RESUMO
Solvent organization is a key but underexploited contributor to the thermodynamics of protein-ligand recognition, with implications for ligand discovery, drug resistance, and protein engineering. Here, we explore the contribution of solvent to ligand binding in the Haemophilus influenzae virulence protein SiaP. By introducing a single mutation without direct ligand contacts, we observed a >1000-fold change in sialic acid binding affinity. Crystallographic and calorimetric data of wild-type and mutant SiaP showed that this change results from an enthalpically unfavorable perturbation of the solvent network. This disruption is reflected by changes in the normalized atomic displacement parameters of crystallographic water molecules. In SiaP's enclosed cavity, relative differences in water-network dynamics serve as a simple predictor of changes in the free energy of binding upon changing protein, ligand, or both. This suggests that solvent structure is an evolutionary constraint on protein sequence that contributes to ligand affinity and selectivity.
Assuntos
Solventes/química , Proteínas Virais/química , Fatores de Virulência/química , Água/química , Sítios de Ligação , Ligantes , Ligação Proteica , Conformação Proteica , TermodinâmicaRESUMO
African sleeping sickness or human African trypanosomiasis, caused by Trypanosoma brucei spp., is responsible for approximately 30,000 deaths each year. Available treatments for this disease are poor, with unacceptable efficacy and safety profiles, particularly in the late stage of the disease when the parasite has infected the central nervous system. Here we report the validation of a molecular target and the discovery of associated lead compounds with the potential to address this lack of suitable treatments. Inhibition of this target-T. brucei N-myristoyltransferase-leads to rapid killing of trypanosomes both in vitro and in vivo and cures trypanosomiasis in mice. These high-affinity inhibitors bind into the peptide substrate pocket of the enzyme and inhibit protein N-myristoylation in trypanosomes. The compounds identified have promising pharmaceutical properties and represent an opportunity to develop oral drugs to treat this devastating disease. Our studies validate T. brucei N-myristoyltransferase as a promising therapeutic target for human African trypanosomiasis.
Assuntos
Aciltransferases/antagonistas & inibidores , Antiparasitários/farmacologia , Antiparasitários/uso terapêutico , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologia , Tripanossomíase Africana/tratamento farmacológico , Tripanossomíase Africana/parasitologia , Aciltransferases/metabolismo , Aminopiridinas/química , Aminopiridinas/metabolismo , Aminopiridinas/farmacologia , Aminopiridinas/uso terapêutico , Animais , Antiparasitários/química , Antiparasitários/metabolismo , Ensaios Enzimáticos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Camundongos , Estrutura Molecular , Pirazóis/química , Pirazóis/metabolismo , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Ratos , Sulfonamidas/química , Sulfonamidas/metabolismo , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Fatores de Tempo , Trypanosoma brucei brucei/crescimento & desenvolvimentoRESUMO
Pathogenic bacteria are endowed with an arsenal of specialized enzymes to convert nutrient compounds from their cell hosts. The essential N-acetylmannosamine-6-phosphate 2-epimerase (NanE) belongs to a convergent glycolytic pathway for utilization of the three amino sugars, GlcNAc, ManNAc, and sialic acid. The crystal structure of ligand-free NanE from Clostridium perfringens reveals a modified triose-phosphate isomerase (ß/α)8 barrel in which a stable dimer is formed by exchanging the C-terminal helix. By retaining catalytic activity in the crystalline state, the structure of the enzyme bound to the GlcNAc-6P product identifies the topology of the active site pocket and points to invariant residues Lys(66) as a putative single catalyst, supported by the structure of the catalytically inactive K66A mutant in complex with substrate ManNAc-6P. (1)H NMR-based time course assays of native NanE and mutated variants demonstrate the essential role of Lys(66) for the epimerization reaction with participation of neighboring Arg(43), Asp(126), and Glu(180) residues. These findings unveil a one-base catalytic mechanism of C2 deprotonation/reprotonation via an enolate intermediate and provide the structural basis for the development of new antimicrobial agents against this family of bacterial 2-epimerases.
Assuntos
Proteínas de Bactérias/metabolismo , Carboidratos Epimerases/metabolismo , Clostridium perfringens/enzimologia , Ácido N-Acetilneuramínico/metabolismo , Acetilglucosamina/análogos & derivados , Acetilglucosamina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Carboidratos Epimerases/química , Carboidratos Epimerases/genética , Clostridium perfringens/genética , Cristalografia por Raios X , Hexosaminas/metabolismo , Cinética , Lisina/genética , Lisina/metabolismo , Espectroscopia de Ressonância Magnética , Redes e Vias Metabólicas , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Prótons , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Fosfatos Açúcares/metabolismoRESUMO
N-Myristoyltransferase (NMT) has been shown to be essential in Leishmania and subsequently validated as a drug target in Plasmodium. Herein, we discuss the use of antifungal NMT inhibitors as a basis for inhibitor development resulting in the first sub-micromolar peptidomimetic inhibitors of Plasmodium and Leishmania NMTs. High-resolution structures of these inhibitors with Plasmodium and Leishmania NMTs permit a comparative analysis of binding modes, and provide the first crystal structure evidence for a ternary NMT-Coenzyme A/myristoylated peptide product complex.
Assuntos
Aciltransferases/antagonistas & inibidores , Antifúngicos/farmacologia , Inibidores Enzimáticos/farmacologia , Leishmania/enzimologia , Peptidomiméticos/farmacologia , Plasmodium/enzimologia , Aciltransferases/metabolismo , Antifúngicos/síntese química , Antifúngicos/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Leishmania/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , Testes de Sensibilidade Parasitária , Peptidomiméticos/síntese química , Peptidomiméticos/química , Plasmodium/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Kluyvera citrophila penicillin G acylase (KcPGA) has recently attracted increased attention relative to the well studied and commonly used Escherichia coli PGA (EcPGA) because KcPGA is more resilient to harsh conditions and is easier to immobilize for the industrial hydrolysis of natural penicillins to generate the 6-aminopenicillin (6-APA) nucleus, which is the starting material for semi-synthetic antibiotic production. Like other penicillin acylases, KcPGA is synthesized as a single-chain inactive pro-PGA, which upon autocatalytic processing becomes an active heterodimer of α and ß chains. Here, the cloning of the pac gene encoding KcPGA and the preparation of a slow-processing mutant precursor are reported. The purification, crystallization and preliminary X-ray analysis of crystals of this precursor protein are described. The protein crystallized in two different space groups, P1, with unit-cell parameters a = 54.0, b = 124.6, c = 135.1 Å, α = 104.1, ß = 101.4, γ = 96.5°, and C2, with unit-cell parameters a = 265.1, b = 54.0, c = 249.2 Å, ß = 104.4°, using the sitting-drop vapour-diffusion method. Diffraction data were collected at 100 K and the phases were determined using the molecular-replacement method. The initial maps revealed electron density for the spacer peptide.
Assuntos
Proteínas de Bactérias/genética , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Kluyvera/genética , Mutação/genética , Penicilina Amidase/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Clonagem Molecular/métodos , Cristalização , Cristalografia por Raios X , Kluyvera/enzimologia , Penicilina Amidase/biossíntese , Penicilina Amidase/química , Dobramento de ProteínaRESUMO
N-myristoylation is the irreversible attachment of a C(14) fatty acid, myristic acid, to the N-terminal glycine of a protein via formation of an amide bond. This modification is catalyzed by myristoyl-coenzyme A (CoA):protein N-myristoyltransferase (NMT), an enzyme ubiquitous in eukaryotes that is up-regulated in several cancers. Here we report a sensitive fluorescence-based assay to study the enzymatic activity of human NMT1 and NMT2 based on detection of CoA by 7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin. We also describe expression and characterization of NMT1 and NMT2 and assay validation with small molecule inhibitors. This assay should be broadly applicable to NMTs from a range of organisms.
Assuntos
Aciltransferases/análise , Aciltransferases/antagonistas & inibidores , Aciltransferases/genética , Aciltransferases/metabolismo , Coenzima A , Cumarínicos , Fluorescência , Corantes Fluorescentes , Humanos , Cinética , Ácidos Mirísticos/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Plasmodium falciparum is the causative agent of malaria, a disease where new drug targets are required due to increasing resistance to current anti-malarials. TMPK (thymidylate kinase) is a good candidate as it is essential for the synthesis of dTTP, a critical precursor of DNA and has been much studied due to its role in prodrug activation and as a drug target. Type I TMPKs, such as the human enzyme, phosphorylate the substrate AZT (3'-azido-3'-deoxythymidine)-MP (monophosphate) inefficiently compared with type II TMPKs (e.g. Escherichia coli TMPK). In the present paper we report that eukaryotic PfTMPK (P. falciparum TMPK) presents sequence features of a type I enzyme yet the kinetic parameters for AZT-MP phosphorylation are similar to those of the highly efficient E. coli enzyme. Structural information shows that this is explained by a different juxtaposition of the P-loop and the azide of AZT-MP. Subsequent formation of the transition state requires no further movement of the PfTMPK P-loop, with no steric conflicts for the azide moiety, allowing efficient phosphate transfer. Likewise, we present results that confirm the ability of the enzyme to uniquely accept dGMP as a substrate and shed light on the basis for its wider substrate specificity. Information resulting from two ternary complexes (dTMP-ADP and AZT-MP-ADP) and a binary complex with the transition state analogue AP5dT [P1-(5'-adenosyl)-P5-(5'-thymidyl) pentaphosphate] all reveal significant differences with the human enzyme, notably in the lid region and in the P-loop which may be exploited in the rational design of Plasmodium-specific TMPK inhibitors with therapeutic potential.
Assuntos
Nucleotídeos de Desoxiguanina/metabolismo , Didesoxinucleotídeos/química , Didesoxinucleotídeos/metabolismo , Núcleosídeo-Fosfato Quinase/química , Plasmodium falciparum/enzimologia , Nucleotídeos de Timina/química , Nucleotídeos de Timina/metabolismo , Zidovudina/análogos & derivados , Nucleotídeos de Desoxiguanina/química , Cinética , Núcleosídeo-Fosfato Quinase/metabolismo , Fosforilação , Plasmodium falciparum/metabolismo , Especificidade por Substrato , Zidovudina/química , Zidovudina/metabolismoRESUMO
The leishmaniases are infectious diseases caused by a number of species of obligate intracellular protozoa of the genus Leishmania with disease manifesting as cutaneous, mucocutaneous and visceral forms. Despite being endemic in more than 80 countries and its being the cause of high morbidity and mortality, leishmaniasis remains a neglected tropical disease. Chemotherapy is the frontline treatment, but drugs in current use suffer from toxic side effects, difficulties in administration and extended treatment times - moreover, resistance is emerging. New anti-leishmanial drugs are a recognised international priority. Here, we review investigations into N-myristoyltransferase (NMT) as a potential drug target. NMT catalyses the co-translational transfer of a C14 fatty acid from myristoyl-CoA onto the N-terminal glycine residue of a significant subset of proteins in eukaryotic cells. This covalent modification influences the stability and interactions of substrate proteins with lipids and partner proteins. Structure-guided development of new lead compounds emerging from high-throughput screening campaigns targeting Leishmania donovani NMT has led to the discovery of potent inhibitors which have been used to gain insights into the role of protein myristoylation in these parasites and to validate NMT as a drug target.
RESUMO
Malaria pathology is caused by multiplication of asexual parasites within erythrocytes, whereas mosquito transmission of malaria is mediated by sexual precursor cells (gametocytes). Microarray analysis identified glycerol kinase (GK) as the second most highly upregulated gene in Plasmodium falciparum gametocytes with no expression detectable in asexual blood stage parasites. Phosphorylation of glycerol by GK is the rate-limiting step in glycerol utilization. Deletion of this gene from P. falciparum had no effect on asexual parasite growth, but surprisingly also had no effect on gametocyte development or exflagellation, suggesting that these life cycle stages do not utilize host-derived glycerol as a carbon source. Kinetic studies of purified PfGK showed that the enzyme is not regulated by fructose 1,6 bisphosphate. The high-resolution crystal structure of P. falciparum GK, the first of a eukaryotic GK, reveals two domains embracing a capacious ligand-binding groove. In the complexes of PfGK with glycerol and ADP, we observed closed and open forms of the active site respectively. The 27 degree domain opening is larger than in orthologous systems and exposes an extensive surface with potential for exploitation in selective inhibitor design should the enzyme prove to be essential in vivo either in the human or in the mosquito.
Assuntos
Glicerol Quinase/metabolismo , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Genótipo , Glicerol/metabolismo , Glicerol Quinase/genética , Modelos Moleculares , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Fosforilação , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Estrutura Terciária de Proteína , Proteínas de Protozoários/genética , RNA de Protozoário/genética , Alinhamento de SequênciaRESUMO
CotE is a coat protein that is present in the spores of Clostridium difficile, an obligate anaerobic bacterium and a pathogen that is a leading cause of antibiotic-associated diarrhoea in hospital patients. Spores serve as the agents of disease transmission, and CotE has been implicated in their attachment to the gut epithelium and subsequent colonization of the host. CotE consists of an N-terminal peroxiredoxin domain and a C-terminal chitinase domain. Here, a C-terminal fragment of CotE comprising residues 349-712 has been crystallized and its structure has been determined to reveal a core eight-stranded ß-barrel fold with a neighbouring subdomain containing a five-stranded ß-sheet. A prominent groove running across the top of the barrel is lined by residues that are conserved in family 18 glycosyl hydrolases and which participate in catalysis. Electron density identified in the groove defines the pentapeptide Gly-Pro-Ala-Met-Lys derived from the N-terminus of the protein following proteolytic cleavage to remove an affinity-purification tag. These observations suggest the possibility of designing peptidomimetics to block C. difficile transmission.
Assuntos
Proteínas de Bactérias/química , Quitinases/química , Clostridioides difficile/metabolismo , Peroxirredoxinas/química , Proteínas de Plantas/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Quitinases/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Peroxirredoxinas/metabolismo , Proteínas de Plantas/metabolismo , Conformação ProteicaRESUMO
The leishmaniases, caused by Leishmania species of protozoan parasites, are neglected tropical diseases with millions of cases worldwide. Current therapeutic approaches are limited by toxicity, resistance, and cost. N-Myristoyltransferase (NMT), an enzyme ubiquitous and essential in all eukaryotes, has been validated via genetic and pharmacological methods as a promising anti-leishmanial target. Here we describe a comprehensive structure-activity relationship (SAR) study of a thienopyrimidine series previously identified in a high-throughput screen against Leishmania NMT, across 68 compounds in enzyme- and cell-based assay formats. Using a chemical tagging target engagement biomarker assay, we identify the first inhibitor in this series with on-target NMT activity in leishmania parasites. Furthermore, crystal structure analyses of 12 derivatives in complex with Leishmania major NMT revealed key factors important for future structure-guided optimization delivering IMP-105 (43), a compound with modest activity against Leishmania donovani intracellular amastigotes and excellent selectivity (>660-fold) for Leishmania NMT over human NMTs.
Assuntos
Aciltransferases/antagonistas & inibidores , Antiprotozoários/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Pirimidinas/farmacologia , Tiofenos/farmacologia , Aciltransferases/química , Aciltransferases/metabolismo , Antiprotozoários/síntese química , Antiprotozoários/metabolismo , Sítios de Ligação , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Leishmania donovani/enzimologia , Leishmania major/enzimologia , Estrutura Molecular , Testes de Sensibilidade Parasitária , Ligação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Pirimidinas/síntese química , Pirimidinas/metabolismo , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/metabolismoRESUMO
The bioconjugation of proteins with small molecules has proved an invaluable strategy for probing and perturbing biological mechanisms. The general use of chemical methods for protein functionalisation can be limited however by the requirement for complicated reaction partners to be present in large excess, and harsh conditions which are incompatible with many protein scaffolds. Herein we describe a site-selective organocatalyst-mediated protein aldol ligation (OPAL) that affords stable carbon-carbon linked bioconjugates at neutral pH. OPAL enables rapid modification of proteins using simple aldehyde probes in minimal excess, and is utilised here in the affinity tagging of proteins in cell lysate. Furthermore we demonstrate that the ß-hydroxy aldehyde OPAL product can be functionalised again at neutral pH in a tandem organocatalyst-mediated oxime ligation. This tandem strategy is showcased in the 'chemical mimicry' of a previously inaccessible natural dual post-translationally modified protein integral to the pathogenesis of the neglected tropical disease Leishmaniasis.
RESUMO
Rhinoviruses (RVs) are the pathogens most often responsible for the common cold, and are a frequent cause of exacerbations in asthma, chronic obstructive pulmonary disease and cystic fibrosis. Here we report the discovery of IMP-1088, a picomolar dual inhibitor of the human N-myristoyltransferases NMT1 and NMT2, and use it to demonstrate that pharmacological inhibition of host-cell N-myristoylation rapidly and completely prevents rhinoviral replication without inducing cytotoxicity. The identification of cooperative binding between weak-binding fragments led to rapid inhibitor optimization through fragment reconstruction, structure-guided fragment linking and conformational control over linker geometry. We show that inhibition of the co-translational myristoylation of a specific virus-encoded protein (VP0) by IMP-1088 potently blocks a key step in viral capsid assembly, to deliver a low nanomolar antiviral activity against multiple RV strains, poliovirus and foot and-mouth disease virus, and protection of cells against virus-induced killing, highlighting the potential of host myristoylation as a drug target in picornaviral infections.
Assuntos
Aciltransferases/antagonistas & inibidores , Antivirais/farmacologia , Capsídeo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Rhinovirus/efeitos dos fármacos , Montagem de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Antivirais/química , Inibidores Enzimáticos/química , Células HeLa , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Rhinovirus/enzimologia , Rhinovirus/fisiologiaRESUMO
The parasite Plasmodium vivax is the most widely distributed cause of recurring malaria. N-Myristoyltransferase (NMT), an enzyme that catalyses the covalent attachment of myristate to the N-terminal glycine of substrate proteins, has been described as a potential target for the treatment of this disease. Herein, we report the synthesis and the structure-guided optimization of a series of quinolines with balanced activity against both Plasmodium vivax and Plasmodium falciparum N-myristoyltransferase (NMT).
RESUMO
Besides their role as a source of amino acids for Bacillus subtilis, exogenous peptides play important roles in the signalling pathways leading to the development of competence and sporulation. B.subtilis has three peptide transport systems all belonging to the ATP-binding cassette family, a dipeptide permease (Dpp) and two oligopeptide permeases (Opp and App) with overlapping specificity. These comprise a membrane-spanning channel through which the peptide passes, a pair of ATPases which couple ATP hydrolysis to peptide translocation and a lipid-modified, membrane-anchored extracellular "binding-protein" that serves as the receptor for the system. Here, we present the crystal structure of a soluble form of the peptide-binding protein AppA, which has been solved to 1.6 A spacing by anomalous scattering and molecular replacement methods. The structure reveals a protein made of two distinct lobes with a topology similar to those of DppA from Escherichia coli and OppA from Salmonella typhimurium. Examination of the interlobe region reveals an enlarged pocket, containing electron density defining a nonapeptide ligand. The main-chain of the peptide is well defined and makes a series of polar contacts with the protein including salt-bridges at both its termini. The side-chain density is ambiguous in places, consistent with the interpretation that a population of peptides is bound, whose average electron density resembles the amino acid sequence N-VDSKNTSSW-C.
Assuntos
Bacillus subtilis/química , Bacillus subtilis/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Lipoproteínas/química , Lipoproteínas/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Ligantes , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Conformação ProteicaRESUMO
BACKGROUND: Superoxide dismutases (SODs) are important enzymes in defence against oxidative stress. In Plasmodium falciparum, they may be expected to have special significance since part of the parasite life cycle is spent in red blood cells where the formation of reactive oxygen species is likely to be promoted by the products of haemoglobin breakdown. Thus, inhibitors of P. falciparum SODs have potential as anti-malarial compounds. As a step towards their development we have determined the crystal structure of the parasite's cytosolic iron superoxide dismutase. RESULTS: The cytosolic iron superoxide dismutase from P. falciparum (PfFeSOD) has been overexpressed in E. coli in a catalytically active form. Its crystal structure has been solved by molecular replacement and refined against data extending to 2.5 A resolution. The structure reveals a two-domain organisation and an iron centre in which the metal is coordinated by three histidines, an aspartate and a solvent molecule. Consistent with ultracentrifugation analysis the enzyme is a dimer in which a hydrogen bonding lattice links the two active centres. CONCLUSION: The tertiary structure of PfFeSOD is very similar to those of a number of other iron-and manganese-dependent superoxide dismutases, moreover the active site residues are conserved suggesting a common mechanism of action. Comparison of the dimer interfaces of PfFeSOD with the human manganese-dependent superoxide dismutase reveals a number of differences, which may underpin the design of parasite-selective superoxide dismutase inhibitors.
Assuntos
Plasmodium falciparum/enzimologia , Superóxido Dismutase/química , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Dimerização , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Homologia de Sequência de Aminoácidos , Superóxido Dismutase/genéticaRESUMO
The DNA-binding protein GerE acts as both a repressor and an activator of transcription of genes transcribed by sigma(K)-RNA polymerase (RNA-P) during the later stages of endospore formation in Bacillus subtilis. GerE represses transcription from the sigK promoter, and activates transcription from other promoters, including cotC and cotX. Two different regions of GerE (AR1 and AR2) are required for activation of cotC and cotX, respectively. We used a genetic screen to seek mutations that would define additional regions of GerE required for promoter activation. We found that a substitution of proline for leucine at position 12 of GerE (L12P) decreased cotC promoter activity but did not interfere with GerE-dependent repression of the sigK promoter or with activation of the cotX promoter in vivo. We also found that the L12P substitution had no effect on binding to cotC in vitro. However, the L12P-substituted GerE failed to stimulate cotC transcription in vitro, whereas it stimulated transcription from PcotX. The crystal structure of GerE suggests that L12 is not exposed on the surface of the molecule. Therefore, we propose that the L12P substitution reduces the flexibility of the N-terminal arm, preventing an interaction of AR1 with RNA-P that is essential for activation of the cotC promoter.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Fator sigma , Alelos , Substituição de Aminoácidos , Pegada de DNA , Dimerização , Modelos Moleculares , Mutação , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The response regulator Spo0A is the master control element in the initiation of sporulation in Bacillus subtilis. Like many other multi-domain response regulators, the latent activity of the effector, C-terminal domain is stimulated by phosphorylation on a conserved aspartic acid residue in the regulatory, N-terminal domain. If a threshold concentration of phosphorylated Spo0A is achieved, the transcription of genes required for sporulation is activated, whereas the genes encoding stationary phase sentinels are repressed, and sporulation proceeds. Despite detailed genetic, biochemical and structural characterisation, it is not understood how the phosphorylation signal in the receiver domain is transduced into DNA binding and transcription activation in the distal effector domain. An obstacle to our understanding of Spo0A function is the uncertainty concerning changes in quaternary structure that accompany phosphorylation. Here we have revisited this question and shown unequivocally that Spo0A forms dimers upon phosphorylation and that the subunit interactions in the dimer are mediated principally by the receiver domain. Purified dimers of two mutants of Spo0A, in which the phosphorylatable aspartic acid residue has been substituted, activate transcription from the spoIIG promoter in vitro, whereas monomers do not. This suggests that dimers represent the activated form of Spo0A.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Fator sigma , Esporos Bacterianos/genética , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Bacillus subtilis/enzimologia , Bacillus subtilis/fisiologia , Proteínas de Bactérias/genética , Cromatografia em Gel , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Dimerização , Genes Bacterianos/genética , Genes Reguladores/genética , Modelos Biológicos , Modelos Moleculares , Peso Molecular , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas , Fatores de Transcrição/genética , UltracentrifugaçãoRESUMO
yloQ is one of 11 essential genes in Bacillus subtilis with unknown roles in the physiology of the cell. It encodes a polypeptide of 298 residues with motifs characteristic of GTPases. As a contribution to elucidating its indispensable cellular function, we have solved the crystal structure of YloQ to 1.6 A spacing, revealing a three-domain organisation. At the heart of the molecule is the putative GTPase domain, which exhibits a classical alpha/beta nucleotide-binding fold with a topology very similar to that of Ras and Era. However, as anticipated from the order in which the conserved G protein motifs appear in the sequence, the GTPase domain fold in YloQ is circularly permuted with respect to the classical GTPases. The nucleotide-binding pocket in YloQ is unoccupied, and analysis of the phosphate-binding (P) loop indicates that conformational changes in this region would be needed to accommodate GTP. The GTPase domain is flanked at its N terminus by a beta-barrel domain with an oligonucleotide/oligosaccharide-binding (OB) fold, and at its C terminus by an alpha-helical domain containing a coordinated zinc ion. This combination of protein modules is unique to YloQ and its orthologues. Sequence comparisons reveal a clustering of conserved basic and aromatic residues on one face of the OB domain, perhaps pointing to a role for YloQ in nucleic acid binding. The zinc ion in the alpha-helical domain is coordinated by three cysteine residues and a histidine residue in a novel ligand organisation. The juxtaposition of the switch I and switch II regions of the G domain and the OB and zinc-binding domains suggests that chemical events at the GTPase active site may be transduced into relative movements of these domains. The pattern of conserved residues and electrostatic surface potential calculations suggest that the OB and/or Zn-binding domains participate in nucleic acid binding consistent with a possible role for YloQ at some stage during mRNA translation.
Assuntos
Bacillus subtilis/enzimologia , Cristalografia por Raios X , GTP Fosfo-Hidrolases/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Evolução Molecular , GTP Fosfo-Hidrolases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Zinco/químicaRESUMO
Esterases and deacetylases active on carbohydrate ligands have been classified into 14 families based upon amino acid sequence similarities. Enzymes from carbohydrate esterase family seven (CE-7) are unusual in that they display activity towards both acetylated xylooligosaccharides and the antibiotic, cephalosporin C. The 1.9A structure of the multifunctional CE-7 esterase (hereinafter CAH) from Bacillus subtilis 168 reveals a classical alpha/beta hydrolase fold encased within a 32 hexamer. This is the first example of a hexameric alpha/beta hydrolase and is further evidence of the versatility of this particular fold, which is used in a wide variety of biological contexts. A narrow entrance tunnel leads to the centre of the molecule, where the six active-centre catalytic triads point towards the tunnel interior and thus are sequestered away from cytoplasmic contents. By analogy to self-compartmentalising proteases, the tunnel entrance may function to hinder access of large substrates to the poly-specific active centre. This would explain the observation that the enzyme is active on a variety of small, acetylated molecules. The structure of an active site mutant in complex with the reaction product, acetate, reveals details of the putative oxyanion binding site, and suggests that substrates bind predominantly through non-specific contacts with protein hydrophobic residues. Protein residues involved in catalysis are tethered by interactions with protein excursions from the canonical alpha/beta hydrolase fold. These excursions also mediate quaternary structure maintenance, so it would appear that catalytic competence is only achieved on protein multimerisation. We suggest that the acetyl xylan esterase (EC 3.1.1.72) and cephalosporin C deacetylase (EC 3.1.1.41) enzymes of the CE-7 family represent a single class of proteins with a multifunctional deacetylase activity against a range of small substrates.