RESUMO
Penicillin binding proteins (PBPs) catalyzing transpeptidation reactions that stabilize the peptidoglycan component of the bacterial cell wall are the targets of ß-lactams, the most clinically successful antibiotics to date. However, PBP-transpeptidation enzymology has evaded detailed analysis, because of the historical unavailability of kinetically competent assays with physiologically relevant substrates and the previously unappreciated contribution of protein cofactors to PBP activity. By re-engineering peptidoglycan synthesis, we have constructed a continuous spectrophotometric assay for transpeptidation of native or near native peptidoglycan precursors and fragments by Escherichia coli PBP1B, allowing us to (a) identify recognition elements of transpeptidase substrates, (b) reveal a novel mechanism of stereochemical editing within peptidoglycan transpeptidation, (c) assess the impact of peptidoglycan substrates on ß-lactam targeting of transpeptidation, and (d) demonstrate that both substrates have to be bound before transpeptidation occurs. The results allow characterization of high molecular weight PBPs as enzymes and not merely the targets of ß-lactam acylation.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Proteínas de Ligação às Penicilinas/química , Peptidoglicano Glicosiltransferase/química , Peptidoglicano/química , Monossacarídeos de Poli-Isoprenil Fosfato/química , Oligossacarídeos de Poli-Isoprenil Fosfato/química , D-Ala-D-Ala Carboxipeptidase Tipo Serina/química , Proteínas da Membrana Bacteriana Externa/química , Biocatálise , Ensaios Enzimáticos/métodos , Cinética , Estereoisomerismo , Especificidade por SubstratoRESUMO
RlmJ catalyzes the m(6)A2030 methylation of 23S rRNA during ribosome biogenesis in Escherichia coli. Here, we present crystal structures of RlmJ in apo form, in complex with the cofactor S-adenosyl-methionine and in complex with S-adenosyl-homocysteine plus the substrate analogue adenosine monophosphate (AMP). RlmJ displays a variant of the Rossmann-like methyltransferase (MTase) fold with an inserted helical subdomain. Binding of cofactor and substrate induces a large shift of the N-terminal motif X tail to make it cover the cofactor binding site and trigger active-site changes in motifs IV and VIII. Adenosine monophosphate binds in a partly accommodated state with the target N6 atom 7 Å away from the sulphur of AdoHcy. The active site of RlmJ with motif IV sequence 164DPPY167 is more similar to DNA m(6)A MTases than to RNA m(6)2A MTases, and structural comparison suggests that RlmJ binds its substrate base similarly to DNA MTases T4Dam and M.TaqI. RlmJ methylates in vitro transcribed 23S rRNA, as well as a minimal substrate corresponding to helix 72, demonstrating independence of previous modifications and tertiary interactions in the RNA substrate. RlmJ displays specificity for adenosine, and mutagenesis experiments demonstrate the critical roles of residues Y4, H6, K18 and D164 in methyl transfer.
Assuntos
Adenina/análogos & derivados , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Metiltransferases/química , RNA Ribossômico 23S/metabolismo , Adenina/química , Adenina/metabolismo , Monofosfato de Adenosina/química , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Coenzimas/química , Coenzimas/metabolismo , Proteínas de Escherichia coli/metabolismo , Metiltransferases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , RNA Ribossômico 23S/química , S-Adenosil-Homocisteína/química , S-Adenosilmetionina/química , Alinhamento de SequênciaRESUMO
RlmM (YgdE) catalyzes the S-adenosyl methionine (AdoMet)-dependent 2'O methylation of C2498 in 23S ribosomal RNA (rRNA) of Escherichia coli. Previous experiments have shown that RlmM is active on 23S rRNA from an RlmM knockout strain but not on mature 50S subunits from the same strain. Here, we demonstrate RlmM methyltransferase (MTase) activity on in vitro transcribed 23S rRNA and its domain V. We have solved crystal structures of E. coli RlmM at 1.9 Å resolution and of an RlmM-AdoMet complex at 2.6 Å resolution. RlmM consists of an N-terminal THUMP domain and a C-terminal catalytic Rossmann-like fold MTase domain in a novel arrangement. The catalytic domain of RlmM is closely related to YiiB, TlyA and fibrillarins, with the second K of the catalytic tetrad KDKE shifted by two residues at the C-terminal end of a beta strand compared with most 2'O MTases. The AdoMet-binding site is open and shallow, suggesting that RNA substrate binding may be required to form a conformation needed for catalysis. A continuous surface of conserved positive charge indicates that RlmM uses one side of the two domains and the inter-domain linker to recognize its RNA substrate.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Metiltransferases/química , RNA Ribossômico 23S/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Metiltransferases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , RNA Ribossômico 23S/química , S-Adenosilmetionina/química , Alinhamento de SequênciaRESUMO
Methyltransferase RlmJ uses the cofactor S-adenosylmethionine to methylate the exocyclic nitrogen N6 of nucleotide A2030 in 23S rRNA during ribosome assembly in Escherichia coli. RlmJ with a C-terminal hexahistidine tag was overexpressed in E. coli and purified as a monomer using Ni(2+)-affinity and size-exclusion chromatography. The recombinant RlmJ was crystallized using the sitting-drop vapour-diffusion method and a full data set was collected to 1.85â Å resolution from a single apo crystal. The crystals belonged to space group P2(1), with unit-cell parameters a = 46.9, b = 77.8, c = 82.5â Å, ß = 104°. Data analysis suggested two molecules per asymmetric unit and a Matthews coefficient of 2.20â Å(3)â Da(-1).
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/química , Metiltransferases/química , RNA Ribossômico 23S/química , S-Adenosilmetionina/química , Sequência de Aminoácidos , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Expressão Gênica , Metiltransferases/genética , Metiltransferases/isolamento & purificação , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Ribossomos/genética , Ribossomos/metabolismoRESUMO
There is an urgent need for new tuberculosis (TB) treatments, with novel modes of action, to reduce the incidence/mortality of TB and to combat resistance to current treatments. Through both chemical and genetic methodologies, polyketide synthase 13 (Pks13) has been validated as essential for mycobacterial survival and as an attractive target for Mycobacterium tuberculosis growth inhibitors. A benzofuran series of inhibitors that targeted the Pks13 thioesterase domain, failed to progress to preclinical development due to concerns over cardiotoxicity. Herein, we report the identification of a novel oxadiazole series of Pks13 inhibitors, derived from a high-throughput screening hit and structure-guided optimization. This new series binds in the Pks13 thioesterase domain, with a distinct binding mode compared to the benzofuran series. Through iterative rounds of design, assisted by structural information, lead compounds were identified with improved antitubercular potencies (MIC < 1 µM) and in vitro ADMET profiles.
Assuntos
Benzofuranos , Mycobacterium tuberculosis , Policetídeo Sintases , Antituberculosos/química , Mycobacterium tuberculosis/metabolismo , Benzofuranos/química , Testes de Sensibilidade MicrobianaRESUMO
We identify the Plasmodium falciparum acetyl-coenzyme A synthetase (PfAcAS) as a druggable target, using genetic and chemical validation. In vitro evolution of resistance with two antiplasmodial drug-like compounds (MMV019721 and MMV084978) selects for mutations in PfAcAS. Metabolic profiling of compound-treated parasites reveals changes in acetyl-CoA levels for both compounds. Genome editing confirms that mutations in PfAcAS are sufficient to confer resistance. Knockdown studies demonstrate that PfAcAS is essential for asexual growth, and partial knockdown induces hypersensitivity to both compounds. In vitro biochemical assays using recombinantly expressed PfAcAS validates that MMV019721 and MMV084978 directly inhibit the enzyme by preventing CoA and acetate binding, respectively. Immunolocalization studies reveal that PfAcAS is primarily localized to the nucleus. Functional studies demonstrate inhibition of histone acetylation in compound-treated wild-type, but not in resistant parasites. Our findings identify and validate PfAcAS as an essential, druggable target involved in the epigenetic regulation of gene expression.
Assuntos
Acetato-CoA Ligase/antagonistas & inibidores , Antimaláricos/farmacologia , Inibidores Enzimáticos/farmacologia , Malária/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Acetato-CoA Ligase/metabolismo , Antimaláricos/química , Inibidores Enzimáticos/química , Humanos , Malária/metabolismo , Modelos Moleculares , Estrutura Molecular , Testes de Sensibilidade Parasitária , Plasmodium falciparum/enzimologiaRESUMO
Heat shock factor 1 (HSF1) and 2 (HSF2) play distinct but overlapping regulatory roles in maintaining cellular proteostasis or mediating cell differentiation and development. Upon activation, both HSFs trimerize and bind to heat shock elements (HSEs) present in the promoter region of target genes. Despite structural insights gained from recent studies, structures reflecting the physiological architecture of this transcriptional machinery remains to be determined. Here, we present co-crystal structures of human HSF1 and HSF2 trimers bound to DNA, which reveal a triangular arrangement of the three DNA-binding domains (DBDs) with protein-protein interactions largely mediated by the wing domain. Two structural properties, different flexibility of the wing domain and local DNA conformational changes induced by HSF binding, seem likely to contribute to the subtle differential specificity between HSF1 and HSF2. Besides, two more structures showing DBDs bound to "two-site" head-to-head HSEs were determined as additions to the published tail-to-tail dimer-binding structures.
RESUMO
Aminoacyl-tRNA synthetases are ubiquitous and essential enzymes for protein synthesis and also a variety of other metabolic processes, especially in bacterial species. Bacterial aminoacyl-tRNA synthetases represent attractive and validated targets for antimicrobial drug discovery if issues of prokaryotic versus eukaryotic selectivity and antibiotic resistance generation can be addressed. We have determined high-resolution X-ray crystal structures of the Escherichia coli and Staphylococcus aureus seryl-tRNA synthetases in complex with aminoacyl adenylate analogues and applied a structure-based drug discovery approach to explore and identify a series of small molecule inhibitors that selectively inhibit bacterial seryl-tRNA synthetases with greater than 2 orders of magnitude compared to their human homologue, demonstrating a route to the selective chemical inhibition of these bacterial targets.
Assuntos
Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Sondas Moleculares/química , Serina-tRNA Ligase/antagonistas & inibidores , Staphylococcus aureus/enzimologia , Cristalografia por Raios X , Inibidores Enzimáticos/química , Estrutura Molecular , Serina-tRNA Ligase/químicaRESUMO
Bacterial peptidoglycan glycosyltransferases (PGT) catalyse the essential polymerization of lipid II into linear glycan chains required for peptidoglycan biosynthesis. The PGT domain is composed of a large head subdomain and a smaller jaw subdomain and can be potently inhibited by the antibiotic moenomycin A (MoeA). We present an X-ray structure of the MoeA-bound Staphylococcus aureus monofunctional PGT enzyme, revealing electron density for a second MoeA bound to the jaw subdomain as well as the PGT donor site. Isothermal titration calorimetry confirms two drug-binding sites with markedly different affinities and positive cooperativity. Hydrophobic cluster analysis suggests that the membrane-interacting surface of the jaw subdomain has structural and physicochemical properties similar to amphipathic cationic α -helical antimicrobial peptides for lipid II recognition and binding. Furthermore, molecular dynamics simulations of the drug-free and -bound forms of the enzyme demonstrate the importance of the jaw subdomain movement for lipid II selection and polymerization process and provide molecular-level insights into the mechanism of peptidoglycan biosynthesis by PGTs.
RESUMO
MetR, a LysR-type transcriptional regulator (LTTR), has been extensively studied owing to its role in the control of methionine biosynthesis in proteobacteria. A MetR homodimer binds to a 24-base-pair operator region of the met genes and specifically recognizes the interrupted palindromic sequence 5'-TGAA-N5-TTCA-3'. Mechanistic details underlying the interaction of MetR with its target DNA at the molecular level remain unknown. In this work, the crystal structure of the DNA-binding domain (DBD) of MetR was determined at 2.16â Å resolution. MetR-DBD adopts a winged-helix-turn-helix (wHTH) motif and shares significant fold similarity with the DBD of the LTTR protein BenM. Furthermore, a data-driven macromolecular-docking strategy was used to model the structure of MetR-DBD bound to DNA, which revealed that a bent conformation of DNA is required for the recognition helix α3 and the wing loop of the wHTH motif to interact with the major and minor grooves, respectively. Comparison of the MetR-DBD-DNA complex with the crystal structures of other LTTR-DBD-DNA complexes revealed residues that may confer operator-sequence binding specificity for MetR. Taken together, the results show that MetR-DBD uses a combination of direct base-specific interactions and indirect shape recognition of the promoter to regulate the transcription of met genes.
Assuntos
Proteínas de Bactérias/química , DNA/química , Transativadores/química , Transcrição Gênica , Cristalização , Conformação de Ácido NucleicoRESUMO
The affinity and specificity of protein-ligand interactions are influenced by energetic crosstalk within the protein domain. However, the molecular details of such intradomain allostery are still unclear. Here, we have experimentally detected and computationally predicted interaction pathways in the postsynaptic density 95/discs large/zonula occludens 1 (PDZ)-peptide ligand model system using wild-type and circularly permuted PDZ proteins. The circular permutant introduced small perturbations in the tertiary structure and a concomitant rewiring of allosteric pathways, allowing us to describe how subtle changes may reshape energetic signaling. The results were analyzed in the context of other members of the PDZ family, which were found to contain distinct interaction pathways for different peptide ligands. The data reveal a fascinating scenario whereby several energetic pathways are sampled within one single domain and distinct pathways are activated by specific protein ligands.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas de Membrana/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Sítio Alostérico , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Proteína 1 Homóloga a Discs-Large , Humanos , Ligação de Hidrogênio , Cinética , Proteínas de Membrana/genética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Domínios PDZ , Ligação Proteica , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
Circular permutation is a common molecular mechanism for evolution of proteins. However, such re-arrangement of secondary structure connectivity may interfere with the folding mechanism causing accumulation of folding intermediates, which in turn can lead to misfolding. We solved the crystal structure and investigated the folding pathway of a circularly permuted variant of a PDZ domain, SAP97 PDZ2. Our data illustrate how well circular permutation may work as a mechanism for molecular evolution. The circular permutant retains the overall structure and function of the native protein domain. Further, unlike most examples in the literature, this circular permutant displays a folding mechanism that is virtually identical to that of the wild type. This observation contrasts with previous data on the circularly permuted PDZ2 domain from PTP-BL, for which the folding pathway was remarkably affected by the same mutation in sequence connectivity. The different effects of this circular permutation in two homologous proteins show the strong influence of sequence as compared to topology. Circular permutation, when peripheral to the major folding nucleus, may have little effect on folding pathways and could explain why, despite the dramatic change in primary structure, it is frequently tolerated by different protein folds.