RESUMEN
Structures trapping a variety of functional and conformational states of HIV-1 reverse transcriptase (RT) have been determined by X-ray crystallography. These structures have played important roles in explaining the mechanisms of catalysis, inhibition, and drug resistance and in driving drug design. However, structures of several desired complexes of RT could not be obtained even after many crystallization or crystal soaking experiments. The ternary complexes of doravirine and rilpivirine with RT/DNA are such examples. Structural study of HIV-1 RT by single-particle cryo-electron microscopy (cryo-EM) has been challenging due to the enzyme's relatively smaller size and higher flexibility. We optimized a protocol for rapid structure determination of RT complexes by cryo-EM and determined six structures of wild-type and E138K/M184I mutant RT/DNA in complexes with the nonnucleoside inhibitors rilpivirine, doravirine, and nevirapine. RT/DNA/rilpivirine and RT/DNA/doravirine complexes have structural differences between them and differ from the typical conformation of nonnucleoside RT inhibitor (NNRTI)-bound RT/double-stranded DNA (dsDNA), RT/RNA-DNA, and RT/dsRNA complexes; the primer grip in RT/DNA/doravirine and the YMDD motif in RT/DNA/rilpivirine have large shifts. The DNA primer 3'-end in the doravirine-bound structure is positioned at the active site, but the complex is in a nonproductive state. In the mutant RT/DNA/rilpivirine structure, I184 is stacked with the DNA such that their relative positioning can influence rilpivirine in the pocket. Simultaneously, E138K mutation opens the NNRTI-binding pocket entrance, potentially contributing to a faster rate of rilpivirine dissociation by E138K/M184I mutant RT, as reported by an earlier kinetic study. These structural differences have implications for understanding molecular mechanisms of drug resistance and for drug design.
Asunto(s)
Fármacos Anti-VIH , Farmacorresistencia Viral , Transcriptasa Inversa del VIH , VIH-1 , Piridonas , Inhibidores de la Transcriptasa Inversa , Rilpivirina , Triazoles , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Microscopía por Crioelectrón , Farmacorresistencia Viral/genética , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/genética , VIH-1/enzimología , Mutación , Nitrilos/farmacología , Conformación Proteica , Piridonas/química , Piridonas/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología , Rilpivirina/química , Rilpivirina/farmacología , Triazoles/química , Triazoles/farmacologíaRESUMEN
Inhibitors against Trypanosoma brucei phosphodiesterase B1 (TbrPDEB1) and B2 (TbrPDEB2) have gained interest as new treatments for human African trypanosomiasis. The recently reported alkynamide tetrahydrophthalazinones, which show submicromolar activities against TbrPDEB1 and anti-T. brucei activity, have been used as starting point for the discovery of new TbrPDEB1 inhibitors. Structure-based design indicated that the alkynamide-nitrogen atom can be readily decorated, leading to the discovery of 37, a potent TbrPDEB1 inhibitor with submicromolar activities against T. brucei parasites. Furthermore, 37 is more potent against TbrPDEB1 than hPDE4 and shows no cytotoxicity on human MRC-5 cells. The crystal structures of the catalytic domain of TbrPDEB1 co-crystalized with several different alkynamides show a bidentate interaction with key-residue Gln874, but no interaction with the parasite-specific P-pocket, despite being (uniquely) a more potent inhibitor for the parasite PDE. Incubation of blood stream form trypanosomes by 37 increases intracellular cAMP levels and results in the distortion of the cell cycle and cell death, validating phosphodiesterase inhibition as mode of action.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/efectos de los fármacos , Inhibidores de Fosfodiesterasa/uso terapéutico , Proteínas Protozoarias/efectos de los fármacos , Humanos , Inhibidores de Fosfodiesterasa/farmacología , Relación Estructura-ActividadRESUMEN
Several 3',5'-cyclic nucleotide phosphodiesterases (PDEs) have been validated as good drug targets for a large variety of diseases. Trypanosoma brucei PDEB1 (TbrPDEB1) has been designated as a promising drug target for the treatment of human African trypanosomiasis. Recently, the first class of selective nanomolar TbrPDEB1 inhibitors was obtained by targeting the parasite specific P-pocket. However, these biphenyl-substituted tetrahydrophthalazinone-based inhibitors did not show potent cellular activity against Trypanosoma brucei (T. brucei) parasites, leaving room for further optimization. Herein, we report the discovery of a new class of potent TbrPDEB1 inhibitors that display improved activities against T. brucei parasites. Exploring different linkers between the reported tetrahydrophthalazinone core scaffold and the amide tail group resulted in the discovery of alkynamide phthalazinones as new TbrPDEB1 inhibitors, which exhibit submicromolar activities versus T. brucei parasites and no cytotoxicity to human MRC-5 cells. Elucidation of the crystal structure of alkynamide 8b (NPD-048) bound to the catalytic domain of TbrPDEB1 shows a bidentate interaction with the key-residue Gln874 and good directionality towards the P-pocket. Incubation of trypanosomes with alkynamide 8b results in an increase of intracellular cAMP, validating a PDE-mediated effect in vitro and providing a new interesting compound series for further studies towards selective TbrPDEB1 inhibitors with potent phenotypic activity.
Asunto(s)
Inhibidores de Fosfodiesterasa/uso terapéutico , Trypanosoma brucei brucei/efectos de los fármacos , Humanos , Inhibidores de Fosfodiesterasa/farmacología , Relación Estructura-ActividadRESUMEN
The functional and biological significance of the selected CASP12 targets are described by the authors of the structures. The crystallographers discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP12 experiment.
Asunto(s)
Biología Computacional/métodos , Modelos Moleculares , Conformación Proteica , Proteínas/química , Animales , Bacterias/química , Cristalografía por Rayos X , Humanos , Pliegue de Proteína , Programas InformáticosRESUMEN
Murine adenovirus 2 (MAdV-2) infects cells of the mouse gastrointestinal tract. Like human adenoviruses, it is a member of the genus Mastadenovirus, family Adenoviridae. The MAdV-2 genome has a single fibre gene that expresses a 787 residue-long protein. Through analogy to other adenovirus fibre proteins, it is expected that the carboxy-terminal virus-distal head domain of the fibre is responsible for binding to the host cell, although the natural receptor is unknown. The putative head domain has little sequence identity to adenovirus fibres of known structure. In this report, we present high-resolution crystal structures of the carboxy-terminal part of the MAdV-2 fibre. The structures reveal a domain with the typical adenovirus fibre head topology and a domain containing two triple ß-spiral repeats of the shaft domain. Through glycan microarray profiling, saturation transfer difference nuclear magnetic resonance spectroscopy, isothermal titration calorimetry and site-directed mutagenesis, we show that the fibre specifically binds to the monosaccharide N-acetylglucosamine (GlcNAc). The crystal structure of the complex reveals that GlcNAc binds between the AB and CD loops at the top of each of the three monomers of the MAdV-2 fibre head. However, infection competition assays show that soluble GlcNAc monosaccharide and natural GlcNAc-containing polymers do not inhibit infection by MAdV-2. Furthermore, site-directed mutation of the GlcNAc-binding residues does not prevent the inhibition of infection by soluble fibre protein. On the other hand, we show that the MAdV-2 fibre protein binds GlcNAc-containing mucin glycans, which suggests that the MAdV-2 fibre protein may play a role in viral mucin penetration in the mouse gut.
Asunto(s)
Acetilglucosamina/metabolismo , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Dominios Proteicos , Receptores Virales/metabolismo , Animales , Cristalografía por Rayos X , Ratones , Unión Proteica , Conformación ProteicaRESUMEN
The Critical Assessment of protein Structure Prediction (CASP) experiment would not have been possible without the prediction targets provided by the experimental structural biology community. In this article, selected crystallographers providing targets for the CASP11 experiment discuss the functional and biological significance of the target proteins, highlight their most interesting structural features, and assess whether these features were correctly reproduced in the predictions submitted to CASP11. Proteins 2016; 84(Suppl 1):34-50. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.
Asunto(s)
Biología Computacional/estadística & datos numéricos , Modelos Moleculares , Modelos Estadísticos , Proteínas/química , Programas Informáticos , Bacterias/química , Biología Computacional/métodos , Gráficos por Computador , Cristalografía por Rayos X , Bases de Datos de Proteínas , Humanos , Cooperación Internacional , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Virus/químicaRESUMEN
BACKGROUND: In adenoviruses, primary host cell recognition is generally performed by the head domains of their homo-trimeric fibre proteins. This first interaction is reversible. A secondary, irreversible interaction subsequently takes place via other adenovirus capsid proteins and leads to a productive infection. Although many fibre head structures are known for human mastadenoviruses, not many animal adenovirus fibre head structures have been determined, especially not from those belonging to adenovirus genera other than Mastadenovirus. METHODS: We constructed an expression vector for the fibre head domain from a ruminant atadenovirus, bovine adenovirus 4 (BAdV-4), consisting of amino acids 414-535, expressed the protein in Escherichia coli, purified it by metal affinity and cation exchange chromatography and crystallized it. The structure was solved using single isomorphous replacement plus anomalous dispersion of a mercury derivative and refined against native data that extended to 1.2 Å resolution. RESULTS: Like in other adenoviruses, the BAdV-4 fibre head monomer contains a beta-sandwich consisting of ABCJ and GHID sheets. The topology is identical to the fibre head of the other studied atadenovirus, snake adenovirus 1 (SnAdV-1), including the alpha-helix in the DG-loop, despite of them having a sequence identity of only 15 %. There are also differences which may have implications for ligand binding. Beta-strands G and H are longer and differences in several surface-loops and surface charge are observed. CONCLUSIONS: Chimeric adenovirus fibres have been used to retarget adenovirus-based anti-cancer and gene therapy vectors. Ovine adenovirus 7 (OAdV-7), another ruminant atadenovirus, is intensively tested as a basis for such a vector. Here, we present the high-resolution atomic structure of the BAdV-4 fibre head domain, the second atadenovirus fibre head structure known and the first of an atadenovirus that infects a mammalian host. Future research should focus on the receptor-binding properties of these fibre head domains.
Asunto(s)
Atadenovirus/química , Proteínas de la Cápside/química , Animales , Bovinos , Cristalografía por Rayos X , Modelos Moleculares , Conformación ProteicaRESUMEN
In search of new opportunities to develop Trypanosoma brucei phosphodiesterase B1 (TbrPDEB1) inhibitors that have selectivity over the off-target human PDE4 (hPDE4), different stages of a fragment-growing campaign were studied using a variety of biochemical, structural, thermodynamic, and kinetic binding assays. Remarkable differences in binding kinetics were identified and this kinetic selectivity was explored with computational methods, including molecular dynamics and interaction fingerprint analyses. These studies indicate that a key hydrogen bond between GlnQ.50 and the inhibitors is exposed to a water channel in TbrPDEB1, leading to fast unbinding. This water channel is not present in hPDE4, leading to inhibitors with a longer residence time. The computer-aided drug design protocols were applied to a recently disclosed TbrPDEB1 inhibitor with a different scaffold and our results confirm that shielding this key hydrogen bond through disruption of the water channel represents a viable design strategy to develop more selective inhibitors of TbrPDEB1. Our work shows how computational protocols can be used to understand the contribution of solvent dynamics to inhibitor binding, and our results can be applied in the design of selective inhibitors for homologous PDEs found in related parasites.
RESUMEN
Turkey adenovirus 3 belongs to the genus Siadenovirus. Its predicted fibre protein consists of an N-terminal virus-attachment domain, a central shaft domain and a head domain at the C-terminus. The head domain has little sequence identity to known adenovirus fibre head structures. Crystals of the fibre head domain consisting of amino acids 304-454 with an N-terminal purification tag were produced. Crystals of native and selenomethionine-derivatized protein belonged to space group I23 (unit-cell parameter 99â Å). They diffracted synchrotron radiation to 2.0 and 2.14â Å resolution, respectively, and are expected to contain one monomer in the asymmetric unit.
Asunto(s)
Siadenovirus/metabolismo , Proteínas Virales/química , Secuencia de Aminoácidos , Cristalización , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas Virales/aislamiento & purificaciónRESUMEN
Adenovirus fibre proteins play an important role in determining viral tropism. The C-terminal domain of the fibre protein from snake adenovirus type 1, a member of the Atadenovirus genus, has been expressed, purified and crystallized. Crystals were obtained belonging to space groups P2(1)2(1)2(1) (two different forms), I2(1)3 and F23. The best of these diffracted synchrotron radiation to a resolution of 1.4 Å. As the protein lacks methionines or cysteines, site-directed mutagenesis was performed to change two leucine residues to methionines. Crystals of selenomethionine-derivatized crystals of the I2(1)3 form were also obtained and a multi-wavelength anomalous dispersion data set was collected.
Asunto(s)
Atadenovirus/química , Proteínas de la Cápside/química , Leucina/química , Metionina/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Atadenovirus/genética , Atadenovirus/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Cristalización , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Iguanas/virología , Leucina/genética , Metionina/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Miocitos Cardíacos/citología , Miocitos Cardíacos/virología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Selenometionina/química , Selenometionina/metabolismo , Serpientes/virología , SincrotronesRESUMEN
The enzyme reverse transcriptase (RT) plays a central role in the life cycle of human immunodeficiency virus (HIV), and RT has been an important drug target. Elucidations of the RT structures trapping and detailing the enzyme at various functional and conformational states by X-ray crystallography have been instrumental for understanding RT activities, inhibition, and drug resistance. The structures have contributed to anti-HIV drug development. Currently, two classes of RT inhibitors are in clinical use. These are nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). However, the error-prone viral replication generates variants that frequently develop resistance to the available drugs, thus warranting a continued effort to seek more effective treatment options. RT also provides multiple additional potential druggable sites. Recently, the use of single-particle cryogenic electron microscopy (cryo-EM) enabled obtaining structures of NNRTI-inhibited HIV-1 RT/dsRNA initiation and RT/dsDNA elongation complexes that were unsuccessful by X-ray crystallography. The cryo-EM platform for the structural study of RT has been established to aid drug design. In this article, we review the roles of structural biology in understanding and targeting HIV RT in the past three decades and the recent structural insights of RT, using cryo-EM.
Asunto(s)
Farmacorresistencia Viral , Infecciones por VIH , VIH-1 , Inhibidores de la Transcriptasa Inversa , Transcriptasa Inversa del VIH/genética , VIH-1/efectos de los fármacos , Humanos , Inhibidores de la Transcriptasa Inversa/farmacología , Inhibidores de la Transcriptasa Inversa/uso terapéuticoRESUMEN
HIV-1 reverse transcriptase (RT) slides over an RNA/DNA or dsDNA substrate while copying the viral RNA to a proviral DNA. We report a crystal structure of RT/dsDNA complex in which RT overstepped the primer 3'-end of a dsDNA substrate and created a transient P-pocket at the priming site. We performed a high-throughput screening of 300 drug-like fragments by X-ray crystallography that identifies two leads that bind the P-pocket, which is composed of structural elements from polymerase active site, primer grip, and template-primer that are resilient to drug-resistance mutations. Analogs of a fragment were synthesized, two of which show noticeable RT inhibition. An engineered RT/DNA aptamer complex could trap the transient P-pocket in solution, and structures of the RT/DNA complex were determined in the presence of an inhibitory fragment. A synthesized analog bound at P-pocket is further analyzed by single-particle cryo-EM. Identification of the P-pocket within HIV RT and the developed structure-based platform provide an opportunity for the design new types of polymerase inhibitors.
Asunto(s)
ADN/química , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología , Sitios de Unión , Microscopía por Crioelectrón , Cristalografía por Rayos X , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Transcriptasa Inversa del VIH/metabolismo , VIH-1/genética , Modelos Moleculares , Conformación Proteica , ARNRESUMEN
HIV-1 reverse transcriptase (RT) plays a central role in the viral life cycle, and roughly half of the FDA-approved anti-HIV drugs are targeting RT. Nucleoside analogs (NRTIs) require cellular phosphorylation for binding to RT, and to bypass this rate-limiting path, we designed a new series of acyclic nucleoside phosphonate analogs as nucleoside triphosphate mimics, aiming at the chelation of the catalytic Mg2+ ions via a phosphonate and/or a carboxylic acid group. Novel synthetic procedures were developed to access these nucleoside phosphonate analogs. X-ray structures in complex with HIV-1 RT/dsDNA demonstrated that their binding modes are distinct from that of our previously reported compound series. The impact of chain length, chirality and linker atom have been discussed. The detailed structural understanding of these new compounds provides opportunities for designing new class of HIV-1 RT inhibitors.
Asunto(s)
Fármacos Anti-VIH/farmacología , Diseño de Fármacos , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/efectos de los fármacos , Nucleótidos/antagonistas & inhibidores , Inhibidores de la Transcriptasa Inversa/farmacología , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/química , Sitios de Unión/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Nucleótidos/metabolismo , Inhibidores de la Transcriptasa Inversa/síntesis química , Inhibidores de la Transcriptasa Inversa/química , Relación Estructura-ActividadRESUMEN
Talin is a mechanosensitive adapter protein that couples integrins to the cytoskeleton. Talin rod domain-containing protein 1 (TLNRD1) shares 22% homology with the talin R7R8 rod domains, and is highly conserved throughout vertebrate evolution, although little is known about its function. Here we show that TLNRD1 is an α-helical protein structurally homologous to talin R7R8. Like talin R7R8, TLNRD1 binds F-actin, but because it forms a novel antiparallel dimer, it also bundles F-actin. In addition, it binds the same LD motif-containing proteins, RIAM and KANK, as talin R7R8. In cells, TLNRD1 localizes to actin bundles as well as to filopodia. Increasing TLNRD1 expression enhances filopodia formation and cell migration on 2D substrates, while TLNRD1 down-regulation has the opposite effect. Together, our results suggest that TLNRD1 has retained the diverse interactions of talin R7R8, but has developed distinct functionality as an actin-bundling protein that promotes filopodia assembly.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Chaperonas Moleculares/metabolismo , Seudópodos/metabolismo , Talina/metabolismo , Citoesqueleto de Actina/ultraestructura , Actinas/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular Tumoral , Movimiento Celular , Clonación Molecular , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Chaperonas Moleculares/antagonistas & inhibidores , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Osteoblastos/citología , Osteoblastos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Multimerización de Proteína , Seudópodos/ultraestructura , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Talina/genéticaRESUMEN
G protein-coupled receptors (GPCRs) are involved in a plethora of different diseases. Consequently, these proteins are considered as an important class of drug targets. Measuring detailed kinetic information on these types of proteins has been challenging. Surface plasmon resonance (SPR) can provide this information, however, the use of SPR on GPCRs remains a complex issue. Here, we report an SPR assay to investigate the interactions between the full-length chemokine receptor CXCR4 and nanobody-Fc (Nb-Fc) ligands. Nb-Fcs consist of two monovalent VHH domains fused with an Fc domain of a human IgG molecule. The CXCR4 protein used in this assay was produced with a C-terminal 10x-histidine tag and was immobilized on a nitrilotriacetic acid chip. In order to verify the sensitivity and effectiveness of this assay, the results were compared to data obtained from cellular assays as well as from another SPR assay using CXCR4 virus-like particles (VLPs). CXCR4 remained intact and stable for at least 12 h, and the kinetic results correlated well with both the cellular assays and the VLP SPR assay results. Apart from determining the binding kinetics of Nb-Fc with CXCR4, our results contributed to understanding CXCR4 interaction dynamics. In conclusion, this assay provides a viable experimental platform that has high potential to be expanded for studying other molecules as well as other histidine-tagged GPCRs.
Asunto(s)
Resonancia por Plasmón de Superficie/métodos , Bioensayo , Humanos , Ligandos , Unión Proteica , Receptores CXCR4 , Transducción de SeñalRESUMEN
Human African trypanosomiasis is causing thousands of deaths every year in the rural areas of Africa. In this manuscript we describe the optimization of a family of phtalazinone derivatives. Phosphodiesterases have emerged as attractive molecular targets for a novel treatment for a variety of neglected parasitic diseases. Compound 1 resulted in being a potent TbrPDEB1 inhibitor with interesting activity against T. brucei in a phenotypic screen. Derivative 1 was studied in an acute in vivo mouse disease model but unfortunately showed no efficacy due to low metabolic stability. We report structural modifications to achieve compounds with an improved metabolic stability while maintaining high potency against TbrPDEB1 and T. brucei. Compound 14 presented a good microsomal stability in mouse and human microsomes and provides a good starting point for future efforts.
Asunto(s)
Inhibidores de Fosfodiesterasa/farmacología , Ftalazinas/farmacología , Tripanocidas/farmacología , Animales , Cristalografía por Rayos X , Estabilidad de Medicamentos , Humanos , Ratones , Microsomas Hepáticos/metabolismo , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Inhibidores de Fosfodiesterasa/síntesis química , Inhibidores de Fosfodiesterasa/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Ftalazinas/síntesis química , Ftalazinas/metabolismo , Unión Proteica , Proteínas Protozoarias/metabolismo , Relación Estructura-Actividad , Tripanocidas/síntesis química , Tripanocidas/metabolismo , Trypanosoma brucei brucei/efectos de los fármacosRESUMEN
Vinculin is a central component of mechanosensitive adhesive complexes that form between cells and the extracellular matrix. A myriad of infectious agents mimic vinculin binding sites (VBS), enabling them to hijack the adhesion machinery and facilitate cellular entry. Here, we report the structural and biochemical characterisation of VBS from the chlamydial virulence factor TarP. Whilst the affinities of isolated VBS peptides from TarP and talin for vinculin are similar, their behaviour in larger fragments is markedly different. In talin, VBS are cryptic and require mechanical activation to bind vinculin, whereas the TarP VBS are located in disordered regions, and so are constitutively active. We demonstrate that the TarP VBS can uncouple talin:vinculin complexes, which may lead to adhesion destabilisation.
Asunto(s)
Proteínas Bacterianas/fisiología , Chlamydia/patogenicidad , Imitación Molecular , Talina/fisiología , Vinculina/metabolismo , Factores de Virulencia/fisiología , Secuencia de Aminoácidos , Cristalografía por Rayos X , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica , Homología de Secuencia de Aminoácido , Talina/química , Talina/metabolismo , Vinculina/químicaRESUMEN
Several trypanosomatid cyclic nucleotide phosphodiesterases (PDEs) possess a unique, parasite-specific cavity near the ligand-binding region that is referred to as the P-pocket. One of these enzymes, Trypanosoma brucei PDE B1 (TbrPDEB1), is considered a drug target for the treatment of African sleeping sickness. Here, we elucidate the molecular determinants of inhibitor binding and reveal that the P-pocket is amenable to directed design. By iterative cycles of design, synthesis, and pharmacological evaluation and by elucidating the structures of inhibitor-bound TbrPDEB1, hPDE4B, and hPDE4D complexes, we have developed 4a,5,8,8a-tetrahydrophthalazinones as the first selective TbrPDEB1 inhibitor series. Two of these, 8 (NPD-008) and 9 (NPD-039), were potent ( Ki = 100 nM) TbrPDEB1 inhibitors with antitrypanosomal effects (IC50 = 5.5 and 6.7 µM, respectively). Treatment of parasites with 8 caused an increase in intracellular cyclic adenosine monophosphate (cAMP) levels and severe disruption of T. brucei cellular organization, chemically validating trypanosomal PDEs as therapeutic targets in trypanosomiasis.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Inhibidores de Fosfodiesterasa/química , Inhibidores de Fosfodiesterasa/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Tripanocidas/química , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/enzimología , 3',5'-AMP Cíclico Fosfodiesterasas/química , Amidas/química , Amidas/farmacología , Dominio Catalítico , Concentración 50 Inhibidora , Modelos Moleculares , Terapia Molecular Dirigida , Proteínas Protozoarias/química , Relación Estructura-ActividadRESUMEN
Although non-human adenoviruses (AdVs) might offer solutions to problems posed by human AdVs as therapeutic vectors, little is known about their basic biology. In particular, there are no structural studies on the complete virion of any AdV with a non-mammalian host. We combine mass spectrometry, cryo-electron microscopy, and protein crystallography to characterize the composition and structure of a snake AdV (SnAdV-1, Atadenovirus genus). SnAdV-1 particles contain the genus-specific proteins LH3, p32k, and LH2, a previously unrecognized structural component. Remarkably, the cementing protein LH3 has a trimeric ß helix fold typical of bacteriophage host attachment proteins. The organization of minor coat proteins differs from that in human AdVs, correlating with higher thermostability in SnAdV-1. These findings add a new piece to the intriguing puzzle of virus evolution, hint at the use of cell entry pathways different from those in human AdVs, and will help development of new, thermostable SnAdV-1-based vectors.
Asunto(s)
Atadenovirus/química , Proteínas de la Cápside/química , Cápside/química , Reptiles/virología , Secuencia de Aminoácidos , Animales , Atadenovirus/genética , Proteínas de la Cápside/genética , Microscopía por Crioelectrón , Cristalografía por Rayos X , Espectrometría de Masas , Modelos Moleculares , Estructura Secundaria de ProteínaRESUMEN
The virulent form of turkey adenovirus 3 (TAdV-3), also known as turkey hemorrhagic enteritis virus (THEV), is an economically important poultry pathogen, while the avirulent form is used as a vaccine. TAdV-3 belongs to the genus Siadenovirus. The carboxy-terminal region of its fibre does not have significant sequence similarity to any other adenovirus fibre heads of known structure. Two amino acid sequence differences between virulent and avirulent TAdV-3 map on the fibre head: where virulent TAdV-3 contains Ile354 and Thr376, avirulent TAdV-3 contains Met354 and Met376. We determined the crystal structures of the trimeric virulent and avirulent TAdV-3 fibre head domains at 2.2 Å resolution. Each monomer contains a beta-sandwich, which, surprisingly, resembles reovirus fibre head more than other adenovirus fibres, although the ABCJ-GHID topology is conserved in all. A beta-hairpin insertion in the C-strand of each trimer subunit embraces its neighbouring monomer. The avirulent and virulent TAdV-3 fibre heads are identical apart from the exact orientation of the beta-hairpin insertion. In vitro, sialyllactose was identified as a ligand by glycan microarray analysis, nuclear magnetic resonance spectroscopy, and crystallography. Its dissociation constant was measured to be in the mM range by isothermal titration calorimetry. The ligand binds to the side of the fibre head, involving amino acids Glu392, Thr419, Val420, Lys421, Asn422, and Gly423 binding to the sialic acid group. It binds slightly more strongly to the avirulent form. We propose that, in vivo, the TAdV-3 fibre may bind a sialic acid-containing cell surface component.