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1.
Cell ; 186(19): 4059-4073.e27, 2023 09 14.
Artículo en Inglés | MEDLINE | ID: mdl-37611581

RESUMEN

Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C55PP, lipid II, and lipid IIIWTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development.


Asunto(s)
Antibacterianos , Bacterias , Microbiología del Suelo , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Bioensayo , Difosfatos
2.
Proteins ; 91(12): 1658-1683, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-37905971

RESUMEN

We present the results for CAPRI Round 54, the 5th joint CASP-CAPRI protein assembly prediction challenge. The Round offered 37 targets, including 14 homodimers, 3 homo-trimers, 13 heterodimers including 3 antibody-antigen complexes, and 7 large assemblies. On average ~70 CASP and CAPRI predictor groups, including more than 20 automatics servers, submitted models for each target. A total of 21 941 models submitted by these groups and by 15 CAPRI scorer groups were evaluated using the CAPRI model quality measures and the DockQ score consolidating these measures. The prediction performance was quantified by a weighted score based on the number of models of acceptable quality or higher submitted by each group among their five best models. Results show substantial progress achieved across a significant fraction of the 60+ participating groups. High-quality models were produced for about 40% of the targets compared to 8% two years earlier. This remarkable improvement is due to the wide use of the AlphaFold2 and AlphaFold2-Multimer software and the confidence metrics they provide. Notably, expanded sampling of candidate solutions by manipulating these deep learning inference engines, enriching multiple sequence alignments, or integration of advanced modeling tools, enabled top performing groups to exceed the performance of a standard AlphaFold2-Multimer version used as a yard stick. This notwithstanding, performance remained poor for complexes with antibodies and nanobodies, where evolutionary relationships between the binding partners are lacking, and for complexes featuring conformational flexibility, clearly indicating that the prediction of protein complexes remains a challenging problem.


Asunto(s)
Algoritmos , Mapeo de Interacción de Proteínas , Mapeo de Interacción de Proteínas/métodos , Conformación Proteica , Unión Proteica , Simulación del Acoplamiento Molecular , Biología Computacional/métodos , Programas Informáticos
3.
Proteins ; 88(8): 1029-1036, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-31886559

RESUMEN

Our information-driven docking approach HADDOCK has demonstrated a sustained performance since the start of its participation to CAPRI. This is due, in part, to its ability to integrate data into the modeling process, and to the robustness of its scoring function. We participated in CAPRI both as server and manual predictors. In CAPRI rounds 38-45, we have used various strategies depending on the available information. These ranged from imposing restraints to a few residues identified from literature as being important for the interaction, to binding pockets identified from homologous complexes or template-based refinement/CA-CA restraint-guided docking from identified templates. When relevant, symmetry restraints were used to limit the conformational sampling. We also tested for a large decamer target a new implementation of the MARTINI coarse-grained force field in HADDOCK. Overall, we obtained acceptable or better predictions for 13 and 11 server and manual submissions, respectively, out of the 22 interfaces. Our server performance (acceptable or higher-quality models when considering the top 10) was better (59%) than the manual (50%) one, in which we typically experiment with various combinations of protocols and data sources. Again, our simple scoring function based on a linear combination of intermolecular van der Waals and electrostatic energies and an empirical desolvation term demonstrated a good performance in the scoring experiment with a 63% success rate across all 22 interfaces. An analysis of model quality indicates that, while we are consistently performing well in generating acceptable models, there is room for improvement for generating/identifying higher quality models.


Asunto(s)
Simulación del Acoplamiento Molecular , Péptidos/química , Proteínas/química , Programas Informáticos , Secuencia de Aminoácidos , Sitios de Unión , Humanos , Ligandos , Péptidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Multimerización de Proteína , Proteínas/metabolismo , Proyectos de Investigación , Homología Estructural de Proteína , Termodinámica
4.
J Biol Chem ; 292(17): 7023-7039, 2017 04 28.
Artículo en Inglés | MEDLINE | ID: mdl-28292930

RESUMEN

Leishmania parasites have evolved a number of strategies to cope with the harsh environmental changes during mammalian infection. One of these mechanisms involves the functional gain that allows mitochondrial 2-Cys peroxiredoxins to act as molecular chaperones when forming decamers. This function is critical for parasite infectivity in mammals, and its activation has been considered to be controlled exclusively by the enzyme redox state under physiological conditions. Herein, we have revealed that magnesium and calcium ions play a major role in modulating the ability of these enzymes to act as molecular chaperones, surpassing the redox effect. These ions are directly involved in mitochondrial metabolism and participate in a novel mechanism to stabilize the decameric form of 2-Cys peroxiredoxins in Leishmania mitochondria. Moreover, we have demonstrated that a constitutively dimeric Prx1m mutant impairs the survival of Leishmania under heat stress, supporting the central role of the chaperone function of Prx1m for Leishmania parasites during the transition from insect to mammalian hosts.


Asunto(s)
Calcio/metabolismo , Leishmania/metabolismo , Magnesio/metabolismo , Proteínas Mitocondriales/metabolismo , Peroxirredoxinas/metabolismo , Proteínas Protozoarias/metabolismo , Anisotropía , Cromatografía , Disulfuros/química , Fluorometría , Regulación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Luz , Mitocondrias/metabolismo , Chaperonas Moleculares/metabolismo , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Oxígeno/química , Multimerización de Proteína , Dispersión de Radiación , Temperatura
5.
J Biol Chem ; 289(11): 7362-73, 2014 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-24469445

RESUMEN

Arabinanases (ABNs, EC 3.2.1.99) are promising catalysts for environmentally friendly biomass conversion into energy and chemicals. These enzymes catalyze the hydrolysis of the α-1,5-linked L-arabinofuranoside backbone of plant cell wall arabinans releasing arabino-oligosaccharides and arabinose, the second most abundant pentose in nature. In this work, new findings about the molecular mechanisms governing activation, functional differentiation, and catalysis of GH43 ABNs are presented. Biophysical, mutational, and biochemical studies with the hyperthermostable two-domain endo-acting ABN from Thermotoga petrophila (TpABN) revealed how some GH43 ABNs are activated by calcium ions via hyperpolarization of the catalytically relevant histidine and the importance of the ancillary domain for catalysis and conformational stability. On the other hand, the two GH43 ABNs from rumen metagenome, ARN2 and ARN3, presented a calcium-independent mechanism in which sodium is the most likely substituent for calcium ions. The crystal structure of the two-domain endo-acting ARN2 showed that its ability to efficiently degrade branched substrates is due to a larger catalytic interface with higher accessibility than that observed in other ABNs with preference for linear arabinan. Moreover, crystallographic characterization of the single-domain exo-acting ARN3 indicated that its cleavage pattern producing arabinose is associated with the chemical recognition of the reducing end of the substrate imposed by steric impediments at the aglycone-binding site. By structure-guided rational design, ARN3 was converted into a classical endo enzyme, confirming the role of the extended Arg(203)-Ala(230) loop in determining its action mode. These results reveal novel molecular aspects concerning the functioning of GH43 ABNs and provide new strategies for arabinan degradation.


Asunto(s)
Arabinosa/química , Proteínas Bacterianas/metabolismo , Catálisis , Glicósido Hidrolasas/metabolismo , Bacilos Gramnegativos Anaerobios Rectos, Curvos y Espirales/enzimología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Biotecnología , Calcio/química , Bovinos , Clonación Molecular , Cristalografía por Rayos X , Análisis Mutacional de ADN , Hidrólisis , Iones/química , Cinética , Ligandos , Metagenoma , Metales/química , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Ingeniería de Proteínas , Estructura Terciaria de Proteína , Rumen/microbiología , Homología de Secuencia de Aminoácido , Solventes/química
6.
Biochim Biophys Acta ; 1844(3): 545-52, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24373874

RESUMEN

Snake venom metalloproteinases (SVMPs) belonging to P-I class are able to hydrolyze extracellular matrix proteins and coagulation factors triggering local and systemic reactions by multiple molecular mechanisms that are not fully understood. BmooMPα-I, a P-I class SMVP from Bothrops moojeni venom, was active upon neuro- and vaso-active peptides including angiotensin I, bradykinin, neurotensin, oxytocin and substance P. Interestingly, BmooMPα-I showed a strong bias towards hydrolysis after proline residues, which is unusual for most of characterized peptidases. Moreover, the enzyme showed kininogenase activity similar to that observed in plasma and cells by kallikrein. FRET peptide assays indicated a relative promiscuity at its S2-S'2 subsites, with proline determining the scissile bond. This unusual post-proline cleaving activity was confirmed by the efficient hydrolysis of the synthetic combinatorial library MCA-GXXPXXQ-EDDnp, described as resistant for canonical peptidases, only after Pro residues. Structural analysis of the tripeptide LPL complexed with BmooMPα-I, generated by molecular dynamics simulations, assisted in defining the subsites and provided the structural basis for subsite preferences such as the restriction of basic residues at the S2 subsite due to repulsive electrostatic effects and the steric impediment for large aliphatic or aromatic side chains at the S1 subsite. These new functional and structural findings provided a further understanding of the molecular mechanisms governing the physiological effects of this important class of enzymes in envenomation process.


Asunto(s)
Venenos de Crotálidos/enzimología , Calicreínas/metabolismo , Metaloproteasas/metabolismo , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Animales , Bothrops , Hidrólisis , Cinética , Simulación de Dinámica Molecular , Péptidos/química , Péptidos/metabolismo , Prolil Oligopeptidasas , Radioinmunoensayo , Especificidad por Sustrato
7.
BMC Bioinformatics ; 15: 197, 2014 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-24938294

RESUMEN

BACKGROUND: The characterization of protein binding sites is a major challenge in computational biology. Proteins interact with a wide variety of molecules and understanding of such complex interactions is essential to gain deeper knowledge of protein function. Shape complementarity is known to be important in determining protein-ligand interactions. Furthermore, these protein structural features have been shown to be useful in assisting medicinal chemists during lead discovery and optimization. RESULTS: We developed KVFinder, a highly versatile and easy-to-use tool for cavity prospection and spatial characterization. KVFinder is a geometry-based method that has an innovative customization of the search space. This feature provides the possibility of cavity segmentation, which alongside with the large set of customizable parameters, allows detailed cavity analyses. Although the main focus of KVFinder is the steered prospection of cavities, we tested it against a benchmark dataset of 198 known drug targets in order to validate our software and compare it with some of the largely accepted methods. Using the one click mode, we performed better than most of the other methods, staying behind only of hybrid prospection methods. When using just one of KVFinder's customizable features, we were able to outperform all other compared methods. KVFinder is also user friendly, as it is available as a PyMOL plugin, or command-line version. CONCLUSION: KVFinder presents novel usability features, granting full customizable and highly detailed cavity prospection on proteins, alongside with a friendly graphical interface. KVFinder is freely available on http://lnbio.cnpem.br/bioinformatics/main/software/.


Asunto(s)
Biología Computacional/métodos , Proteínas/química , Programas Informáticos , Algoritmos , Sitios de Unión , Ligandos , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína
8.
J Biol Chem ; 288(47): 34131-34145, 2013 Nov 22.
Artículo en Inglés | MEDLINE | ID: mdl-24097982

RESUMEN

Myosin V (MyoV) motors have been implicated in the intracellular transport of diverse cargoes including vesicles, organelles, RNA-protein complexes, and regulatory proteins. Here, we have solved the cargo-binding domain (CBD) structures of the three human MyoV paralogs (Va, Vb, and Vc), revealing subtle structural changes that drive functional differentiation and a novel redox mechanism controlling the CBD dimerization process, which is unique for the MyoVc subclass. Moreover, the cargo- and motor-binding sites were structurally assigned, indicating the conservation of residues involved in the recognition of adaptors for peroxisome transport and providing high resolution insights into motor domain inhibition by CBD. These results contribute to understanding the structural requirements for cargo transport, autoinhibition, and regulatory mechanisms in myosin V motors.


Asunto(s)
Miosina Tipo V/química , Sitios de Unión , Transporte Biológico Activo/fisiología , Humanos , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , Peroxisomas/química , Peroxisomas/genética , Peroxisomas/metabolismo , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Homología Estructural de Proteína
9.
Commun Biol ; 7(1): 49, 2024 01 06.
Artículo en Inglés | MEDLINE | ID: mdl-38184711

RESUMEN

The formation of a stable complex between proteins lies at the core of a wide variety of biological processes and has been the focus of countless experiments. The huge amount of information contained in the protein structural interactome in the Protein Data Bank can now be used to characterise and classify the existing biological interfaces. We here introduce ARCTIC-3D, a fast and user-friendly data mining and clustering software to retrieve data and rationalise the interface information associated with the protein input data. We demonstrate its use by various examples ranging from showing the increased interaction complexity of eukaryotic proteins, 20% of which on average have more than 3 different interfaces compared to only 10% for prokaryotes, to associating different functions to different interfaces. In the context of modelling biomolecular assemblies, we introduce the concept of "recognition entropy", related to the number of possible interfaces of the components of a protein-protein complex, which we demonstrate to correlate with the modelling difficulty in classical docking approaches. The identified interface clusters can also be used to generate various combinations of interface-specific restraints for integrative modelling. The ARCTIC-3D software is freely available at github.com/haddocking/arctic3d and can be accessed as a web-service at wenmr.science.uu.nl/arctic3d.


Asunto(s)
Minería de Datos , Células Procariotas , Análisis por Conglomerados , Bases de Datos de Proteínas , Entropía
10.
Nat Protoc ; 2024 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-38886530

RESUMEN

Interactions between macromolecules, such as proteins and nucleic acids, are essential for cellular functions. Experimental methods can fail to provide all the information required to fully model biomolecular complexes at atomic resolution, particularly for large and heterogeneous assemblies. Integrative computational approaches have, therefore, gained popularity, complementing traditional experimental methods in structural biology. Here, we introduce HADDOCK2.4, an integrative modeling platform, and its updated web interface ( https://wenmr.science.uu.nl/haddock2.4 ). The platform seamlessly integrates diverse experimental and theoretical data to generate high-quality models of macromolecular complexes. The user-friendly web server offers automated parameter settings, access to distributed computing resources, and pre- and post-processing steps that enhance the user experience. To present the web server's various interfaces and features, we demonstrate two different applications: (i) we predict the structure of an antibody-antigen complex by using NMR data for the antigen and knowledge of the hypervariable loops for the antibody, and (ii) we perform coarse-grained modeling of PRC1 with a nucleosome particle guided by mutagenesis and functional data. The described protocols require some basic familiarity with molecular modeling and the Linux command shell. This new version of our widely used HADDOCK web server allows structural biologists and non-experts to explore intricate macromolecular assemblies encompassing various molecule types.

11.
J Biol Chem ; 287(51): 43071-82, 2012 Dec 14.
Artículo en Inglés | MEDLINE | ID: mdl-23105116

RESUMEN

ADAM17, which is also known as TNFα-converting enzyme, is the major sheddase for the EGF receptor ligands and is considered to be one of the main proteases responsible for the ectodomain shedding of surface proteins. How a membrane-anchored proteinase with an extracellular catalytic domain can be activated by inside-out regulation is not completely understood. We characterized thioredoxin-1 (Trx-1) as a partner of the ADAM17 cytoplasmic domain that could be involved in the regulation of ADAM17 activity. We induced the overexpression of the ADAM17 cytoplasmic domain in HEK293 cells, and ligands able to bind this domain were identified by MS after protein immunoprecipitation. Trx-1 was also validated as a ligand of the ADAM17 cytoplasmic domain and full-length ADAM17 recombinant proteins by immunoblotting, immunolocalization, and solid phase binding assay. In addition, using nuclear magnetic resonance, it was shown in vitro that the titration of the ADAM17 cytoplasmic domain promotes changes in the conformation of Trx-1. The MS analysis of the cross-linked complexes showed cross-linking between the two proteins by lysine residues. To further evaluate the functional role of Trx-1, we used a heparin-binding EGF shedding cell model and observed that the overexpression of Trx-1 in HEK293 cells could decrease the activity of ADAM17, activated by either phorbol 12-myristate 13-acetate or EGF. This study identifies Trx-1 as a novel interaction partner of the ADAM17 cytoplasmic domain and suggests that Trx-1 is a potential candidate that could be involved in ADAM17 activity regulation.


Asunto(s)
Proteínas ADAM/metabolismo , Tiorredoxinas/metabolismo , Proteínas ADAM/química , Proteína ADAM17 , Secuencia de Aminoácidos , Animales , Cromatografía Liquida , Reactivos de Enlaces Cruzados/farmacología , Activación Enzimática/efectos de los fármacos , Células HEK293 , Células HeLa , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Immunoblotting , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ligandos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , Acetato de Tetradecanoilforbol/farmacología , Tiorredoxinas/química
12.
Arch Biochem Biophys ; 539(1): 20-30, 2013 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-24035743

RESUMEN

The periplasmic-binding proteins in ATP-binding cassette systems (ABC Transporters) are responsible for the capture and delivery of ligands to their specific transporters, triggering a series of ATP-driven conformational changes that leads to the transport of the ligand. Structurally consisting of two lobes, the proteins change conformation after interaction with the ligand. The structure of the molybdate-binding protein (ModA) from Xanthomonas citri, bound to molybdate, was previously solved by our group and an interdomain interaction, mediated by a salt bridge between K127 and D59, apparently supports the binding properties and keeps the domains closed. To determinate the importance of this interaction, we built two ModA mutants, K127S and D59A, and analysed their functional and structural properties. Based on a set of spectroscopic experiments, crystallisation trials, structure determination and molecular dynamics (MD) simulations, we showed that the salt bridge is essential to maintain the structure and binding properties. Additionally, the MD simulations revealed that this mutant adopted a more compact structure that packed down the ligand-binding pocket. From the closed bound to open structure, the positioning of the helices forming the dipole and the salt bridge are essential to induce an intermediate state.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Xanthomonas , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Sitios de Unión , Permeabilidad de la Membrana Celular , Disulfuros/química , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Molibdeno/metabolismo , Mutación , Unión Proteica , Estabilidad Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , Temperatura
13.
bioRxiv ; 2023 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-37292624

RESUMEN

Antimicrobial resistance is a leading mortality factor worldwide. Here we report the discovery of clovibactin, a new antibiotic, isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant bacterial pathogens without detectable resistance. Using biochemical assays, solid-state NMR, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C 55 PP, Lipid II, Lipid WTA ). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate, but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the irreversible sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. Uncultured bacteria offer a rich reservoir of antibiotics with new mechanisms of action that could replenish the antimicrobial discovery pipeline.

14.
Curr Opin Struct Biol ; 70: 70-77, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34139639

RESUMEN

Proteins play crucial roles in every cellular process by interacting with each other, nucleic acids, metabolites, and other molecules. The resulting assemblies can be very large and intricate and pose challenges to experimental methods. In the current era of integrative modeling, it is often only by a combination of various experimental techniques and computations that three-dimensional models of those molecular machines can be obtained. Among the various computational approaches available, molecular docking is often the method of choice when it comes to predicting three-dimensional structures of complexes. Docking can generate particularly accurate models when taking into account the available information on the complex of interest. We review here the use of experimental and bioinformatics data in protein-protein docking, describing recent software developments and highlighting applications for the modeling of antibody-antigen complexes and membrane protein complexes, and the use of evolutionary and shape information.


Asunto(s)
Biología Computacional , Programas Informáticos , Simulación del Acoplamiento Molecular , Unión Proteica , Proteínas/metabolismo
15.
Front Mol Biosci ; 8: 729513, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34395534

RESUMEN

Structural biology aims at characterizing the structural and dynamic properties of biological macromolecules at atomic details. Gaining insight into three dimensional structures of biomolecules and their interactions is critical for understanding the vast majority of cellular processes, with direct applications in health and food sciences. Since 2010, the WeNMR project (www.wenmr.eu) has implemented numerous web-based services to facilitate the use of advanced computational tools by researchers in the field, using the high throughput computing infrastructure provided by EGI. These services have been further developed in subsequent initiatives under H2020 projects and are now operating as Thematic Services in the European Open Science Cloud portal (www.eosc-portal.eu), sending >12 millions of jobs and using around 4,000 CPU-years per year. Here we review 10 years of successful e-infrastructure solutions serving a large worldwide community of over 23,000 users to date, providing them with user-friendly, web-based solutions that run complex workflows in structural biology. The current set of active WeNMR portals are described, together with the complex backend machinery that allows distributed computing resources to be harvested efficiently.

16.
Front Mol Biosci ; 6: 102, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31632986

RESUMEN

Modeling biomolecular assemblies is an important field in computational structural biology. The inherent complexity of their energy landscape and the computational cost associated with modeling large and complex assemblies are major drawbacks for integrative modeling approaches. The so-called coarse-graining approaches, which reduce the degrees of freedom of the system by grouping several atoms into larger "pseudo-atoms," have been shown to alleviate some of those limitations, facilitating the identification of the global energy minima assumed to correspond to the native state of the complex, while making the calculations more efficient. Here, we describe and assess the implementation of the MARTINI force field for DNA into HADDOCK, our integrative modeling platform. We combine it with our previous implementation for protein-protein coarse-grained docking, enabling coarse-grained modeling of protein-nucleic acid complexes. The system is modeled using MARTINI topologies and interaction parameters during the rigid body docking and semi-flexible refinement stages of HADDOCK, and the resulting models are then converted back to atomistic resolution by an atom-to-bead distance restraints-guided protocol. We first demonstrate the performance of this protocol using 44 complexes from the protein-DNA docking benchmark, which shows an overall ~6-fold speed increase and maintains similar accuracy as compared to standard atomistic calculations. As a proof of concept, we then model the interaction between the PRC1 and the nucleosome (a former CAPRI target in round 31), using the same information available at the time the target was offered, and compare all-atom and coarse-grained models.

17.
Antioxid Redox Signal ; 29(8): 717-734, 2018 09 10.
Artículo en Inglés | MEDLINE | ID: mdl-29334756

RESUMEN

AIMS: A disintegrin and metalloprotease 17 (ADAM17) modulates signaling events by releasing surface protein ectodomains such as TNFa and the EGFR-ligands. We have previously characterized cytoplasmic thioredoxin-1 (Trx-1) as a partner of ADAM17 cytoplasmic domain. Still, the mechanism of ADAM17 regulation by Trx-1 is unknown, and it has become of paramount importance to assess the degree of influence that Trx-1 has on metalloproteinase ADAM17. RESULTS: Combining discovery and targeted proteomic approaches, we uncovered that Trx-1 negatively regulates ADAM17 by direct and indirect effect. We performed cell-based assays with synthetic peptides and site-directed mutagenesis, and we demonstrated that the interaction interface of Trx-1 and ADAM17 is important for the negative regulation of ADAM17 activity. However, both Trx-1K72A and catalytic site mutant Trx-1C32/35S rescued ADAM17 activity, although the interaction with Trx-1C32/35S was unaffected, suggesting an indirect effect of Trx-1. We confirmed that the Trx-1C32/35S mutant showed diminished reductive capacity, explaining this indirect effect on increasing ADAM17 activity through oxidant levels. Interestingly, Trx-1K72A mutant showed similar oxidant levels to Trx-1C32/35S, even though its catalytic site was preserved. We further demonstrated that the general reactive oxygen species inhibitor, Nacetylcysteine (NAC), maintained the regulation of ADAM17 dependent of Trx-1 reductase activity levels; whereas the electron transport chain modulator, rotenone, abolished Trx-1 effect on ADAM17 activity. INNOVATION: We show for the first time that the mechanism of ADAM17 regulation, Trx-1 dependent, can be by direct interaction and indirect effect, bringing new insights into the cross-talk between isomerases and mammalian metalloproteinases. CONCLUSION: This unexpected Trx-1K72A behavior was due to more dimer formation and, consequently, the reduction of its Trx-1 reductase activity, evaluated through dimer verification, by gel filtration and mass spectrometry analysis. Antioxid. Redox Signal. 29, 717-734.


Asunto(s)
Proteína ADAM17/metabolismo , Tiorredoxinas/metabolismo , Sitios de Unión , Células HEK293 , Humanos , Modelos Moleculares , Oxidación-Reducción , Tiorredoxinas/análisis , Tiorredoxinas/genética , Células Tumorales Cultivadas
18.
Biophys Chem ; 184: 44-53, 2013 Dec 31.
Artículo en Inglés | MEDLINE | ID: mdl-24056191

RESUMEN

The flavoprotein old yellow enzyme of Trypanosoma cruzi (TcOYE) is an oxidoreductase that uses NAD(P)H as cofactor. This enzyme is clinically relevant due to its role in the action mechanism of some trypanocidal drugs used in the treatment of Chagas' disease by producing reactive oxygen species. In this work, the recombinant enzyme TcOYE was produced and collectively, X-ray crystallography, small angle X-ray scattering, analytical ultracentrifugation and molecular dynamics provided a detailed description of its structure, specificity and hydrodynamic behavior. The crystallographic structure at 1.27Å showed a classical (α/ß)8 fold with the FMN prosthetic group buried at the positively-charged active-site cleft. In solution, TcOYE behaved as a globular monomer, but it exhibited a molecular envelope larger than that observed in the crystal structure, suggesting intrinsic protein flexibility. Moreover, the binding mode of ß-lapachone, a trypanocidal agent, and other naphthoquinones was investigated by molecular docking and dynamics suggesting that their binding to TcOYE are stabilized mainly by interactions with the isoalloxazine ring from FMN and residues from the active-site pocket.


Asunto(s)
NADPH Deshidrogenasa/química , NADPH Deshidrogenasa/metabolismo , Trypanosoma cruzi/enzimología , Cristalografía por Rayos X , Modelos Moleculares , NADPH Deshidrogenasa/genética , Conformación Proteica , Especificidad por Sustrato , Termodinámica
19.
Protein J ; 30(5): 340-50, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21691771

RESUMEN

Pathogenic bacteria constitute an important cause of hospital-acquired infections. However, the misuse of available bactericidal agents has led to the appearance of antibiotic-resistant strains. Thus, efforts to seek new antimicrobials with different action mechanisms would have an enormous impact. Here, a novel antimicrobial protein (SiAMP2) belonging to the 2S albumin family was isolated from Sesamum indicum kernels and evaluated against several bacteria and fungi. Furthermore, in silico analysis was conducted in order to identify conserved residues through other 2S albumin antimicrobial proteins (2S-AMPs). SiAMP2 specifically inhibited Klebsiella sp. Specific regions in the molecule surface where cationic (RR/RRRK) and hydrophobic (MEYWPR) residues are exposed and conserved were proposed as being involved in antimicrobial activity. This study reinforces the hypothesis that plant storage proteins might also play as pathogen protection providing an insight into the mechanism of action for this novel 2S-AMP and evolutionary relations between antimicrobial activity and 2S albumins.


Asunto(s)
Albuminas 2S de Plantas/química , Albuminas 2S de Plantas/farmacología , Antibacterianos/química , Antibacterianos/farmacología , Sesamum/química , Albuminas 2S de Plantas/aislamiento & purificación , Antibacterianos/aislamiento & purificación , Simulación por Computador , Enterobacteriaceae/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Hongos Mitospóricos/efectos de los fármacos , Filogenia , Semillas/química , Relación Estructura-Actividad
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