RESUMO
The rise in antimicrobial resistance is a global health crisis and necessitates the development of novel strategies to treat infections. For example, in 2022 tuberculosis (TB) was the second leading infectious killer after COVID-19, with multi-drug-resistant strains of TB having an â¼40% fatality rate. Targeting essential biosynthetic pathways in pathogens has proven to be successful for the development of novel antimicrobial treatments. Fatty-acid synthesis (FAS) in bacteria proceeds via the type II pathway, which is substantially different from the type I pathway utilized in animals. This makes bacterial fatty-acid biosynthesis (Fab) enzymes appealing as drug targets. FabG is an essential FASII enzyme, and some bacteria, such as Mycobacterium tuberculosis, the causative agent of TB, harbor multiple homologs. FabG4 is a conserved, high-molecular-weight FabG (HMwFabG) that was first identified in M. tuberculosis and is distinct from the canonical low-molecular-weight FabG. Here, structural and functional analyses of Mycolicibacterium smegmatis FabG4, the third HMwFabG studied to date, are reported. Crystal structures of NAD+ and apo MsFabG4, along with kinetic analyses, show that MsFabG4 preferentially binds and uses NADH when reducing CoA substrates. As M. smegmatis is often used as a model organism for M. tuberculosis, these studies may aid the development of drugs to treat TB and add to the growing body of research that distinguish HMwFabGs from the archetypal low-molecular-weight FabG.
Assuntos
Proteínas de Bactérias , Mycobacterium smegmatis , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Cristalografia por Raios X , Modelos Moleculares , Sequência de Aminoácidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
The name of one of the authors in Beard et al. [(2022), Acta Cryst. F78, 59-65] is corrected.
RESUMO
Giardiasis is the most prevalent diarrheal disease globally and affects humans and animals. It is a significant problem in developing countries, the number one cause of travelers' diarrhea and affects children and immunocompromised individuals, especially HIV-infected individuals. Giardiasis is treated with antibiotics (tinidazole and metronidazole) that are also used for other infections such as trichomoniasis. The ongoing search for new therapeutics for giardiasis includes characterizing the structure and function of proteins from the causative protozoan Giardia lamblia. These proteins include hypothetical proteins that share 30% sequence identity or less with proteins of known structure. Here, the atomic resolution structure of a 15.6â kDa protein was determined by molecular replacement. The structure has the two-layer αß-sandwich topology observed in the prototypical endoribonucleases L-PSPs (liver perchloric acid-soluble proteins) with conserved allosteric active sites containing small molecules from the crystallization solution. This article is an educational collaboration between Hampton University and the Seattle Structural Genomics Center for Infectious Disease.
Assuntos
Giardia lamblia/química , Proteínas de Protozoários/química , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , Proteínas de Protozoários/metabolismoRESUMO
Tumour necrosis factor (TNF) is a cytokine belonging to a family of trimeric proteins; it has been shown to be a key mediator in autoimmune diseases such as rheumatoid arthritis and Crohn's disease. While TNF is the target of several successful biologic drugs, attempts to design small molecule therapies directed to this cytokine have not led to approved products. Here we report the discovery of potent small molecule inhibitors of TNF that stabilise an asymmetrical form of the soluble TNF trimer, compromising signalling and inhibiting the functions of TNF in vitro and in vivo. This discovery paves the way for a class of small molecule drugs capable of modulating TNF function by stabilising a naturally sampled, receptor-incompetent conformation of TNF. Furthermore, this approach may prove to be a more general mechanism for inhibiting protein-protein interactions.
Assuntos
Anti-Inflamatórios/farmacologia , Artrite Experimental/tratamento farmacológico , Multimerização Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Anti-Inflamatórios/uso terapêutico , Artrite Experimental/imunologia , Linhagem Celular , Cristalografia por Raios X , Descoberta de Drogas , Masculino , Camundongos , Simulação de Dinâmica Molecular , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Estabilidade Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína/efeitos dos fármacos , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Transdução de Sinais/imunologia , Relação Estrutura-Atividade , Resultado do Tratamento , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/isolamento & purificação , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/ultraestruturaRESUMO
Cat scratch fever (also known as cat scratch disease and bartonellosis) is an infectious disease caused by the proteobacterium Bartonella henselae following a cat scratch. Although the infection usually resolves spontaneously without treatment in healthy adults, bartonellosis may lead to severe complications in young children and immunocompromised patients, and there is new evidence suggesting that B. henselae may be associated with a broader range of clinical symptoms then previously believed. The genome of B. henselae contains genes for two putative Nudix hydrolases, BH02020 and BH01640 (KEGG). Nudix proteins play an important role in regulating the intracellular concentration of nucleotide cofactors and signaling molecules. The amino-acid sequence of BH02020 is similar to that of the prototypical member of the Nudix superfamily, Escherichia coli MutT, a protein that is best known for its ability to neutralize the promutagenic compound 7,8-dihydro-8-oxoguanosine triphosphate. Here, the crystal structure of BH02020 (Bh-MutT) in the Mg(2+)-bound state was determined at 2.1 Å resolution (PDB entry 3hhj). As observed in all Nudix hydrolase structures, the α-helix of the highly conserved `Nudix box' in Bh-MutT is one of two helices that sandwich a four-stranded mixed ß-sheet with the central two ß-strands parallel to each other. The catalytically essential divalent cation observed in the Bh-MutT structure, Mg(2+), is coordinated to the side chains of Glu57 and Glu61. The structure is not especially robust; a temperature melt obtained using circular dichroism spectroscopy shows that Bh-MutT irreversibly unfolds and precipitates out of solution upon heating, with a T(m) of 333 K.
Assuntos
Bartonella henselae/enzimologia , Magnésio/química , Pirofosfatases/química , Magnésio/metabolismo , Modelos Moleculares , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Pirofosfatases/metabolismo , Homologia Estrutural de Proteína , Nudix HidrolasesRESUMO
Glycogen synthase kinase-3 (GSK-3) is a drug target under intense investigation in pharmaceutical companies and constitutes an attractive piggyback target for eukaryotic pathogens. Two different GSKs are found in trypanosomatids, one about 150 residues shorter than the other. GSK-3 short (GeneDB: Tb927.10.13780) has previously been validated genetically as a drug target in Trypanosoma brucei by RNAi induced growth retardation; and chemically by correlation between enzyme and in vitro growth inhibition. Here, we report investigation of the equivalent GSK-3 short enzymes of L. major (LmjF18.0270) and L. infantum (LinJ18_V3.0270, identical in amino acid sequences to LdonGSK-3 short) and a crystal structure of LmajGSK-3 short at 2 Å resolution. The inhibitor structure-activity relationships (SARs) of L. major and L. infantum are virtually identical, suggesting that inhibitors could be useful for both cutaneous and visceral leishmaniasis. Leishmania spp. GSK-3 short has different inhibitor SARs than TbruGSK-3 short, which can be explained mostly by two variant residues in the ATP-binding pocket. Indeed, mutating these residues in the ATP-binding site of LmajGSK-3 short to the TbruGSK-3 short equivalents results in a mutant LmajGSK-3 short enzyme with SAR more similar to that of TbruGSK-3 short. The differences between human GSK-3ß (HsGSK-3ß) and LmajGSK-3 short SAR suggest that compounds which selectively inhibit LmajGSK-3 short may be found.
Assuntos
Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Isoformas de Proteínas/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Recombinantes/antagonistas & inibidores , Motivos de Aminoácidos/efeitos dos fármacos , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli , Expressão Gênica , Quinase 3 da Glicogênio Sintase/química , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Cinética , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/genética , Leishmania infantum/metabolismo , Leishmania major/efeitos dos fármacos , Leishmania major/genética , Leishmania major/metabolismo , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Cutânea/genética , Leishmaniose Cutânea/metabolismo , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/genética , Leishmaniose Visceral/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica/efeitos dos fármacos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade da Espécie , Relação Estrutura-Atividade , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMO
Tryptophanyl-tRNA synthetase (TrpRS) is an essential enzyme that is recognizably conserved across all forms of life. It is responsible for activating and attaching tryptophan to a cognate tRNA(Trp) molecule for use in protein synthesis. In some eukaryotes this original core function has been supplemented or modified through the addition of extra domains or the expression of variant TrpRS isoforms. The three TrpRS structures from pathogenic protozoa described here represent three illustrations of this malleability in eukaryotes. The Cryptosporidium parvum genome contains a single TrpRS gene, which codes for an N-terminal domain of uncertain function in addition to the conserved core TrpRS domains. Sequence analysis indicates that this extra domain, conserved among several apicomplexans, is related to the editing domain of some AlaRS and ThrRS. The C. parvum enzyme remains fully active in charging tRNA(Trp) after truncation of this extra domain. The crystal structure of the active, truncated enzyme is presented here at 2.4Å resolution. The Trypanosoma brucei genome contains separate cytosolic and mitochondrial isoforms of TrpRS that have diverged in their respective tRNA recognition domains. The crystal structure of the T. brucei cytosolic isoform is presented here at 2.8Å resolution. The Entamoeba histolytica genome contains three sequences that appear to be TrpRS homologs. However one of these, whose structure is presented here at 3.0Å resolution, has lost the active site motifs characteristic of the Class I aminoacyl-tRNA synthetase catalytic domain while retaining the conserved features of a fully formed tRNA(Trp) recognition domain. The biological function of this variant E. histolytica TrpRS remains unknown, but, on the basis of a completely conserved tRNA recognition region and evidence for ATP but not tryptophan binding, it is tempting to speculate that it may perform an editing function. Together with a previously reported structure of an unusual TrpRS from Giardia, these protozoan structures broaden our perspective on the extent of structural variation found in eukaryotic TrpRS homologs.
Assuntos
Cryptosporidium parvum/enzimologia , Entamoeba histolytica/enzimologia , Proteínas de Protozoários/química , Trypanosoma brucei brucei/enzimologia , Triptofano-tRNA Ligase/química , Sequência de Aminoácidos , Sítios de Ligação , Cryptosporidium parvum/química , Cryptosporidium parvum/genética , Cristalografia por Raios X , Entamoeba histolytica/química , Entamoeba histolytica/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/genética , Triptofano-tRNA Ligase/genética , Triptofano-tRNA Ligase/metabolismoRESUMO
New drugs are needed to treat toxoplasmosis. Toxoplasma gondii calcium-dependent protein kinases (TgCDPKs) are attractive targets because they are absent in mammals. We show that TgCDPK1 is inhibited by low nanomolar levels of bumped kinase inhibitors (BKIs), compounds inactive against mammalian kinases. Cocrystal structures of TgCDPK1 with BKIs confirm that the structural basis for selectivity is due to the unique glycine gatekeeper residue in the ATP-binding site. We show that BKIs interfere with an early step in T. gondii infection of human cells in culture. Furthermore, we show that TgCDPK1 is the in vivo target of BKIs because T. gondii expressing a glycine to methionine gatekeeper mutant enzyme show significantly decreased sensitivity to BKIs. Thus, design of selective TgCDPK1 inhibitors with low host toxicity may be achievable.
Assuntos
Antiparasitários/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Toxoplasma/enzimologia , Toxoplasmose/tratamento farmacológico , Sequência de Aminoácidos , Animais , Antiparasitários/química , Cristalografia por Raios X , Fibroblastos/parasitologia , Interações Hospedeiro-Parasita/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Conformação Proteica , Inibidores de Proteínas Quinases/química , Proteínas Quinases/análise , Toxoplasma/citologia , Toxoplasma/efeitos dos fármacos , Toxoplasma/crescimento & desenvolvimentoRESUMO
The 2.1A crystal structure of tryptophanyl-tRNA synthetase (TrpRS) from the diplomonad Giardia lamblia reveals that the N-terminus of this class I aminoacyl-tRNA synthetase forms a 16-residue alpha-helix. This helix replaces a beta-hairpin that is required by human TrpRS for normal activity and has been inferred to play a similar role in all eukaryotic TrpRS. The primary sequences of TrpRS homologs from several basal eukaryotes including Giardia lack a set of three residues observed to stabilize interactions with this beta-hairpin in the human TrpRS. Thus the present structure suggests that the activation reaction mechanism of TrpRS from the basal eukaryote G. lamblia differs from that of higher eukaryotes. Furthermore, the protein as observed in the crystal forms an (alpha(2))(2) homotetramer. The canonical dimer interface observed in all previous structures of tryptophanyl-tRNA synthetases is maintained, but in addition each N-terminal alpha-helix reciprocally interlocks with the equivalent helix from a second dimer to form a dimer of dimers. Although we have no evidence for tetramer formation in vivo, modeling indicates that the crystallographically observed tetrameric structure would be compatible with the tRNA binding mode used by dimeric TrpRS and TyrRS.
Assuntos
Giardia lamblia/enzimologia , Triptofano-tRNA Ligase/química , Humanos , Modelos Moleculares , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Difração de Raios XRESUMO
The great power of protein crystallography to reveal biological structure is often limited by the tremendous effort required to produce suitable crystals. A hybrid crystal growth predictive model is presented that combines both experimental and sequence-derived data from target proteins, including novel variables derived from physico-chemical characterization such as R(30), the ratio between a protein's DSF intensity at 30°C and at T(m). This hybrid model is shown to be more powerful than sequence-based prediction alone - and more likely to be useful for prioritizing and directing the efforts of structural genomics and individual structural biology laboratories.
Assuntos
Modelos Moleculares , Proteínas/química , Cristalização , Cristalografia por Raios X , Interpretação Estatística de Dados , Análise de Sequência de ProteínaRESUMO
Crystal structures of histidyl-tRNA synthetase (HisRS) from the eukaryotic parasites Trypanosoma brucei and Trypanosoma cruzi provide a first structural view of a eukaryotic form of this enzyme and reveal differences from bacterial homologs. HisRSs in general contain an extra domain inserted between conserved motifs 2 and 3 of the Class II aminoacyl-tRNA synthetase catalytic core. The current structures show that the three-dimensional topology of this domain is very different in bacterial and archaeal/eukaryotic forms of the enzyme. Comparison of apo and histidine-bound trypanosomal structures indicates substantial active-site rearrangement upon histidine binding but relatively little subsequent rearrangement after reaction of histidine with ATP to form the enzyme's first reaction product, histidyladenylate. The specific residues involved in forming the binding pocket for the adenine moiety differ substantially both from the previously characterized binding site in bacterial structures and from the homologous residues in human HisRSs. The essentiality of the single HisRS gene in T. brucei is shown by a severe depression of parasite growth rate that results from even partial suppression of expression by RNA interference.
Assuntos
Histidina-tRNA Ligase/química , Trypanosoma brucei brucei/enzimologia , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Domínio Catalítico , Cristalografia por Raios X , Técnicas de Silenciamento de Genes , Genes Essenciais , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Alinhamento de Sequência , Trypanosoma brucei brucei/química , Trypanosoma cruzi/químicaRESUMO
Purine nucleoside phosphorylases (PNPs) and uridine phosphorylases (UPs) are closely related enzymes involved in purine and pyrimidine salvage, respectively, which catalyze the removal of the ribosyl moiety from nucleosides so that the nucleotide base may be recycled. Parasitic protozoa generally are incapable of de novo purine biosynthesis; hence, the purine salvage pathway is of potential therapeutic interest. Information about pyrimidine biosynthesis in these organisms is much more limited. Though all seem to carry at least a subset of enzymes from each pathway, the dependency on de novo pyrimidine synthesis versus salvage varies from organism to organism and even from one growth stage to another. We have structurally and biochemically characterized a putative nucleoside phosphorylase (NP) from the pathogenic protozoan Trypanosoma brucei and find that it is a homodimeric UP. This is the first characterization of a UP from a trypanosomal source despite this activity being observed decades ago. Although this gene was broadly annotated as a putative NP, it was widely inferred to be a purine nucleoside phosphorylase. Our characterization of this trypanosomal enzyme shows that it is possible to distinguish between PNP and UP activity at the sequence level based on the absence or presence of a characteristic UP-specificity insert. We suggest that this recognizable feature may aid in proper annotation of the substrate specificity of enzymes in the NP family.
Assuntos
Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/enzimologia , Uridina Fosforilase/química , Uridina Fosforilase/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Primers do DNA/genética , DNA de Protozoário/genética , Genes de Protozoários , Metais/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Interferência de RNA , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Trypanosoma brucei brucei/genética , Uridina Fosforilase/antagonistas & inibidores , Uridina Fosforilase/genéticaRESUMO
The crystal structure of the aspartyl-tRNA synthetase from the eukaryotic parasite Entamoeba histolytica has been determined at 2.8Aresolution. Relative to homologous sequences, the E. histolytica protein contains a 43-residue insertion between the N-terminal anticodon binding domain and the C-terminal catalytic domain. The present structure reveals that this insertion extends an arm of the hinge region that has previously been shown to mediate interaction of aspartyl-tRNA synthetase with the cognate tRNA D-stem. Modeling indicates that this Entamoeba-specific insertion is likely to increase the interaction surface with the cognate tRNA(Asp). In doing so it may substitute functionally for an RNA-binding motif located in N-terminal extensions found in AspRS sequences from lower eukaryotes but absent in Entamoeba. The E. histolytica AspRS structure shows a well-ordered N-terminus that contributes to the AspRS dimer interface.
Assuntos
Aspartato-tRNA Ligase/química , Entamoeba histolytica/química , Entamoeba histolytica/enzimologia , Proteínas de Protozoários/química , Sequência de Aminoácidos , Animais , Aspartato-tRNA Ligase/genética , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
The history of fragment-based drug discovery, with an emphasis on crystallographic methods, is sketched, illuminating various contributions, including our own, which preceded the industrial development of the method. Subsequently, the creation of the BMSC fragment cocktails library is described. The BMSC collection currently comprises 68 cocktails of 10 compounds that are shape-wise diverse. The utility of these cocktails for initiating lead discovery in structure-based drug design has been explored by soaking numerous protein crystals obtained by our MSGPP (Medical Structural Genomics of Pathogenic Protozoa) consortium. Details of the fragment selection and cocktail design procedures, as well as examples of the successes obtained are given. The BMSC Fragment Cocktail recipes are available free of charge and are in use in over 20 academic labs.
Assuntos
Descoberta de Drogas/métodos , Infecções por Protozoários/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/química , Cristalografia por Raios X , Desenho de Fármacos , Genoma de Protozoário , Humanos , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
The Structural Genomics of Pathogenic Protozoa (SGPP) Consortium aimed to determine crystal structures of proteins from trypanosomatid and malaria parasites in a high throughput manner. The pipeline of target selection, protein production, crystallization, and structure determination, is sketched. Special emphasis is given to a number of technology developments including domain prediction, the use of "co-crystallants," and capillary crystallization. "Fragment cocktail crystallography" for medical structural genomics is also described.
Assuntos
Genômica/métodos , Plasmodium/genética , Proteínas de Protozoários/química , Trypanosomatina/genética , Animais , Cristalização , Cristalografia por Raios X/métodosRESUMO
Nucleotide biosynthesis pathways have been reported to be essential in some protozoan pathogens. Hence, we evaluated the essentiality of one enzyme in the pyrimidine biosynthetic pathway, dihydroorotate dehydrogenase (DHODH) from the eukaryotic parasite Trypanosoma brucei through gene knockdown studies. RNAi knockdown of DHODH expression in bloodstream form T. brucei did not inhibit growth in normal medium, but profoundly retarded growth in pyrimidine-depleted media or in the presence of the known pyrimidine uptake antagonist 5-fluorouracil (5-FU). These results have significant implications for the development of therapeutics to combat T. brucei infection. Specifically, a combination therapy including a T. brucei-specific DHODH inhibitor plus 5-FU may prove to be an effective therapeutic strategy. We also show that this trypanosomal enzyme is inhibited by known inhibitors of bacterial Class 1A DHODH, in distinction to the sensitivity of DHODH from human and other higher eukaryotes. This selectivity is supported by the crystal structure of the T. brucei enzyme, which is reported here at a resolution of 1.95 A. Additional research, guided by the crystal structure described herein, is needed to identify potent inhibitors of T. brucei DHODH.
Assuntos
Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Proteínas de Protozoários/genética , Interferência de RNA , Trypanosoma brucei brucei/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Cristalografia por Raios X , Di-Hidro-Orotato Desidrogenase , Desenho de Fármacos , Fluoruracila/farmacologia , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Pirimidinas/metabolismo , Homologia de Sequência de Aminoácidos , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologiaRESUMO
The structure of a putative Raf kinase inhibitor protein (RKIP) homolog from the eukaryotic parasite Plasmodium vivax has been studied to a resolution of 1.3 A using multiple-wavelength anomalous diffraction at the Se K edge. This protozoan protein is topologically similar to previously studied members of the phosphatidylethanolamine-binding protein (PEBP) sequence family, but exhibits a distinctive left-handed alpha-helical region at one side of the canonical phospholipid-binding site. Re-examination of previously determined PEBP structures suggests that the P. vivax protein and yeast carboxypeptidase Y inhibitor may represent a structurally distinct subfamily of the diverse PEBP-sequence family.
Assuntos
Proteína de Ligação a Fosfatidiletanolamina/química , Proteína de Ligação a Fosfatidiletanolamina/fisiologia , Plasmodium vivax/química , Motivos de Aminoácidos/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação/fisiologia , Cristalografia por Raios X , Dados de Sequência Molecular , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Plasmodium vivax/metabolismo , Conformação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Estrutura Secundária de Proteína/fisiologia , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/fisiologiaRESUMO
The gene product of structural genomics target Lmaj006129 from Leishmania major codes for a 164-residue protein of unknown function. When SeMet expression of the full-length gene product failed, several truncation variants were created with the aid of Ginzu, a domain-prediction method. 11 truncations were selected for expression, purification and crystallization based upon secondary-structure elements and disorder. The structure of one of these variants, Lmaj006129AAH, was solved by multiple-wavelength anomalous diffraction (MAD) using ELVES, an automatic protein crystal structure-determination system. This model was then successfully used as a molecular-replacement probe for the parent full-length target, Lmaj006129AAA. The final structure of Lmaj006129AAA was refined to an R value of 0.185 (Rfree = 0.229) at 1.60 A resolution. Structure and sequence comparisons based on Lmaj006129AAA suggest that proteins belonging to Pfam sequence families PF04543 and PF01878 may share a common ligand-binding motif.
Assuntos
Leishmania major/química , Proteínas de Protozoários/química , Animais , Cristalização , Cristalografia por Raios XRESUMO
Fructose-1,6-(bis)phosphate aldolase is a ubiquitous enzyme that catalyzes the reversible aldol cleavage of fructose-1,6-(bis)phosphate and fructose 1-phosphate to dihydroxyacetone phosphate and either glyceral-dehyde-3-phosphate or glyceraldehyde, respectively. Vertebrate aldolases exist as three isozymes with different tissue distributions and kinetics: aldolase A (muscle and red blood cell), aldolase B (liver, kidney, and small intestine), and aldolase C (brain and neuronal tissue). The structures of human aldolases A and B are known and herein we report the first structure of the human aldolase C, solved by X-ray crystallography at 3.0 A resolution. Structural differences between the isozymes were expected to account for isozyme-specific activity. However, the structures of isozymes A, B, and C are the same in their overall fold and active site structure. The subtle changes observed in active site residues Arg42, Lys146, and Arg303 are insufficient to completely account for the tissue-specific isozymic differences. Consequently, the structural analysis has been extended to the isozyme-specific residues (ISRs), those residues conserved among paralogs. A complete analysis of the ISRs in the context of this structure demonstrates that in several cases an amino acid residue that is conserved among aldolase C orthologs prevents an interaction that occurs in paralogs. In addition, the structure confirms the clustering of ISRs into discrete patches on the surface and reveals the existence in aldolase C of a patch of electronegative residues localized near the C terminus. Together, these structural changes highlight the differences required for the tissue and kinetic specificity among aldolase isozymes.
Assuntos
Encéfalo/enzimologia , Frutose-Bifosfato Aldolase/química , Sequência de Bases , Sítios de Ligação , Cristalografia por Raios X , Frutose-Bifosfato Aldolase/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
An active form of the Dengue virus protease NS3 (CF40.Gly.NS3pro) was expressed in Escherichia coli. This construct consists of a critical 40 amino acid cofactor domain from NS2B fused to the N-terminal 184 amino acid protease domain of NS3 via a flexible, covalent linker (Gly(4)SerGly(4)). The recombinantly produced protein is soluble and has a hexa-histidine tag engineered at the N-terminus for ease of purification using metal affinity chromatography. However, the presence of lower molecular weight impurities after affinity chromatography indicated the need for additional purification steps. The consistent appearance of these impurities suggested that they may be the products of proteolysis and/or auto-proteolysis. The latter possibility was subsequently excluded by the observation of the same impurities in a purified, catalytically inactive form of the recombinant protease (CF40.Gly.NS3pro.SA). Further analysis indicated that these impurities may represent premature translation termination products. Regardless of their origin, they were shown to form various sized aggregates with full-length CF40.Gly.NS3pro that can be separated by size exclusion chromatography, yielding fractions of active protease of sufficient purity for crystallisation trials. The ultimate goal of these studies is to obtain a crystal structure of a catalytically active form of the Dengue virus NS3 protease for structure-based drug design.