RESUMEN
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a worldwide scourge with more than 10 million people affected yearly. Among the proteins essential for the survival of Mtb, InhA has been and is still clinically validated as a therapeutic target. A new family of direct diaryl ether inhibitors, not requiring prior activation by the catalase peroxidase enzyme KatG, has been designed with the ambition of fully occupying the InhA substrate-binding site. Thus, eleven compounds, featuring three pharmacophores within the same molecule, were synthesized. One of them, 5-(((4-(2-hydroxyphenoxy)benzyl)(octyl)amino)methyl)-2-phenoxyphenol (compound 21), showed good inhibitory activity against InhA with IC50 of 0.70 µM. The crystal structure of compound 21 in complex with InhA/NAD+ showed how the molecule fills the substrate-binding site as well as the minor portal of InhA. This study represents a further step towards the design of new inhibitors of InhA.
Asunto(s)
Antituberculosos , Imidazoles , Mycobacterium tuberculosis , Sulfonamidas , Tiofenos , Humanos , Antituberculosos/farmacología , Antituberculosos/química , Éter , Éteres , Sitios de Unión , Éteres de Etila , Proteínas Bacterianas/metabolismoRESUMEN
Tuberculosis (TB) remains a global health crisis, further exacerbated by the slow pace of new treatment options, and the emergence of extreme and total drug resistance to existing drugs. The challenge to developing new antibacterial compounds with activity against Mycobacterium tuberculosis (Mtb), the causative agent of TB, is in part due to unique features of this pathogen, especially the composition and structure of its complex cell envelope. Therefore, targeting enzymes involved in cell envelope synthesis has been of major interest for anti-TB drug discovery. FAAL32 is a fatty acyl-AMP ligase involved in the biosynthesis of the cell wall mycolic acids, and a potential target for drug discovery. To rapidly advance research in this area, we initiated a drug repurposing campaign and screened a collection of 1280 approved human or veterinary drugs (Prestwick Chemical Library) using a biochemical assay that reads out FAAL32 inhibition. These efforts led to the discovery of salicylanilide closantel, and some of its derivatives as inhibitors with potent in vitro activity against M. tuberculosis. These results suggest that salicylanilide represents a potentially promising pharmacophore for the conception of novel anti-tubercular candidates targeting FAAL32 that would open new targeting opportunities. Moreover, this work illustrates the value of drug repurposing campaigns to discover new leads in challenging drug discovery fields.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Adenosina Monofosfato/uso terapéutico , Antituberculosos/química , Evaluación Preclínica de Medicamentos , Humanos , Salicilanilidas , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
With the advent of structural biology in the drug discovery process, medicinal chemists gained the opportunity to use detailed structural information in order to progress screening hits into leads or drug candidates. X-ray crystallography has proven to be an invaluable tool in this respect, as it is able to provide exquisitely comprehensive structural information about the interaction of a ligand with a pharmacological target. As fragment-based drug discovery emerged in the recent years, X-ray crystallography has also become a powerful screening technology, able to provide structural information on complexes involving low-molecular weight compounds, despite weak binding affinities. Given the low numbers of compounds needed in a fragment library, compared to the hundreds of thousand usually present in drug-like compound libraries, it now becomes feasible to screen a whole fragment library using X-ray crystallography, providing a wealth of structural details that will fuel the fragment to drug process. Here, we review theoretical and practical aspects as well as the pros and cons of using X-ray crystallography in the drug discovery process.
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Descubrimiento de Drogas , Proteínas/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Humanos , Ligandos , Proteínas/uso terapéuticoRESUMEN
Mycolic acids are essential components of the mycobacterial cell envelope, and their biosynthetic pathway is one of the targets of first-line antituberculous drugs. This pathway contains a number of potential targets, including some that have been identified only recently and have yet to be explored. One such target, FadD32, is required for activation of the long meromycolic chain and is essential for mycobacterial growth. We report here an in-depth biochemical, biophysical, and structural characterization of four FadD32 orthologs, including the very homologous enzymes fromMycobacterium tuberculosisandMycobacterium marinum Determination of the structures of two complexes with alkyl adenylate inhibitors has provided direct information, with unprecedented detail, about the active site of the enzyme and the associated hydrophobic tunnel, shedding new light on structure-function relationships and inhibition mechanisms by alkyl adenylates and diarylated coumarins. This work should pave the way for the rational design of inhibitors of FadD32, a highly promising drug target.
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Antituberculosos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Diseño de Fármacos , Ligasas/química , Ligasas/metabolismo , Mycobacterium tuberculosis/enzimología , Mycobacterium/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/antagonistas & inhibidores , Ligasas de Carbono-Azufre , Cristalografía por Rayos X , Ligasas/antagonistas & inhibidores , Modelos Moleculares , Datos de Secuencia Molecular , Mycobacterium/química , Mycobacterium/efectos de los fármacos , Infecciones por Mycobacterium/tratamiento farmacológico , Infecciones por Mycobacterium/microbiología , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/efectos de los fármacos , Ácidos Micólicos/metabolismo , Conformación Proteica , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
Thanatos associated protein 11 (THAP11) is a cell cycle and cell growth regulator differentially expressed in cancer cells. THAP11 belongs to a distinct family of transcription factors recognizing specific DNA sequences via an atypical zinc finger motif and regulating diverse cellular processes. Outside the extensively characterized DNA-binding domain, THAP proteins vary in size and predicted domains, for which structural data are still lacking. We report here the crystal structure of the C-terminal region of human THAP11 protein, providing the first 3D structure of a coiled-coil motif from a THAP family member. We further investigate the stability, dynamics and oligomeric properties of the determined structure combining molecular dynamics simulations and biophysical experiments. Our results show that the C-ter region of THAP11 forms a left-handed parallel homo-dimeric coiled-coil structure possessing several unusual features.
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Multimerización de Proteína , Proteínas Represoras/química , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Dominios Proteicos/fisiología , Estabilidad Proteica , Estructura Secundaria de Proteína , Proteínas Represoras/fisiologíaRESUMEN
InhA is an enoyl-ACP reductase of Mycobacterium tuberculosis implicated in the biosynthesis of mycolic acids, essential constituents of the mycobacterial cell wall. To date, this enzyme is considered as a promising target for the discovery of novel antitubercular drugs. In this work, we describe the first crystal structure of the apo form of the wild-type InhA at 1.80Å resolution as well as the crystal structure of InhA in complex with the synthetic metabolite of the antitubercular drug isoniazid refined to 1.40Å. This metabolite, synthesized in the absence of InhA, is able to displace and replace the cofactor NADH in the enzyme active site. This work provides a unique opportunity to enlighten the structural adaptation of apo-InhA to the binding of the NADH cofactor or of the isoniazid adduct. In addition, a differential scanning fluorimetry study of InhA, in the apo-form as well as in the presence of NAD(+), NADH and INH-NADH was performed showing that binding of the INH-NADH adduct had a strong stabilizing effect.
Asunto(s)
Proteínas Bacterianas/química , Isoniazida/química , Mycobacterium tuberculosis/enzimología , Oxidorreductasas/química , Biomimética/métodos , Dominio Catalítico , NAD/química , Unión Proteica/fisiologíaRESUMEN
The mechanisms permitting nonpolymorphic CD1 molecules to present lipid antigens that differ considerably in polar head and aliphatic tails remain elusive. It is also unclear why hydrophobic motifs in the aliphatic tails of some antigens, which presumably embed inside CD1 pockets, contribute to determinants for T-cell recognition. The 1.9-Å crystal structure of an active complex of CD1b and a mycobacterial diacylsulfoglycolipid presented here provides some clues. Upon antigen binding, endogenous spacers of CD1b, which consist of a mixture of diradylglycerols, moved considerably within the lipid-binding groove. Spacer displacement was accompanied by F' pocket closure and an extensive rearrangement of residues exposed to T-cell receptors. Such structural reorganization resulted in reduction of the A' pocket capacity and led to incomplete embedding of the methyl-ramified portion of the phthioceranoyl chain of the antigen, explaining why such hydrophobic motifs are critical for T-cell receptor recognition. Mutagenesis experiments supported the functional importance of the observed structural alterations for T-cell stimulation. Overall, our data delineate a complex molecular mechanism combining spacer repositioning and ligand-induced conformational changes that, together with pocket intricacy, endows CD1b with the required molecular plasticity to present a broad range of structurally diverse antigens.
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Antígenos CD1/química , Glucolípidos/química , Modelos Moleculares , Mycobacterium tuberculosis/química , Conformación Proteica , Antígenos CD1/metabolismo , Cromatografía en Capa Delgada , Cristalografía por Rayos X , Análisis de Fourier , Glucolípidos/metabolismo , Humanos , Mutagénesis , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
CD1e is the only human CD1 protein existing in soluble form in the late endosomes of dendritic cells, where it facilitates the processing of glycolipid antigens that are ultimately recognized by CD1b-restricted T cells. The precise function of CD1e remains undefined, thus impeding efforts to predict the participation of this protein in the presentation of other antigens. To gain insight into its function, we determined the crystal structure of recombinant CD1e expressed in human cells at 2.90-Å resolution. The structure revealed a groove less intricate than in other CD1 proteins, with a significantly wider portal characterized by a 2 Å-larger spacing between the α1 and α2 helices. No electron density corresponding to endogenous ligands was detected within the groove, despite the presence of ligands unequivocally established by native mass spectrometry in recombinant CD1e. Our structural data indicate that the water-exposed CD1e groove could ensure the establishment of loose contacts with lipids. In agreement with this possibility, lipid association and dissociation processes were found to be considerably faster with CD1e than with CD1b. Moreover, CD1e was found to mediate in vitro the transfer of lipids to CD1b and the displacement of lipids from stable CD1b-antigen complexes. Altogether, these data support that CD1e could have evolved to mediate lipid-exchange/editing processes with CD1b and point to a pathway whereby the repertoire of lipid antigens presented by human dendritic cells might be expanded.
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Antígenos CD1/química , Antígenos CD1/metabolismo , Metabolismo de los Lípidos , Lípidos/química , Acilación , Cristalografía por Rayos X , Humanos , Ligandos , Modelos Moleculares , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Knr4/Smi1 proteins are specific to the fungal kingdom and their deletion in the model yeast Saccharomyces cerevisiae and the human pathogen Candida albicans results in hypersensitivity to specific antifungal agents and a wide range of parietal stresses. In S. cerevisiae, Knr4 is located at the crossroads of several signalling pathways, including the conserved cell wall integrity and calcineurin pathways. Knr4 interacts genetically and physically with several protein members of those pathways. Its sequence suggests that it contains large intrinsically disordered regions. Here, a combination of small-angle X-ray scattering (SAXS) and crystallographic analysis led to a comprehensive structural view of Knr4. This experimental work unambiguously showed that Knr4 comprises two large intrinsically disordered regions flanking a central globular domain whose structure has been established. The structured domain is itself interrupted by a disordered loop. Using the CRISPR/Cas9 genome editing technique, strains expressing KNR4 genes deleted from different domains were constructed. The N-terminal domain and the loop are essential for optimal resistance to cell wall-binding stressors. The C-terminal disordered domain, on the other hand, acts as a negative regulator of this function of Knr4. The identification of molecular recognition features, the possible presence of secondary structure in these disordered domains and the functional importance of the disordered domains revealed here designate these domains as putative interacting spots with partners in either pathway. Targeting these interacting regions is a promising route to the discovery of inhibitory molecules that could increase the susceptibility of pathogens to the antifungals currently in clinical use.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Humanos , Pared Celular/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Dispersión del Ángulo Pequeño , Factores de Transcripción/metabolismo , Difracción de Rayos XRESUMEN
Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) affects 10 million people each year and the emergence of resistant TB augurs for a growing incidence. In the last 60 years, only three new drugs were approved for TB treatment, for which resistances are already emerging. Therefore, there is a crucial need for new chemotherapeutic agents capable of eradicating TB. Enzymes belonging to the type II fatty acid synthase system (FAS-II) are involved in the biosynthesis of mycolic acids, cell envelope components essential for mycobacterial survival. Among them, InhA is the primary target of isoniazid (INH), one of the most effective compounds to treat TB. INH acts as a prodrug requiring activation by the catalase-peroxidase KatG, whose mutations are the major cause for INH resistance. Herein, a new series of direct InhA inhibitors were designed based on a molecular hybridization approach. They exhibit potent inhibitory activities of InhA and, for some of them, good antitubercular activities. Moreover, they display a low toxicity on human cells. A study of the mechanism of action of the most effective molecules shows that they inhibit the biosynthesis of mycolic acids. The X-ray structures of two InhA/NAD+/inhibitor complexes have been obtained showing a binding mode of a part of the molecule in the minor portal, rarely seen in the InhA structures reported so far.
Asunto(s)
Antituberculosos , Mycobacterium tuberculosis , Humanos , Antituberculosos/farmacología , Antituberculosos/química , Proteínas Bacterianas/metabolismo , Éter , Éteres/farmacología , Éteres de Etila/farmacología , Isoniazida/farmacología , Mutación , Ácidos MicólicosRESUMEN
Soluble forms of recombinant LukE protein (expressed in Escherichia coli) and of wild-type LukD protein (expressed in Staphylococcus aureus), which together form the staphylococcal LukE-LukD leukotoxin, were purified to homogeneity and crystallized using the sitting-drop vapour-diffusion method. The crystals of LukE belonged to space group I4, with unit-cell parameters a = b = 134.50, c = 64.43 Å, and diffracted X-rays to 1.6 Å resolution. The crystals of LukD belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 48.04, b = 50.99, c = 137.40 Å, and diffracted to 1.9 Å resolution. Molecular replacement using the LukF-PV structure (PDB entry 1pvl) as a template model allowed the identification of an initial structure solution for the LukD data. In the case of LukE, a solution comprising only a single copy of the search model (LukS-PV; PDB entry 1t5r) was found, although the unit-cell parameters indicated that up to three molecules could be accommodated in the asymmetric unit.
Asunto(s)
Proteínas Bacterianas/química , Exotoxinas/química , Staphylococcus aureus/química , Cristalización , Cristalografía por Rayos XRESUMEN
The mycolic acid biosynthetic pathway represents a promising source of pharmacological targets in the fight against tuberculosis. In Mycobacterium tuberculosis, mycolic acids are subject to specific chemical modifications introduced by a set of eight S-adenosylmethionine dependent methyltransferases. Among these, Hma (MmaA4) is responsible for the introduction of oxygenated modifications. Crystallographic screening of a library of fragments allowed the identification of seven ligands of Hma. Two mutually exclusive binding modes were identified, depending on the conformation of residues 147-154. These residues are disordered in apo-Hma but fold upon binding of the S-adenosylmethionine (SAM) cofactor as well as of analogues, resulting in the formation of the short η1-helix. One of the observed conformations would be incompatible with the presence of the cofactor, suggesting that allosteric inhibitors could be designed against Hma. Chimeric compounds were designed by fusing some of the bound fragments, and the relative binding affinities of initial fragments and evolved compounds were investigated using molecular dynamics simulation and generalised Born and Poisson-Boltzmann calculations coupled to the surface area continuum solvation method. Molecular dynamics simulations were also performed on apo-Hma to assess the structural plasticity of the unliganded protein. Our results indicate a significant improvement in the binding properties of the designed compounds, suggesting that they could be further optimised to inhibit Hma activity.
RESUMEN
Owing to their role in activating enzymes essential for bacterial viability and pathogenicity, phosphopantetheinyl transferases represent novel and attractive drug targets. In this work, we examined the inhibitory effect of the aminido-urea 8918 compound against the phosphopantetheinyl transferases PptAb from Mycobacterium abscessus and PcpS from Pseudomonas aeruginosa, two pathogenic bacteria associated with cystic fibrosis and bronchiectasis, respectively. Compound 8918 exhibits inhibitory activity against PptAb but displays no activity against PcpS in vitro, while no antimicrobial activity against Mycobacterium abscessus or Pseudomonas aeruginosa could be detected. X-ray crystallographic analysis of 8918 bound to PptAb-CoA alone and in complex with an acyl carrier protein domain in addition to the crystal structure of PcpS in complex with CoA revealed the structural basis for the inhibition mechanism of PptAb by 8918 and its ineffectiveness against PcpS. Finally, in crystallo screening of potent inhibitors from the National Cancer Institute library identified a hydroxypyrimidinethione derivative that binds PptAb. Both compounds could serve as scaffolds for the future development of phosphopantetheinyl transferases inhibitors.
Asunto(s)
Proteínas Bacterianas/química , Inhibidores Enzimáticos/química , Pirimidinonas/química , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Urea/química , Proteínas Bacterianas/antagonistas & inhibidores , Sitios de Unión , Inhibidores Enzimáticos/farmacología , Conformación Molecular , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mycobacterium abscessus/enzimología , Unión Proteica , Pseudomonas aeruginosa/enzimología , Proteínas Recombinantes , Relación Estructura-Actividad , Especificidad por Sustrato , Transferasas (Grupos de Otros Fosfatos Sustitutos)/antagonistas & inhibidores , Urea/análogos & derivados , Urea/farmacologíaRESUMEN
Mycobacterium tuberculosis is the causative agent of the tuberculosis disease, which claims more human lives each year than any other bacterial pathogen. M. tuberculosis and other mycobacterial pathogens have developed a range of unique features that enhance their virulence and promote their survival in the human host. Among these features lies the particular cell envelope with high lipid content, which plays a substantial role in mycobacterial pathogenicity. Several envelope components of M. tuberculosis and other mycobacteria, e.g., mycolic acids, phthiocerol dimycocerosates, and phenolic glycolipids, belong to the "family" of polyketides, secondary metabolites synthesized by fascinating versatile enzymes-polyketide synthases. These megasynthases consist of multiple catalytic domains, among which the acyltransferase domain plays a key role in selecting and transferring the substrates required for polyketide extension. Here, we present three new crystal structures of acyltransferase domains of mycobacterial polyketide synthases and, for one of them, provide evidence for the identification of residues determining extender unit specificity. Unravelling the molecular basis for such specificity is of high importance considering the role played by extender units for the final structure of key mycobacterial components. This work provides major advances for the use of mycobacterial polyketide synthases as potential therapeutic targets and, more generally, contributes to the prediction and bioengineering of polyketide synthases with desired specificity.
Asunto(s)
Mycobacterium/enzimología , Sintasas Poliquetidas/metabolismo , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Sintasas Poliquetidas/química , Conformación Proteica , Especificidad por SustratoRESUMEN
Bark of elderberry (Sambucus nigra) contains a galactose (Gal)/N-acetylgalactosamine (GalNAc)-specific lectin (SNA-II) corresponding to slightly truncated B-chains of a genuine Type-II ribosome-inactivating protein (Type-II RIPs, SNA-V), found in the same species. The three-dimensional X-ray structure of SNA-II has been determined in two distinct crystal forms, hexagonal and tetragonal, at 1.90 A and 1.35 A, respectively. In both crystal forms, the SNA-II molecule folds into two linked beta-trefoil domains, with an overall conformation similar to that of the B-chains of ricin and other Type-II RIPs. Glycosylation is observed at four sites along the polypeptide chain, accounting for 14 saccharide units. The high-resolution structures of SNA-II in complex with Gal and five Gal-related saccharides (GalNAc, lactose, alpha1-methylgalactose, fucose, and the carcinoma-specific Tn antigen) were determined at 1.55 A resolution or better. Binding is observed in two saccharide-binding sites for most of the sugars: a conserved aspartate residue interacts simultaneously with the O3 and O4 atoms of saccharides. In one of the binding sites, additional interactions with the protein involve the O6 atom. Analytical gel filtration, small angle X-ray scattering studies and crystal packing analysis indicate that, although some oligomeric species are present, the monomeric species predominate in solution.
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Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Galactosa/metabolismo , Lectinas de Plantas/química , Lectinas de Plantas/metabolismo , Proteínas Inactivadoras de Ribosomas/química , Proteínas Inactivadoras de Ribosomas/metabolismo , Sambucus nigra/química , Antígenos de Carbohidratos Asociados a Tumores/química , Sitios de Unión , Cristalografía por Rayos X , Galactosa/análisis , Galactosa/química , Lectinas de Plantas/aislamiento & purificación , Polisacáridos/química , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Proteínas Inactivadoras de Ribosomas/aislamiento & purificación , Sambucus nigra/metabolismo , Dispersión del Ángulo Pequeño , Madera/químicaRESUMEN
The carboxysome is a bacterial micro-compartment (BMC) subtype that encapsulates enzymatic activities necessary for carbon fixation. Carboxysome shells are composed of a relatively complex cocktail of proteins, their precise number and identity being species dependent. Shell components can be classified in two structural families, the most abundant class associating as hexamers (BMC-H) that are supposed to be major players for regulating shell permeability. Up to recently, these proteins were proposed to associate as homo-oligomers. Genomic data, however, demonstrated the existence of paralogs coding for multiple shell subunits. Here, we studied cross-association compatibilities among BMC-H CcmK proteins of Synechocystis sp. PCC6803. Co-expression in Escherichia coli proved a consistent formation of hetero-hexamers combining CcmK1 and CcmK2 or, remarkably, CcmK3 and CcmK4 subunits. Unlike CcmK1/K2 hetero-hexamers, the stoichiometry of incorporation of CcmK3 in associations with CcmK4 was low. Cross-interactions implicating other combinations were weak, highlighting a structural segregation of the two groups that could relate to gene organization. Sequence analysis and structural models permitted the localization of interactions that would favor formation of CcmK3/K4 hetero-hexamers. The crystallization of these CcmK3/K4 associations conducted to the elucidation of a structure corresponding to the CcmK4 homo-hexamer. Yet, subunit exchange could not be demonstrated in vitro. Biophysical measurements showed that hetero-hexamers are thermally less stable than homo-hexamers, and impeded in forming larger assemblies. These novel findings are discussed within the context of reported data to propose a functional scenario in which minor CcmK3/K4 incorporation in shells would introduce sufficient local disorder as to allow shell remodeling necessary to adapt rapidly to environmental changes.
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Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Complejos Multiproteicos/química , Synechocystis/metabolismo , Proteínas Bacterianas/genética , Sitios de Unión , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Modelos Moleculares , Unión Proteica , Ingeniería de Proteínas , Multimerización de Proteína , Estabilidad Proteica , Synechocystis/genética , TermodinámicaRESUMEN
SecB chaperones assist protein export by binding both unfolded proteins and the SecA motor. Certain SecB homologs can also control toxin-antitoxin (TA) systems known to modulate bacterial growth in response to stress. In such TA-chaperone (TAC) systems, SecB assists the folding and prevents degradation of the antitoxin, thus facilitating toxin inhibition. Chaperone dependency is conferred by a C-terminal extension in the antitoxin known as chaperone addiction (ChAD) sequence, which makes the antitoxin aggregation-prone and prevents toxin inhibition. Using TAC of Mycobacterium tuberculosis, we present the structure of a SecB-like chaperone bound to its ChAD peptide. We find differences in the binding interfaces when compared to SecB-SecA or SecB-preprotein complexes, and show that the antitoxin can reach a functional form while bound to the chaperone. This work reveals how chaperones can use discrete surface binding regions to accommodate different clients or partners and thereby expand their substrate repertoire and functions.
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Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Chaperonas Moleculares/metabolismo , Sistemas Toxina-Antitoxina/fisiología , Sitios de Unión , Chaperonas Moleculares/genética , Mycobacterium tuberculosis/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Sistemas Toxina-Antitoxina/genéticaRESUMEN
The original version of this Article contained errors in Figures 1 and 4. In Fig. 1b, the Mtb-SecBTA sequence was displayed incorrectly. In the inset panel within Fig. 4c, the y-axis of the graph incorrectly read (Q.Rg)2 × I(Q)//(0), and should have read (Q.Rg)2 × I(Q)/I(0). These errors have been corrected in both the PDF and HTML versions of the Article.
RESUMEN
Staphylococcal leucotoxins, leucocidins, and gamma-hemolysins are bicomponent beta-barrel pore-forming toxins (beta-PFTs). Their production is associated with several clinical diseases. They have cytotoxic activity due to the synergistic action of a class S component and a class F component, which are secreted as water-soluble monomers and form hetero-oligomeric transmembrane pores, causing the lysis of susceptible cells. Structural information is currently available for the monomeric S and F proteins and the homoheptamer formed by the related alpha-hemolysin. These structures illustrate the start and end points in the mechanistic framework of beta-PFT assembly. Only limited structural data exist for the intermediate stages, including hetero-oligomeric complexes of leucotoxins. We investigated the protein-protein interactions responsible for maintaining the final bipartite molecular architecture and describe here the high-resolution crystal structure and low-resolution solution structure of a site-specific cross-linked heterodimer of gamma-hemolysin (HlgA T28C-HlgB N156C), which were solved by X-ray crystallography and small angle X-ray scattering, respectively. These structures reveal a molecular plasticity of beta-PFTs, which may facilitate the transition from membrane-bound monomers to heterodimers.
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Toxinas Bacterianas/química , Exotoxinas/química , Cristalografía por Rayos X , Dimerización , Unión Proteica , Staphylococcaceae/química , Difracción de Rayos XRESUMEN
Rational drug design is essential for new drugs to emerge, especially when the structure of a target protein or catalytic enzyme is known experimentally. To that purpose, high-throughput virtual ligand screening campaigns aim at discovering computationally new binding molecules or fragments to inhibit a particular protein interaction or biological activity. The virtual ligand screening process often relies on docking methods which allow predicting the binding of a molecule into a biological target structure with a correct conformation and the best possible affinity. The docking method itself is not sufficient as it suffers from several and crucial limitations (lack of protein flexibility information, no solvation effects, poor scoring functions, and unreliable molecular affinity estimation).At the interface of computer techniques and drug discovery, molecular dynamics (MD) allows introducing protein flexibility before or after a docking protocol, refining the structure of protein-drug complexes in the presence of water, ions and even in membrane-like environments, and ranking complexes with more accurate binding energy calculations. In this chapter we describe the up-to-date MD protocols that are mandatory supporting tools in the virtual ligand screening (VS) process. Using docking in combination with MD is one of the best computer-aided drug design protocols nowadays. It has proved its efficiency through many examples, described below.