RESUMO
The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an α2ß2 tetrameric enzyme in which the α-chain harbors the 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) active sites, and the ß-chain provides the 3-ketoacyl-CoA thiolase (KAT) active site. Linear, medium-chain and long-chain 2E-enoyl-CoA molecules are the preferred substrates of MtTFE. Previous crystallographic binding and modeling studies identified binding sites for the acyl-CoA substrates at the three active sites, as well as the NAD binding pocket at the HAD active site. These studies also identified three additional CoA binding sites on the surface of MtTFE that are different from the active sites. It has been proposed that one of these additional sites could be of functional relevance for the substrate channeling (by surface crawling) of reaction intermediates between the three active sites. Here, 226 fragments were screened in a crystallographic fragment-binding study of MtTFE crystals, resulting in the structures of 16 MtTFE-fragment complexes. Analysis of the 121 fragment-binding events shows that the ECH active site is the `binding hotspot' for the tested fragments, with 41 binding events. The mode of binding of the fragments bound at the active sites provides additional insight into how the long-chain acyl moiety of the substrates can be accommodated at their proposed binding pockets. In addition, the 20 fragment-binding events between the active sites identify potential transient binding sites of reaction intermediates relevant to the possible channeling of substrates between these active sites. These results provide a basis for further studies to understand the functional relevance of the latter binding sites and to identify substrates for which channeling is crucial.
Assuntos
Acil Coenzima A , Proteínas de Bactérias , Domínio Catalítico , Mycobacterium tuberculosis , Mycobacterium tuberculosis/enzimologia , Cristalografia por Raios X , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Acil Coenzima A/metabolismo , Acil Coenzima A/química , Especificidade por Substrato , Sítios de Ligação , Modelos Moleculares , Enoil-CoA Hidratase/metabolismo , Enoil-CoA Hidratase/química , Ligação Proteica , 3-Hidroxiacil-CoA Desidrogenases/química , 3-Hidroxiacil-CoA Desidrogenases/metabolismoRESUMO
KDM4 histone demethylases became an exciting target for inhibitor development as the evidence linking them directly to tumorigenesis mounts. In this study, we set out to better understand the binding cavity using an X-ray crystallographic approach to provide a detailed landscape of possible interactions within the under-investigated region of KDM4. Our design strategy was based on utilizing known KDM binding motifs, such as nicotinic acid and tetrazolylhydrazides, as core motifs that we decided to enrich with flexible tails to map the distal histone binding site. The resulting X-ray structures of the novel compounds bound to KDM4D, a representative of the KDM4 family, revealed the interaction pattern with distal residues in the histone-binding site. The most prominent protein rearrangement detected upon ligand binding is the loop movement that blocks the accessibility to the histone binding site. Apart from providing new sites that potential inhibitors can target, the novel compounds may prove helpful in exploring the capacity of ligands to bind in sites distal to the cofactor-binding site of other KDMs or 2-oxoglutarate (2OG)-dependent oxygenases. The case study proves that combining a strong small binding motif with flexible tails to probe the binding pocket will facilitate lead discovery in classical drug-discovery campaigns, given the ease of accessing X-ray quality crystals.
Assuntos
Histonas , Histona Desmetilases com o Domínio Jumonji , Piridinas , Tetrazóis , Histona Desmetilases com o Domínio Jumonji/metabolismo , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/química , Tetrazóis/química , Tetrazóis/farmacologia , Tetrazóis/metabolismo , Tetrazóis/síntese química , Piridinas/química , Piridinas/farmacologia , Piridinas/metabolismo , Piridinas/síntese química , Humanos , Sítios de Ligação , Cristalografia por Raios X , Relação Estrutura-Atividade , Histonas/metabolismo , Histonas/química , Estrutura Molecular , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Modelos Moleculares , Relação Dose-Resposta a DrogaRESUMO
CdaA is the most widespread diadenylate cyclase in many bacterial species, including several multidrug-resistant human pathogens. The enzymatic product of CdaA, cyclic di-AMP, is a secondary messenger that is essential for the viability of many bacteria. Its absence in humans makes CdaA a very promising and attractive target for the development of new antibiotics. Here, the structural results are presented of a crystallographic fragment screen against CdaA from Listeria monocytogenes, a saprophytic Gram-positive bacterium and an opportunistic food-borne pathogen that can cause listeriosis in humans and animals. Two of the eight fragment molecules reported here were localized in the highly conserved ATP-binding site. These fragments could serve as potential starting points for the development of antibiotics against several CdaA-dependent bacterial species.
Assuntos
Listeria monocytogenes , Listeria monocytogenes/enzimologia , Cristalografia por Raios X/métodos , Sítios de Ligação , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Fosfatos de Dinucleosídeos/metabolismo , Fosfatos de Dinucleosídeos/química , Antibacterianos/farmacologia , Humanos , Fósforo-Oxigênio Liases/química , Fósforo-Oxigênio Liases/metabolismo , Conformação ProteicaRESUMO
A 1H-isoindol-3-amine was identified as suitable P1 group for the proprotein convertase furin using a crystallographic screening with a set of 20 fragments known to occupy the S1 pocket of trypsin-like serine proteases. Its binding mode is very similar to that observed for the P1 group of benzamidine-derived peptidic furin inhibitors suggesting an aminomethyl substitution of this fragment to obtain a couplable P1 residue for the synthesis of substrate-analogue furin inhibitors. The obtained inhibitors possess a slightly improved picomolar inhibitory potency compared to their benzamidine-derived analogues. The crystal structures of two inhibitors in complex with furin revealed that the new P1 group is perfectly suited for incorporation in peptidic furin inhibitors. Selected inhibitors were tested for antiviral activity against respiratory syncytial virus (RSV) and a furin-dependent influenza A virus (SC35M/H7N7) in A549 human lung cells and demonstrated an efficient inhibition of virus activation and replication at low micromolar or even submicromolar concentrations. First results suggest that the Mas-related G-protein coupled receptor GPCR-X2 could be a potential off-target for certain benzamidine-derived furin inhibitors.
Assuntos
Antivirais , Desenho de Fármacos , Furina , Furina/antagonistas & inibidores , Furina/metabolismo , Humanos , Antivirais/farmacologia , Antivirais/síntese química , Antivirais/química , Relação Estrutura-Atividade , Células A549 , Vírus da Influenza A/efeitos dos fármacos , Cristalografia por Raios X , Indóis/farmacologia , Indóis/química , Indóis/síntese química , Estrutura Molecular , Modelos Moleculares , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Relação Dose-Resposta a DrogaRESUMO
The main protease Mpro, nsp5, of SARS-CoV-2 (SCoV2) is one of its most attractive drug targets. Here, we report primary screening data using nuclear magnetic resonance spectroscopy (NMR) of four different libraries and detailed follow-up synthesis on the promising uracil-containing fragment Z604 derived from these libraries. Z604 shows time-dependent binding. Its inhibitory effect is sensitive to reducing conditions. Starting with Z604, we synthesized and characterized 13 compounds designed by fragment growth strategies. Each compound was characterized by NMR and/or activity assays to investigate their interaction with Mpro. These investigations resulted in the four-armed compound 35b that binds directly to Mpro. 35b could be cocrystallized with Mpro revealing its noncovalent binding mode, which fills all four active site subpockets. Herein, we describe the NMR-derived fragment-to-hit pipeline and its application for the development of promising starting points for inhibitors of the main protease of SCoV2.
Assuntos
Descoberta de Drogas , SARS-CoV-2 , Descoberta de Drogas/métodos , SARS-CoV-2/metabolismo , Domínio Catalítico , Espectroscopia de Ressonância Magnética , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/metabolismo , Antivirais/farmacologia , Simulação de Acoplamento MolecularRESUMO
The Shigella pathogenicity factor IpgC belongs to the class II of type III secretion system chaperones, whose members are characterized by a tetratricopeptide repeat (TPR) domain consisting of three and a half TPR motifs. Since IpgC is essential for Shigella virulence, we determined a high-resolution crystal structure of this chaperone to facilitate its use as a target for the structure-based design of anti-shigellosis compounds. The crystal structure revealed two possible homodimer assemblies, which strongly differ from the homodimer architectures so far known for IpgC and orthologues thereof. Through crystallographic fragment screening, we identified 10 small molecules that bind to IpgC and, therefore, are available for expansion to generate larger, more potent binders. A follow-up compound, based on one of our fragment hits, binds to a strictly conserved site, which overlaps with the binding site of the chaperone's substrates, IpaB and IpaC. Therefore, it constitutes a promising starting point for the design of functional IpgC inhibitors.
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The increasing number of people dying from tuberculosis and the existence of extensively drug-resistant strains has led to an urgent need for new antituberculotic drugs with alternative modes of action. As part of the thioredoxin system, thioredoxin reductase (TrxR) is essential for the survival of Mycobacterium tuberculosis (Mtb) and shows substantial differences from human TrxR, making it a promising and most likely selective target. As a model organism for Mtb, crystals of Mycobacterium smegmatis TrxR that diffracted to high resolution were used in crystallographic fragment screening to discover binding fragments and new binding sites. The application of the 96 structurally diverse fragments from the F2X-Entry Screen revealed 56 new starting points for fragment-based drug design of new TrxR inhibitors. Over 200 crystal structures were analyzed using FragMAXapp, which includes processing and refinement by largely automated software pipelines and hit identification via the multi-data-set analysis approach PanDDA. The fragments are bound to 11 binding sites, of which four are positioned at binding pockets or important interaction sites and therefore show high potential for possible inhibition of TrxR.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Humanos , Tiorredoxina Dissulfeto Redutase/química , Tiorredoxina Dissulfeto Redutase/metabolismo , Mycobacterium tuberculosis/metabolismo , Sítios de Ligação , Desenho de FármacosRESUMO
Secretory proteins are used by pathogenic bacteria to manipulate the host systems and compete with other microorganisms, thereby enabling their survival in their host. Similar to other bacteria, secretory proteins of Mycobacterium tuberculosis also play a pivotal role in evading immune response within hosts, thereby leading to acute and latent tuberculosis infection. Prokaryotes have several classes of bacterial secretory systems out of which the Sec and Tat pathways are the most conserved in Mtb to transport proteins across the cytoplasmic membrane. Here, we report the crystal structure of a secretory protein, Rv0398c determined to 1.9 Å resolution. The protein comprises a core of antiparallel ß sheets surrounded by α helices adopting a unique ß sandwich fold. Structural comparison with other secretory proteins in Mtb and other pathogenic bacteria reveals that Rv0398c may be secreted via the Sec pathway. Our structural and in silico analyses thus provide mechanistic insights into the pathway adopted by Mtb to transport out secretory protein, Rv0398c which will facilitate the invasion to the host immune system.
Assuntos
Proteínas de Bactérias , Mycobacterium tuberculosis , Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas de Transporte/metabolismo , Transporte BiológicoRESUMO
The ephrin type-A receptor 2 (EPHA2) kinase belongs to the largest family of receptor tyrosine kinases. There are several indications of an involvement of EPHA2 in the development of infectious diseases and cancer. Despite pharmacological potential, EPHA2 is an under-examined target protein. In this study, we synthesized a series of derivatives of the inhibitor NVP-BHG712 and triazine-based compounds. These compounds were evaluated to determine their potential as kinase inhibitors of EPHA2, including elucidation of their binding mode (X-ray crystallography), affinity (microscale thermophoresis), and selectivity (Kinobeads assay). Eight inhibitors showed affinities in the low-nanomolar regime (KD <10â nM). Testing in up to seven colon cancer cell lines that express EPHA2 reveals that several derivatives feature promising effects for the control of human colon carcinoma. Thus, we have developed a set of powerful tool compounds for fundamental new research on the interplay of EPH receptors in a cellular context.
Assuntos
Neoplasias Colorretais , Pirazóis , Humanos , Pirazóis/química , Pirimidinas/farmacologia , Pirimidinas/química , Linhagem Celular , Neoplasias Colorretais/tratamento farmacológico , Linhagem Celular TumoralRESUMO
TfCa, a promiscuous carboxylesterase from Thermobifida fusca, was found to hydrolyze polyethylene terephthalate (PET) degradation intermediates such as bis(2-hydroxyethyl) terephthalate (BHET) and mono-(2-hydroxyethyl)-terephthalate (MHET). In this study, we elucidated the structures of TfCa in its apo form, as well as in complex with a PET monomer analogue and with BHET. The structure-function relationship of TfCa was investigated by comparing its hydrolytic activity on various ortho- and para-phthalate esters of different lengths. Structure-guided rational engineering of amino acid residues in the substrate-binding pocket resulted in the TfCa variant I69W/V376A (WA), which showed 2.6-fold and 3.3-fold higher hydrolytic activity on MHET and BHET, respectively, than the wild-type enzyme. TfCa or its WA variant was mixed with a mesophilic PET depolymerizing enzyme variant [Ideonella sakaiensis PETase (IsPETase) PM] to degrade PET substrates of various crystallinity. The dual enzyme system with the wild-type TfCa or its WA variant produced up to 11-fold and 14-fold more terephthalate (TPA) than the single IsPETase PM, respectively. In comparison to the recently published chimeric fusion protein of IsPETase and MHETase, our system requires 10% IsPETase and one-fourth of the reaction time to yield the same amount of TPA under similar PET degradation conditions. Our simple dual enzyme system reveals further advantages in terms of cost-effectiveness and catalytic efficiency since it does not require time-consuming and expensive cross-linking and immobilization approaches.
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The identification of starting points for compound development is one of the key steps in early-stage drug discovery. Information-rich techniques such as crystallographic fragment screening can potentially increase the efficiency of this step by providing the structural information of the binding mode of the ligands in addition to the mere binding information. Here, we present the crystallographic screening of our 1000-plus-compound F2X-Universal Library against the complex of the yeast spliceosomal Prp8 RNaseH-like domain and the snRNP assembly factor Aar2. The observed 269 hits are distributed over 10 distinct binding sites on the surface of the protein-protein complex. Our work shows that hit clusters from large-scale crystallographic fragment screening campaigns identify known interaction sites with other proteins and suggest putative additional interaction sites. Furthermore, the inherent binding pose validation within the hit clusters may accelerate downstream compound optimization.
Assuntos
Descoberta de Drogas , Proteínas , Cristalografia por Raios X , Ligantes , Descoberta de Drogas/métodos , Sítios de Ligação , Ligação ProteicaRESUMO
In their recent commentary in Protein Science, Jaskolski et al. analyzed three randomly picked diffraction data sets from fragment-screening group depositions from the PDB and, based on that, they claimed that such data are principally problematic. We demonstrate here that if such data are treated properly, none of the proclaimed criticisms persist.
Assuntos
Proteínas , Cristalografia por Raios X , Ligantes , Proteínas/químicaRESUMO
Viral infection in cells triggers a cascade of molecular defense mechanisms to maintain host-cell homoeostasis. One of these mechanisms is ADP-ribosylation, a fundamental post-translational modification (PTM) characterized by the addition of ADP-ribose (ADPr) on substrates. Poly(ADP-ribose) polymerases (PARPs) are implicated in this process and they perform ADP-ribosylation on host and pathogen proteins. Some viral families contain structural motifs that can reverse this PTM. These motifs known as macro domains (MDs) are evolutionarily conserved protein domains found in all kingdoms of life. They are divided in different classes with the viral belonging to Macro-D-type class because of their properties to recognize and revert the ADP-ribosylation. Viral MDs are potential pharmaceutical targets, capable to counteract host immune response. Sequence and structural homology between viral and human MDs are an impediment for the development of new active compounds against their function. Remdesivir, is a drug administrated in viral infections inhibiting viral replication through RNA-dependent RNA polymerase (RdRp). Herein, GS-441524, the active metabolite of the remdesivir, is tested as a hydrolase inhibitor for several viral MDs and for its binding to human homologs found in PARPs. This study presents biochemical and biophysical studies, which indicate that GS-441524 selectively modifies SARS-CoV-2 MD de-MARylation activity, while it does not interact with hPARP14 MD2 and hPARP15 MD2. The structural investigation of MDâ¢GS-441524 complexes, using solution NMR and X-ray crystallography, discloses the impact of certain amino acids in ADPr binding cavity suggesting that F360 and its adjacent residues tune the selective binding of the inhibitor to SARS-CoV-2 MD.
Assuntos
ADP-Ribosilação , Adenosina/análogos & derivados , Inibidores de Protease de Coronavírus , Poli(ADP-Ribose) Polimerases , SARS-CoV-2 , ADP-Ribosilação/efeitos dos fármacos , Adenosina/química , Adenosina/farmacologia , Adenosina Difosfato Ribose/química , Inibidores de Protease de Coronavírus/química , Inibidores de Protease de Coronavírus/farmacologia , Humanos , Poli(ADP-Ribose) Polimerases/química , Ligação Proteica , Domínios Proteicos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologiaRESUMO
Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides. The catalytic activity of most RNRs depends on the formation of a dimer of the catalytic subunits. The active site is located at the interface, and part of the substrate binding site and regulatory mechanisms work across the subunit in the dimer. In this study, we describe and characterize a novel domain responsible for forming the catalytic dimer in several class II RNRs. The 3D structure of the class II RNR from Rhodobacter sphaeroides reveals a so far undescribed α-helical domain in the dimer interface, which is embracing the other subunit. Genetic removal of this HUG domain leads to a severe reduction of activity paired with reduced dimerization capability. In comparison with other described RNRs, the enzyme with this domain is less dependent on the presence of nucleotides to act as allosteric effectors in the formation of dimers. The HUG domain appears to serve as an interlock to keep the dimer intact and functional even at low enzyme and/or effector concentrations.
Assuntos
Ribonucleotídeo Redutases , Regulação Alostérica , Sítios de Ligação , Domínio Catalítico , Modelos Moleculares , Ribonucleotídeo Redutases/químicaRESUMO
Although light is a prominent stimulus for smart materials, the application of photoswitches as light-responsive triggers for phase transitions of porous materials remains poorly explored. Here we incorporate an azobenzene photoswitch in the backbone of a metal-organic framework producing light-induced structural contraction of the porous network in parallel to gas adsorption. Light-stimulation enables non-invasive spatiotemporal control over the mechanical properties of the framework, which ultimately leads to pore contraction and subsequent guest release via negative gas adsorption. The complex mechanism of light-gated breathing is established by a series of in situ diffraction and spectroscopic experiments, supported by quantum mechanical and molecular dynamic simulations. Unexpectedly, this study identifies a novel light-induced deformation mechanism of constrained azobenzene photoswitches relevant to the future design of light-responsive materials.
RESUMO
Protein-mediated redox reactions play a critical role in many biological processes and often occur at centres that contain metal ions as cofactors. In order to understand the exact mechanisms behind these reactions it is important to not only characterize the three-dimensional structures of these proteins and their cofactors, but also to identify the oxidation states of the cofactors involved and to correlate this knowledge with structural information. The only suitable approach for this based on crystallographic measurements is spatially resolved anomalous dispersion (SpReAD) refinement, a method that has been used previously to determine the redox states of metals in iron-sulfur cluster-containing proteins. In this article, the feasibility of this approach for small, non-iron-sulfur redox centres is demonstrated by employing SpReAD analysis to characterize Sulfolobus tokodaii sulerythrin, a ruberythrin-like protein that contains a binuclear metal centre. Differences in oxidation states between the individual iron ions of the binuclear metal centre are revealed in sulerythrin crystals treated with H2O2. Furthermore, data collection at high X-ray doses leads to photoreduction of this metal centre, showing that careful control of the total absorbed dose is a prerequisite for successfully determining the oxidation state through SpReAD analysis.
Assuntos
Proteínas Ferro-Enxofre , Metaloproteínas , Cristalografia por Raios X , Peróxido de Hidrogênio , Metaloproteínas/química , OxirreduçãoAssuntos
Proteínas , Pesquisa Translacional Biomédica , Biologia , Microscopia Crioeletrônica , Humanos , Ligantes , Proteínas/químicaRESUMO
Immunomodulation of airway hyperreactivity by excretory-secretory (ES) products of the first larval stage (L1) of the gastrointestinal nematode Trichuris suis is reported by us and others. Here, we aimed to identify the proteins accounting for the modulatory effects of the T. suis L1 ES proteins and studied six selected T. suis L1 proteins for their immunomodulatory efficacy in a murine OVA-induced allergic airway disease model. In particular, an enzymatically active T. suis chitinase mediated amelioration of clinical signs of airway hyperreactivity, primarily associated with suppression of eosinophil recruitment into the lung, the associated chemokines, and increased numbers of RELMα + interstitial lung macrophages. While there is no indication of T. suis chitinase directly interfering with dendritic cell activation or antigen presentation to CD4 T cells, treatment of allergic mice with the worm chitinase influenced the hosts' own chitinase activity in the inflamed lung. The three-dimensional structure of the T. suis chitinase as determined by high-resolution X-ray crystallography revealed high similarities to mouse acidic mammalian chitinase (AMCase) but a unique ability of T. suis chitinase to form dimers. Our data indicate that the structural similarities between the parasite and host chitinase contribute to the disease-ameliorating effect of the helminth-derived chitinase on allergic lung inflammation.
Assuntos
Quitinases/ultraestrutura , Eosinofilia/tratamento farmacológico , Proteínas de Helminto/administração & dosagem , Agentes de Imunomodulação/administração & dosagem , Hipersensibilidade Respiratória/tratamento farmacológico , Animais , Líquido da Lavagem Broncoalveolar , Cristalografia por Raios X , Modelos Animais de Doenças , Eosinofilia/diagnóstico , Eosinofilia/imunologia , Eosinofilia/patologia , Feminino , Proteínas de Helminto/ultraestrutura , Interações Hospedeiro-Parasita/imunologia , Humanos , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/imunologia , Camundongos , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Hipersensibilidade Respiratória/diagnóstico , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/patologia , Trichuris/enzimologiaRESUMO
RNA editosomes selectively deaminate cytidines to uridines in plant organellar transcripts-mostly to restore protein functionality and consequently facilitate mitochondrial and chloroplast function. The RNA editosomal pentatricopeptide repeat proteins serve target RNA recognition, whereas the intensively studied DYW domain elicits catalysis. Here we present structures and functional data of a DYW domain in an inactive ground state and activated. DYW domains harbour a cytidine deaminase fold and a C-terminal DYW motif, with catalytic and structural zinc atoms, respectively. A conserved gating domain within the deaminase fold regulates the active site sterically and mechanistically in a process that we termed gated zinc shutter. Based on the structures, an autoinhibited ground state and its activation are cross-validated by RNA editing assays and differential scanning fluorimetry. We anticipate that, in vivo, the framework of an active plant RNA editosome triggers the release of DYW autoinhibition to ensure a controlled and coordinated cytidine deamination playing a key role in mitochondrial and chloroplast homeostasis.
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Fungal xylanases belonging to family GH30_7, initially categorized as endo-glucuronoxylanases, are now known to differ both in terms of substrate specificity, as well as mode of action. Recently, TtXyn30A, a GH30_7 xylanase from Thermothelomyces thermophila, was shown to possess dual activity, acting on the xylan backbone in both an endo- and an exo- manner. Here, in an effort to identify the structural characteristics that append these functional properties to the enzyme, we present the biochemical characterization of various TtXyn30A mutants as well as its crystal structure, alone, and in complex with the reaction product. An auxiliary catalytic amino acid has been identified, while it is also shown that glucuronic acid recognition is not mediated by a conserved arginine residue, as shown by previously determined GH30 structures.