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1.
Pathogens ; 13(3)2024 Mar 08.
Artículo en Inglés | MEDLINE | ID: mdl-38535584

RESUMEN

Human T-cell Leukemia Virus type 1 (HTLV-1) is a human retrovirus responsible for leukaemia in 5 to 10% of infected individuals. Among the viral proteins, Tax has been described as directly involved in virus-induced leukemogenesis. Tax is therefore an interesting therapeutic target. However, its 3D structure is still unknown and this hampers the development of drug-design-based therapeutic strategies. Several algorithms are available that can be used to predict the structure of proteins, particularly with the recent appearance of artificial intelligence (AI)-driven pipelines. Here, we review how the structure of Tax is predicted by several algorithms using distinct modelling strategies. We discuss the consequences for the understanding of Tax structure/function relationship, and more generally for the use of structure models for modular and/or flexible proteins, which are frequent in retroviruses.

2.
Vaccines (Basel) ; 12(3)2024 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-38543950

RESUMEN

Dengue virus (DENV) infection continues to be a public health challenge, lacking a specific cure. Vaccination remains the primary strategy against dengue; however, existing live-attenuated vaccines display variable efficacy across four serotypes, influenced by host serostatus and age, and predominantly inducing humoral responses. To address this limitation, this study investigates a multiepitope-based immunogen designed to induce robust cellular immunity across all DENV serotypes. The chimeric immunogen integrates H-2d specific MHC-I binding T-cell epitopes derived from conserved domains within the DENV envelope protein. Immuno-informatics analyses supported its stability, non-allergenic nature, and strong MHC-I binding affinity as an antigen. To assess the immunogenicity of the multiepitope, it was expressed in murine bone-marrow-derived dendritic cells (BMDCs) that were used to prime mice. In this experimental model, simultaneous exposure to T-cell epitopes from all four DENV serotypes initiated distinct IFNγ-CD8 T-cell responses for different serotypes. These results supported the potential of the multiepitope construct as a vaccine candidate. While the optimization of the immunogen design remains a continuous pursuit, this proof-of-concept study provides a starting point for evaluating its protective efficacy against dengue infection in vivo. Moreover, our results support the development of a multiepitope vaccine that could trigger a pan-serotype anti-dengue CD8 response.

3.
Biochim Biophys Acta Biomembr ; 1865(1): 184065, 2023 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-36206830

RESUMEN

Diffraction anisotropy is a phenomenon that impacts more specifically membrane proteins, compared to soluble ones, but the reasons for this discrepancy remained unclear. Often, it is referred to a difference in resolution limits between highest and lowest diffraction limits as a signature for anisotropy. We show in this article that there is no single correlation between anisotropy and difference in resolution limits, with notably a substantial number of structures displaying various anisotropy with no difference in resolution limits. We further investigated diffraction intensity profiles, and observed a peak centred on 4.9 Å resolution more predominant in membrane proteins. Since this peak is in the region corresponding to secondary structures, we investigated the influence of secondary structure ratio. We showed that secondary structure content has little influence on this profile, while secondary structure collinearity in membrane proteins correlate with a stronger peak. Finally, we could further show that the presence of this peak is linked to higher diffraction anisotropy. These results bring to light a specific diffraction of membrane protein crystals, which calls for a specific handling by crystallographic software. It also brings an explanation for investigators struggling with their anisotropic data.


Asunto(s)
Proteínas de la Membrana , Programas Informáticos , Difracción de Rayos X , Proteínas de la Membrana/química , Estructura Secundaria de Proteína , Anisotropía
4.
Int J Mol Sci ; 23(22)2022 Nov 08.
Artículo en Inglés | MEDLINE | ID: mdl-36430221

RESUMEN

Recent evidence indicates that the HIV-1 Integrase (IN) binds the viral genomic RNA (gRNA), playing a critical role in the morphogenesis of the viral particle and in the stability of the gRNA once in the host cell. By combining biophysical, molecular biology, and biochemical approaches, we found that the 18-residues flexible C-terminal tail of IN acts as a sensor of the peculiar apical structure of the trans-activation response element RNA (TAR), interacting with its hexaloop. We show that the binding of the whole IN C-terminal domain modifies TAR structure, exposing critical nucleotides. These modifications favour the subsequent binding of the HIV transcriptional trans-activator Tat to TAR, finally displacing IN from TAR. Based on these results, we propose that IN assists the binding of Tat to TAR RNA. This working model provides a mechanistic sketch accounting for the emerging role of IN in the early stages of proviral transcription and could help in the design of anti-HIV-1 therapeutics against this new target of the viral infectious cycle.


Asunto(s)
Integrasa de VIH , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , ARN Guía de Kinetoplastida , Integrasa de VIH/genética , ARN Viral/genética , ARN Viral/metabolismo , Factores de Transcripción
5.
Viruses ; 14(7)2022 06 27.
Artículo en Inglés | MEDLINE | ID: mdl-35891378

RESUMEN

Retroviral integrase is a multimeric enzyme that catalyzes the integration of reverse-transcribed viral DNA into the cellular genome. Beyond integration, the Human immunodeficiency virus type 1 (HIV-1) integrase is also involved in many other steps of the viral life cycle, such as reverse transcription, nuclear import, virion morphogenesis and proviral transcription. All these additional functions seem to depend on the action of the integrase C-terminal domain (CTD) that works as a molecular hub, interacting with many different viral and cellular partners. In this review, we discuss structural issues concerning the CTD, with particular attention paid to its interaction with nucleic acids. We also provide a detailed map of post-translational modifications and interaction with molecular partners.


Asunto(s)
Integrasa de VIH , VIH-1 , ADN Viral , Integrasa de VIH/metabolismo , VIH-1/química , Humanos , Provirus/genética , Transcripción Reversa , Integración Viral
6.
Pharmaceuticals (Basel) ; 15(6)2022 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-35745675

RESUMEN

Tetanus toxoid (TTd) plays an important role in the pharmaceutical world, especially in vaccines. The toxoid is obtained after formaldehyde treatment of the tetanus toxin. In parallel, current emphasis in the drug discovery field is put on producing well-defined and safer drugs, explaining the interest in finding new alternative proteins. The tetanus toxin fragment C (TTFC) has been extensively studied both as a neuroprotective agent for central nervous system disorders owing to its neuronal properties and as a carrier protein in vaccines. Indeed, it is derived from a part of the tetanus toxin and, as such, retains its immunogenic properties without being toxic. Moreover, this fragment has been well characterized, and its entire structure is known. Here, we propose a systematic review of TTFC by providing information about its structural features, its properties and its methods of production. We also describe the large uses of TTFC in the field of drug discovery. TTFC can therefore be considered as an attractive alternative to TTd and remarkably offers a wide range of uses, including as a carrier, delivery vector, conjugate, booster, inducer, and neuroprotector.

7.
Biochemistry ; 60(24): 1896-1908, 2021 06 22.
Artículo en Inglés | MEDLINE | ID: mdl-34096272

RESUMEN

Feline immunodeficiency virus (FIV) is a veterinary infective agent for which there is currently no efficient drug available. Drugs targeting the lentivirus capsid are currently under development for the treatment of human immunodeficiency virus 1 (HIV-1). Here we describe a lead compound that interacts with the FIV capsid. This compound, 696, modulates the in vitro assembly of and stabilizes the assembled capsid protein. To decipher the mechanism of binding of this compound to the protein, we performed the first nuclear magnetic resonance (NMR) assignment of the FIV p24 capsid protein. Experimental NMR chemical shift perturbations (CSPs) observed after the addition of 696 enabled the characterization of a specific binding site for 696 on p24. This site was further analyzed by molecular modeling of the protein:compound interaction, demonstrating a strong similarity with the binding sites of existing drugs targeting the HIV-1 capsid protein. Taken together, we characterized a promising capsid-interacting compound with a low cost of synthesis, for which derivatives could lead to the development of efficient treatments for FIV infection. More generally, our strategy combining the NMR assignment of FIV p24 with NMR CSPs and molecular modeling will be useful for the analysis of future compounds targeting p24 in the quest to identify an efficient treatment for FIV.


Asunto(s)
Antivirales/farmacología , Bencimidazoles/farmacología , Productos del Gen gag/antagonistas & inhibidores , Virus de la Inmunodeficiencia Felina/efectos de los fármacos , Animales , Sitios de Unión , Cápside/metabolismo , Proteínas de la Cápside/antagonistas & inhibidores , Proteínas de la Cápside/metabolismo , Gatos , Productos del Gen gag/metabolismo , Virus de la Inmunodeficiencia Felina/metabolismo , Plomo/farmacología , Dominios Proteicos
8.
J Biol Chem ; 296: 100305, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33465378

RESUMEN

The type II secretion system (T2SS) transports fully folded proteins of various functions and structures through the outer membrane of Gram-negative bacteria. The molecular mechanisms of substrate recruitment by T2SS remain elusive but a prevailing view is that the secretion determinants could be of a structural nature. The phytopathogenic γ-proteobacteria, Pectobacterium carotovorum and Dickeya dadantii, secrete similar sets of homologous plant cell wall degrading enzymes, mainly pectinases, by similar T2SSs, called Out. However, the orthologous pectate lyases Pel3 and PelI from these bacteria, which share 67% of sequence identity, are not secreted by the counterpart T2SS of each bacterium, indicating a fine-tuned control of protein recruitment. To identify the related secretion determinants, we first performed a structural characterization and comparison of Pel3 with PelI using X-ray crystallography. Then, to assess the biological relevance of the observed structural variations, we conducted a loop-substitution analysis of Pel3 combined with secretion assays. We showed that there is not one element with a definite secondary structure but several distant and structurally flexible loop regions that are essential for the secretion of Pel3 and that these loop regions act together as a composite secretion signal. Interestingly, depending on the crystal contacts, one of these key secretion determinants undergoes disorder-to-order transitions that could reflect its transient structuration upon the contact with the appropriate T2SS components. We hypothesize that such T2SS-induced structuration of some intrinsically disordered zones of secretion substrates could be part of the recruitment mechanism used by T2SS.


Asunto(s)
Proteínas Bacterianas/química , Dickeya/enzimología , Pectobacterium carotovorum/enzimología , Polisacárido Liasas/química , Sistemas de Secreción Tipo II/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Pared Celular/química , Pared Celular/microbiología , Clonación Molecular , Cristalografía por Rayos X , Dickeya/clasificación , Dickeya/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Pectobacterium carotovorum/clasificación , Pectobacterium carotovorum/genética , Filogenia , Células Vegetales/química , Células Vegetales/microbiología , Plantas/química , Plantas/microbiología , Polisacárido Liasas/genética , Polisacárido Liasas/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Sistemas de Secreción Tipo II/genética , Sistemas de Secreción Tipo II/metabolismo
9.
Virology ; 532: 69-81, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31022666

RESUMEN

Retroviral integrase (IN) proteins catalyze the permanent integration of the viral genome into host DNA. They can productively recruit cellular proteins, and the human Bromodomain and Extra-Terminal domain (hBET) proteins have been shown to be co-factors for integration of gamma-retroviruses such as Murine Leukemia Virus (MLV) into human cells. By using two-hybrid, co-immunoprecipitation and in vitro interaction assays, we showed that IN of the gamma- Porcine Endogenous Retrovirus-A/C (PERV IN) interacts through its C-terminal domain (CTD) with hBET proteins. We observed that PERV IN interacts with the BRD2, BRD3 and BRD4 proteins in vitro and that the BRD2 protein specifically binds and co-localizes with PERV IN protein in the nucleus of cells. We further mapped the interaction sites to the conserved Extra-Terminal (ET) domain of the hBET proteins and to several amino acids of the of the C-terminal tail of the PERV IN CTD. Finally, we determined the first experimental structure of an IN CTD - BET ET complex from small-angle X-ray scattering data (SAXS). We showed that the two factors assemble as two distinct modules linked by a short loop which confers partial flexibility. The SAXS-restrained model is structurally compatible with the binding of the PERV intasome to BRD2. Altogether, these data confirm the important role of host BET proteins in the gamma-retroviruses' targeting site and efficiency of integration.


Asunto(s)
Proteínas de Ciclo Celular/química , Retrovirus Endógenos/genética , Interacciones Huésped-Patógeno/genética , Integrasas/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/virología , Cristalografía por Rayos X , Retrovirus Endógenos/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Células HEK293 , Humanos , Integrasas/genética , Integrasas/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Porcinos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Integración Viral
10.
Nucleic Acids Res ; 47(7): 3607-3618, 2019 04 23.
Artículo en Inglés | MEDLINE | ID: mdl-30767014

RESUMEN

The integration of the retroviral genome into the chromatin of the infected cell is catalysed by the integrase (IN)•viral DNA complex (intasome). This process requires functional association between the integration complex and the nucleosomes. Direct intasome/histone contacts have been reported to modulate the interaction between the integration complex and the target DNA (tDNA). Both prototype foamy virus (PFV) and HIV-1 integrases can directly bind histone amino-terminal tails. We have further investigated this final association by studying the effect of isolated histone tails on HIV-1 integration. We show here that the binding of HIV-1 IN to a peptide derived from the H4 tail strongly stimulates integration catalysis in vitro. This stimulation was not observed with peptide tails from other variants or with alpha-retroviral (RAV) and spuma-retroviral PFV integrases. Biochemical analyses show that the peptide tail induces both an increase in the IN oligomerization state and affinity for the target DNA, which are associated with substantial structural rearrangements in the IN carboxy-terminal domain (CTD) observed by NMR. Our data indicate that the H4 peptide tail promotes the formation of active strand transfer complexes (STCs) and support an activation step of the incoming intasome at the contact of the histone tail.


Asunto(s)
Integrasa de VIH/genética , VIH-1/genética , Histonas/genética , Integración Viral/genética , Catálisis , Cromatina/genética , Cromatina/virología , Genoma Viral/genética , VIH-1/patogenicidad , Interacciones Huésped-Patógeno/genética , Humanos , Nucleosomas/genética , Nucleosomas/virología , Spumavirus/genética
11.
Data Brief ; 19: 753-757, 2018 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-30225276

RESUMEN

This data article makes available the informed computation of the whole Protein Data Bank (PDB) to investigate diffraction anisotropy on a large scale and to perform statistics. This data has been investigated in detail in "X-ray diffraction reveals the intrinsic difference in the physical properties of membrane and soluble proteins" [1]. Diffraction anisotropy is traditionally associated with absence of contacts in-between macromolecules within the crystals in a given direction of space. There are however many case that do not follow this empirical rule. To investigate and sort out this discrepancy, we computed diffraction anisotropy for every entry of the PDB, and put them in context of relevant metrics to compare X-ray diffraction in reciprocal space to the crystal packing in real space. These metrics were either extracted from PDB files when available (resolution, space groups, cell parameters, solvent content), or calculated using standard procedures (anisotropy, crystal contacts, presence of ligands). More specifically, we separated entries to compare soluble vs membrane proteins, and further separated the later in subcategories according to their insertion in the membrane, function, or type of crystallization (Type I vs Type II crystal packing). This informed database is being made available to investigators in the raw and curated formats that can be re-used for further downstream studies. This dataset is useful to test ideas and to ascertain hypothesis based on statistical analysis.

12.
Nucleic Acids Res ; 46(W1): W417-W422, 2018 07 02.
Artículo en Inglés | MEDLINE | ID: mdl-29905873

RESUMEN

ArDock (ardock.ibcp.fr) is a structural bioinformatics web server for the prediction and the visualization of potential interaction regions at protein surfaces. ArDock ranks the surface residues of a protein according to their tendency to form interfaces in a set of predefined docking experiments between the query protein and a set of arbitrary protein probes. The ArDock methodology is derived from large scale cross-docking studies where it was observed that randomly chosen proteins tend to dock in a non-random way at protein surfaces. The method predicts interaction site of the protein, or alternate interfaces in the case of proteins with multiple interaction modes. The server takes a protein structure as input and computes a score for each surface residue. Its output focuses on the interactive visualization of results and on interoperability with other services.


Asunto(s)
Algoritmos , Biología Computacional/métodos , Simulación del Acoplamiento Molecular/métodos , Proteínas/química , Programas Informáticos , Homología Estructural de Proteína , Secuencia de Aminoácidos , Benchmarking , Sitios de Unión , Bases de Datos de Proteínas , Humanos , Internet , Ligandos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína
13.
J Biomol Struct Dyn ; 36(14): 3666-3679, 2018 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-29057709

RESUMEN

The unique eukaryotic-like Ser/Thr protein kinases of Streptococcus pneumoniae, StkP, plays a primary role in the cell division process. It is composed of an intracellular kinase domain, a transmembrane helix and four extracellular PASTA subunits. PASTA domains were shown to interact with cell wall fragments but the key questions related to the molecular mechanism governing ligand recognition remain unclear. To address this issue, the full-length structural model of StkP was generated by combining small-angle X-ray scattering data with the results of computer simulations. Docking and molecular dynamics studies on the generated three-dimensional model structure reveal the possibility of peptidoglycan fragment binding at the hinge regions between PASTA subunits with a preference for a bent hinge between PASTA3 and PASTA4.


Asunto(s)
Proteínas Bacterianas/química , Modelos Moleculares , Peptidoglicano/química , Proteínas Serina-Treonina Quinasas/química , Streptococcus pneumoniae/enzimología , Secuencia de Aminoácidos , Conformación Molecular , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes , Relación Estructura-Actividad
14.
Nat Microbiol ; 3(2): 197-209, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29203882

RESUMEN

Eukaryotic-like serine/threonine kinases (eSTKs) with extracellular PASTA repeats are key membrane regulators of bacterial cell division. How PASTA repeats govern eSTK activation and function remains elusive. Using evolution- and structural-guided approaches combined with cell imaging, we disentangle the role of each PASTA repeat of the eSTK StkP from Streptococcus pneumoniae. While the three membrane-proximal PASTA repeats behave as interchangeable modules required for the activation of StkP independently of cell wall binding, they also control the septal cell wall thickness. In contrast, the fourth and membrane-distal PASTA repeat directs StkP localization at the division septum and encompasses a specific motif that is critical for final cell separation through interaction with the cell wall hydrolase LytB. We propose a model in which the extracellular four-PASTA domain of StkP plays a dual function in interconnecting the phosphorylation of StkP endogenous targets along with septal cell wall remodelling to allow cell division of the pneumococcus.


Asunto(s)
División Celular , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Streptococcus pneumoniae/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Modelos Moleculares , N-Acetil Muramoil-L-Alanina Amidasa , Fosforilación , Estructura Terciaria de Proteína , Streptococcus pneumoniae/citología
15.
Sci Rep ; 7(1): 17013, 2017 12 05.
Artículo en Inglés | MEDLINE | ID: mdl-29208950

RESUMEN

Membrane proteins are distinguished from soluble proteins by their insertion into biological membranes. This insertion is achieved via a noticeable arrangement of hydrophobic amino acids that are exposed at the surface of the protein, and renders the interaction with the aliphatic tails of lipids more energetically favorable. This important difference between these two categories of proteins is the source of the need for a specific handling of membrane proteins, which transpired in the creation of new tools for their recombinant expression, purification and even crystallization. Following this line, we show here that crystals of membrane proteins display systematically higher diffraction anisotropy than those of soluble proteins. This phenomenon dramatically hampers structure solution and refinement, and has a strong impact on the quality of electron-density maps. A farther search for origins of this phenomenon showed that the type of crystallization, and thus the crystal packing, has no impact on anisotropy, nor does the nature or function of the membrane protein. Membrane proteins fully embedded within the membrane display equal anisotropy compared to the ones with extra membranous domains or fusions with soluble proteins. Overall, these results overturn common beliefs and call for a specific handling of their diffraction data.


Asunto(s)
Membrana Celular/química , Detergentes/química , Proteínas de la Membrana/química , Difracción de Rayos X , Anisotropía , Membrana Celular/metabolismo , Cristalización , Humanos , Proteínas de la Membrana/metabolismo , Conformación Proteica
16.
Retrovirology ; 14(1): 54, 2017 Nov 28.
Artículo en Inglés | MEDLINE | ID: mdl-29179726

RESUMEN

BACKGROUND: Stable insertion of the retroviral DNA genome into host chromatin requires the functional association between the intasome (integrase·viral DNA complex) and the nucleosome. The data from the literature suggest that direct protein-protein contacts between integrase and histones may be involved in anchoring the intasome to the nucleosome. Since histone tails are candidates for interactions with the incoming intasomes we have investigated whether they could participate in modulating the nucleosomal integration process. RESULTS: We show here that histone tails are required for an optimal association between HIV-1 integrase (IN) and the nucleosome for efficient integration. We also demonstrate direct interactions between IN and the amino-terminal tail of human histone H4 in vitro. Structure/function studies enabled us to identify amino acids in the carboxy-terminal domain of IN that are important for this interaction. Analysis of the nucleosome-binding properties of catalytically active mutated INs confirmed that their ability to engage the nucleosome for integration in vitro was affected. Pseudovirus particles bearing mutations that affect the IN/H4 association also showed impaired replication capacity due to altered integration and re-targeting of their insertion sites toward dynamic regions of the chromatin with lower nucleosome occupancy. CONCLUSIONS: Collectively, our data support a functional association between HIV-1 IN and histone tails that promotes anchoring of the intasome to nucleosomes and optimal integration into chromatin.


Asunto(s)
Integrasa de VIH/metabolismo , VIH-1/metabolismo , Histonas/metabolismo , Nucleosomas/metabolismo , Integración Viral , Línea Celular Transformada , Cromatina/virología , ADN Viral/metabolismo , Células HEK293 , VIH-1/genética , Histonas/química , Interacciones Huésped-Parásitos/fisiología , Humanos , Unión Proteica
17.
J Gen Virol ; 96(10): 3124-3130, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26296914

RESUMEN

Porcine endogenous retroviruses (PERVs) are present in the genomes of pig cells. The PERV-A/C recombinant virus can infect human cells and is a major risk of zoonotic disease in the case of xenotransplantation of pig organs to humans. Raltegravir (RAL) is a viral integrase (IN) inhibitor used in highly active antiretroviral treatment. In the present study, we explored the potential use of RAL against PERV-A/C. We report (i) a three-dimensional model of the PERV-A/C intasome complexed with RAL, (ii) the sensitivity of PERV-A/C IN to RAL in vitro and (iii) the sensitivity of a PERV-A/C-IRES-GFP recombinant virus to RAL in cellulo. We demonstrated that RAL is a potent inhibitor against PERV-A/C IN and PERV-A/C replication with IC50s in the nanomolar range. To date, the use of retroviral inhibitors remains the only way to control the risk of zoonotic PERV infection during pig-to-human xenotransplantation.


Asunto(s)
Antivirales/farmacología , Retrovirus Endógenos/enzimología , Retrovirus Endógenos/fisiología , Integrasas/análisis , Raltegravir Potásico/farmacología , Integración Viral/efectos de los fármacos , Animales , Antivirales/química , Cristalografía por Rayos X , Retrovirus Endógenos/efectos de los fármacos , Concentración 50 Inhibidora , Integrasas/química , Unión Proteica , Conformación Proteica , Raltegravir Potásico/química , Porcinos
18.
J Biol Chem ; 290(20): 12951-63, 2015 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-25837252

RESUMEN

The taurocyamine kinase from the blood fluke Schistosoma mansoni (SmTK) belongs to the phosphagen kinase (PK) family and catalyzes the reversible Mg(2+)-dependent transfer of a phosphoryl group between ATP and taurocyamine. SmTK is derived from gene duplication, as are all known trematode TKs. Our crystallographic study of SmTK reveals the first atomic structure of both a TK and a PK with a bilobal structure. The two unliganded lobes present a canonical open conformation and interact via their respective C- and N-terminal domains at a helix-mediated interface. This spatial arrangement differs from that observed in true dimeric PKs, in which both N-terminal domains make contact. Our structures of SmTK complexed with taurocyamine or l-arginine compounds explain the mechanism by which an arginine residue of the phosphagen specificity loop is crucial for substrate specificity. An SmTK crystal was soaked with the dead end transition state analog (TSA) components taurocyamine-NO3 (2-)-MgADP. One SmTK monomer was observed with two bound TSAs and an asymmetric conformation, with the first lobe semiclosed and the second closed. However, isothermal titration calorimetry and enzyme kinetics experiments showed that the two lobes function independently. A small angle x-ray scattering model of SmTK-TSA in solution with two closed active sites was generated.


Asunto(s)
Proteínas del Helminto/química , Modelos Moleculares , Fosfotransferasas (Aceptor del Grupo Nitrogenado)/química , Schistosoma mansoni/enzimología , Taurina/análogos & derivados , Animales , Cristalografía por Rayos X , Humanos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Taurina/química
19.
Mol Microbiol ; 94(1): 126-40, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25098941

RESUMEN

Type II secretion system (T2SS) is a multiprotein trans-envelope complex that translocates fully folded proteins through the outer membrane of Gram-negative bacteria. Although T2SS is extensively studied in several bacteria pathogenic for humans, animals and plants, the molecular basis for exoprotein recruitment by this secretion machine as well as the underlying targeting motifs remain unknown. To address this question, we used bacterial two-hybrid, surface plasmon resonance, in vivo site-specific photo-cross-linking approaches and functional analyses. We showed that the fibronectin-like Fn3 domain of exoprotein PelI from Dickeya dadantii interacts with four periplasmic domains of the T2SS components GspD and GspC. The interaction between exoprotein and the GspC PDZ domain is positively modulated by the GspD N1 domain, suggesting that exoprotein secretion is driven by a succession of synergistic interactions. We found that an exposed 9-residue-long loop region of PelI interacts with the GspC PDZ domain. This loop acts as a specific secretion signal that controls exoprotein recruitment by the T2SS. Concerted in silico and in vivo approaches reveal the occurrence of equivalent secretion motifs in other exoproteins, suggesting a plausible general mechanism of exoprotein recruitment by the T2SS.


Asunto(s)
Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos , Enterobacteriaceae/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Enterobacteriaceae/química , Enterobacteriaceae/genética , Periplasma/química , Periplasma/genética , Periplasma/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas
20.
Nucleic Acids Res ; 42(Web Server issue): W320-4, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24753421

RESUMEN

ENDscript 2 is a friendly Web server for extracting and rendering a comprehensive analysis of primary to quaternary protein structure information in an automated way. This major upgrade has been fully re-engineered to enhance speed, accuracy and usability with interactive 3D visualization. It takes advantage of the new version 3 of ESPript, our well-known sequence alignment renderer, improved to handle a large number of data with reduced computation time. From a single PDB entry or file, ENDscript produces high quality figures displaying multiple sequence alignment of proteins homologous to the query, colored according to residue conservation. Furthermore, the experimental secondary structure elements and a detailed set of relevant biophysical and structural data are depicted. All this information and more are now mapped on interactive 3D PyMOL representations. Thanks to its adaptive and rigorous algorithm, beginner to expert users can modify settings to fine-tune ENDscript to their needs. ENDscript has also been upgraded as an open platform for the visualization of multiple biochemical and structural data coming from external biotool Web servers, with both 2D and 3D representations. ENDscript 2 and ESPript 3 are freely available at http://endscript.ibcp.fr and http://espript.ibcp.fr, respectively.


Asunto(s)
Conformación Proteica , Programas Informáticos , Algoritmos , Catalasa/química , Internet , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Alineación de Secuencia , Análisis de Secuencia de Proteína
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