabolism , Humans , Leishmania donovani/drug effects , Protein Conformation , User-Computer InterfaceABSTRACT
Coiled coils are ubiquitous structural motifs that serve as a platform for protein-protein interactions and play a central role in myriad physiological processes. Though the formation of a coiled coil requires only the presence of suitably spaced hydrophobic residues, sequence specificities have also been associated with specific oligomeric states. RhXXhE is one such sequence motif, associated with parallel trimers, found in coronins and other proteins. Coronin, present in all eukaryotes, is an actin-associated protein involved in regulating actin turnover. Most eukaryotic coronins possess the RhXXhE trimerization motif. However, a unique feature of parasitic kinetoplastid coronin is that the positions of R and E are swapped within their coiled coil domain, but were still expected to form trimers. To understand the role of swapped motif in oligomeric specificity, we determined the X-ray crystal structure of Leishmania donovani coronin coiled coil domain (LdCoroCC) at 2.2Å, which surprisingly, reveals an anti-parallel tetramer assembly. Small angle X-ray scattering studies and chemical crosslinking confirm the tetramer in solution and is consistent with the oligomerization observed in the full length protein. Structural analyses reveal that LdCoroCC possesses an inherent asymmetry, in that one of the helices of the bundle is axially shifted with respect to the other three. The analysis also identifies steric reasons that cause this asymmetry. The bundle adapts an extended a-d-e core packing, the e residue being polar (with an exception) which results in a thermostable bundle with polar and apolar interfaces, unlike the existing a-d-e core antiparallel homotetramers with apolar core. Functional implications of the anti-parallel association in kinetoplastids are discussed.
Subject(s)
Leishmania donovani/chemistry , Microfilament Proteins/chemistry , Protozoan Proteins/chemistry , Amino Acid Motifs , Crystallography, X-Ray , Protein Domains , Protein Structure, SecondaryABSTRACT
γ-glutamylcysteine synthetase (Gcs) is a vital enzyme catalyzing the first and rate limiting step in the trypanothione biosynthesis pathway, the ATP-dependent ligation of L-Glutamate and L-Cysteine to form gamma-glutamylcysteine. The Trypanothione biosynthesis pathway is unique metabolic pathway essential for trypanosomatid survival rendering Gcs as a potential drug target. Here we report the cloning, expression, purification and characterization of L. donovani Gcs. Three other constructs of Gcs (GcsN, GcsC and GcsT) were designed on the basis of S. cerevisiae and E. coli Gcs crystal structures. The study shows Gcs possesses ATPase activity even in the absence of substrates L-glutamate and L-Cysteine. Divalent ions however plays an indispensable role in LdGcs ATPase activity. Isothermal titration calorimetry and fluorescence studies illustrates that L. donovani Gcs binds substrate in order ATP >L-glutamate>L-cysteine with Glu 92 and Arg 498 involved in ATP hydrolysis and Glu 92, Glu 55 and Arg 498 involved in glutamate binding. Homology modeling and molecular dynamic simulation studies provided the structural rationale of LdGcs catalytic activity and emphasized on the possibility of involvement of three Mg2+ ions along with Glutamates 52, 55, 92, 99, Met 322, Gln 328, Tyr 397, Lys 483, Arg 494 and Arg 498 in the catalytic function of L. donovani Gcs.
ABSTRACT
Polyamine biosynthesis pathway has long been considered an essential drug target for trypanosomatids including Leishmania. S-adenosylmethionine decarboxylase (AdoMetDc) and spermidine synthase (SpdSyn) are enzymes of this pathway that catalyze successive steps, with the product of the former, decarboxylated S-adenosylmethionine (dcSAM), acting as an aminopropyl donor for the latter enzyme. Here we have explored the possibility of and identified the protein-protein interaction between SpdSyn and AdoMetDc. The protein-protein interaction has been identified using GST pull down assay. Isothermal titration calorimetry reveals that the interaction is thermodynamically favorable. Fluorescence spectroscopy studies also confirms the interaction, with SpdSyn exhibiting a change in tertiary structure with increasing concentrations of AdoMetDc. Size exclusion chromatography suggests the presence of the complex as a hetero-oligomer. Taken together, these results suggest that the enzymes indeed form a heteromer. Computational analyses suggest that this complex differs significantly from the corresponding human complex, implying that this complex could be a better therapeutic target than the individual enzymes.
Subject(s)
Adenosylmethionine Decarboxylase/chemistry , Leishmania donovani/enzymology , Protozoan Proteins/chemistry , Spermidine Synthase/chemistry , Adenosylmethionine Decarboxylase/genetics , Adenosylmethionine Decarboxylase/metabolism , Biogenic Polyamines/biosynthesis , Calorimetry , Chromatography, Gel , Cloning, Molecular , Microscopy, Fluorescence , Protein Interaction Mapping , Protozoan Proteins/metabolism , Spermidine Synthase/genetics , Spermidine Synthase/metabolismABSTRACT
Antileishmanial activities of a library of synthetic chalcone analogues have been examined. Among them, five compounds (11, 14, 16, 17, 22, and 24) exhibited better activity than the marketed drug miltefosine in in vitro studies against the intracellular amastigotes form of Leishmania donovani. Three promising compounds, 16, 17, and 22, were tested in a L. donovani/hamster model. Oral administration of chalcone 16, at a concentration of 100 mg/kg of body weight per day for 5 consecutive days, resulted in >84% parasite inhibition at day 7 post-treatment and it retained the activity until day 28. The molecular and immunological studies revealed that compound 16 has a dual nature to act as a direct parasite killing agent and as a host immunostimulant. Pharmacokinetics and serum albumin binding studies also suggest that compound 16 has the potential to be a candidate for the treatment of the nonhealing form of leishmaniasis.
Subject(s)
Antiprotozoal Agents/chemical synthesis , Chalcones/chemical synthesis , Leishmania donovani/drug effects , Animals , Antiprotozoal Agents/pharmacokinetics , Antiprotozoal Agents/pharmacology , Chalcones/pharmacokinetics , Chalcones/pharmacology , Cricetinae , Cytokines/biosynthesis , Drug Stability , Macrophages/immunology , Membrane Potential, Mitochondrial/drug effects , Mesocricetus , Nitric Oxide/biosynthesis , Structure-Activity RelationshipABSTRACT
In addition to the S-adenosylmethionine decarboxylase (AD) present in all organisms, trypanosomatids including Leishmania spp. possess an additional copy, annotated as the putative S-adenosylmethionine decarboxylase-like proenzyme (ADL). Phylogenetic analysis confirms that ADL is unique to trypanosomatids and has several unique features such as lack of autocatalytic cleavage and a distinct evolutionary lineage, even from trypanosomatid ADs. In Trypanosoma ADL was found to be enzymaticaly dead but plays an essential regulatory role by forming a heterodimer complex with AD. However, no structural or functional information is available about ADL from Leishmania spp. Here, in this study, we report the cloning, expression, purification, structural and functional characterization of Leishmania donovani (L. donovani) ADL using biophysical, biochemical and computational techniques. Biophysical studies show that, L. donovani ADL binds S-adenosylmethionine (SAM) and putrescine which are natural substrates of AD. Computational modeling and docking studies showed that in comparison to the ADs of other organisms including human, residues involved in putrescine binding are partially conserved while the SAM binding residues are significantly different. In silico protein-protein interaction study reveals that L. donovani ADL can interact with AD. These results indicate that L. donovani ADL posses a novel substrate binding property and may play an essential role in polyamine biosynthesis with a different mode of function from known proteins of the S-adenosylmethionine decarboxylase super family.
Subject(s)
Adenosylmethionine Decarboxylase/genetics , Adenosylmethionine Decarboxylase/metabolism , Cloning, Molecular/methods , Leishmania donovani/enzymology , Adenosylmethionine Decarboxylase/chemistry , Binding Sites , Evolution, Molecular , Leishmania donovani/chemistry , Leishmania donovani/genetics , Models, Molecular , Molecular Docking Simulation , Phylogeny , Protein Binding , Protein Multimerization , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Putrescine/metabolism , S-Adenosylmethionine/metabolismABSTRACT
The Molybdenum cofactor (Moco) biosynthesis pathway is an evolutionary conserved pathway seen in almost all eukaryotes including the pathogenic species Mycobacterium tuberculosis. This pathway comprises of several novel reactions which include the initial formation of precursor Z from guanosine triphosphate (GTP), catalysed by two enzymes MoaA and MoaC. Although Moco biosynthesis is well understood, the first step is still not clear. In M. tuberculosis H37Rv, three orthologous genes of MoaC have been annotated: moaC1 (Rv3111), moaC2 (Rv0864) and moaC3 (Rv3324c). Rv0864 (MoaC2) is a 17.5 kDa protein and is reported to be down-regulated by â¼3 times in the nutrient starvation model for Mycobacterium tuberculosis. The crystal structure of Moco-biosynthesis protein MoaC2 from Mycobacterium tuberculosis (2.20 Å resolution, space group P213) has been determined. Based on a comparative analysis of structures of homologous proteins, conserved residues were identified and are implicated in structural and functional roles. Molecular docking studies with probable ligands carried out in order to identify its ligand, suggests that pteridinebenzomonophosphate as the most likely ligand. Sequence based interaction study identified MoaA1 to interact with MoaC2. A homology model of MoaA1 was then complexed with MoaC2 and protein-protein interactions are also discussed.
Subject(s)
Bacterial Proteins/chemistry , Coenzymes/biosynthesis , Enzymes/chemistry , Metalloproteins/biosynthesis , Mycobacterium tuberculosis/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Conserved Sequence , Crystallography, X-Ray , Enzymes/genetics , Enzymes/metabolism , Models, Molecular , Molecular Sequence Data , Molybdenum Cofactors , Mycobacterium tuberculosis/genetics , Protein Conformation , Protein Interaction Domains and Motifs , Pteridines , Sequence Homology, Amino Acid , Structural Homology, ProteinABSTRACT
Rv0864 (MoaC2) from Mycobacterium tuberculosis is one of the enzymes in the molybdenum cofactor (Moco) biosynthesis pathway. Together with MoaA, MoaC is involved in the conversion of guanosine triphosphate (GTP) to precursor Z, the first step in Moco synthesis. Full-length MoaC2 (17.5 kDa, 167 residues) was cloned in Escherichia coli and purified to homogeneity. Crystals of recombinant M. tuberculosis MoaC2 were grown by vapour diffusion using a hanging-drop setup. Diffracting crystals grew in a condition in which 3 µl protein solution at 10.5 mg ml(-1) was mixed with 1.5 µl reservoir solution (0.025 M potassium sodium tartrate tetrahydrate pH 8.0) and equilibrated against 1000 µl reservoir solution. Diffraction data extending to 2.5 Å resolution were collected at 100 K. The crystal belonged to the cubic space group P2(1)3, with unit-cell parameter 94.5 Å. Matthews coefficient (V(M)) calculations suggested the presence of two molecules in the asymmetric unit, corresponding to a solvent content of about 39%. Molecular-replacement calculations using the E. coli homologue as the search model gave an unambiguous solution.
Subject(s)
Bacterial Proteins/chemistry , Mycobacterium tuberculosis/chemistry , Nuclear Proteins/chemistry , Amino Acid Sequence , Bacterial Proteins/isolation & purification , Crystallization , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Nuclear Proteins/isolation & purification , Protein Structure, Tertiary , Sequence AlignmentABSTRACT
RbAp46 and RbAp48 (pRB-associated proteins p46 and p48, also known as RBBP7 and RBBP4, respectively) are highly homologous histone chaperones that play key roles in establishing and maintaining chromatin structure. We report here the crystal structure of human RbAp46 bound to histone H4. RbAp46 folds into a seven-bladed beta propeller structure and binds histone H4 in a groove formed between an N-terminal alpha helix and an extended loop inserted into blade six. Surprisingly, histone H4 adopts a different conformation when interacting with RbAp46 than it does in either the nucleosome or in the complex with ASF1, another histone chaperone. Our structural and biochemical results suggest that when a histone H3/H4 dimer (or tetramer) binds to RbAp46 or RbAp48, helix 1 of histone H4 unfolds to interact with the histone chaperone. We discuss the implications of our findings for the assembly and function of RbAp46 and RbAp48 complexes.