RESUMEN
A key challenge for the development of a cure to HIV-1 infection is the persistent viral reservoir established during early infection. Previous studies using Toll-like receptor 7 (TLR7) agonists and broadly neutralizing antibodies (bNAbs) have shown delay or prevention of viral rebound following antiretroviral therapy (ART) discontinuation in simian-human immunodeficiency virus (SHIV)-infected rhesus macaques. In these prior studies, ART was initiated early during acute infection, which limited the size and diversity of the viral reservoir. Here we evaluated in SHIV-infected rhesus macaques that did not initiate ART until 1 year into chronic infection whether the TLR7 agonist vesatolimod in combination with the bNAb PGT121, formatted either as a human IgG1, an effector enhanced IgG1, or an anti-CD3 bispecific antibody, would delay or prevent viral rebound following ART discontinuation. We found that all 3 antibody formats in combination with vesatolimod were able to prevent viral rebound following ART discontinuation in a subset of animals. These data indicate that a TLR7 agonist combined with antibodies may be a promising strategy to achieve long-term ART-free HIV remission in humans.
Asunto(s)
Infecciones por VIH , VIH-1 , Síndrome de Inmunodeficiencia Adquirida del Simio , Virus de la Inmunodeficiencia de los Simios , Animales , Antirretrovirales/farmacología , Antirretrovirales/uso terapéutico , Anticuerpos ampliamente neutralizantes , Anticuerpos Anti-VIH/uso terapéutico , Inmunoglobulina G , Macaca mulatta , Receptor Toll-Like 7/agonistas , Carga ViralRESUMEN
Hexameric helicases couple ATP hydrolysis to processive separation of nucleic acid duplexes, a process critical for gene expression, DNA replication, and repair. All hexameric helicases fall into two families with opposing translocation polarities: the 3'-->5' AAA+ and 5'-->3' RecA-like enzymes. To understand how a RecA-like hexameric helicase engages and translocates along substrate, we determined the structure of the E. coli Rho transcription termination factor bound to RNA and nucleotide. Interior nucleic acid-binding elements spiral around six bases of RNA in a manner unexpectedly reminiscent of an AAA+ helicase, the papillomavirus E1 protein. Four distinct ATP-binding states, representing potential catalytic intermediates, are coupled to RNA positioning through a complex allosteric network. Comparative studies with E1 suggest that RecA and AAA+ hexameric helicases use different portions of their chemomechanical cycle for translocating nucleic acid and track in opposite directions by reversing the firing order of ATPase sites around the hexameric ring. For a video summary of this article, see the PaperFlick file with the Supplemental Data available online.
Asunto(s)
Escherichia coli/enzimología , ARN Helicasas/química , Factor Rho/química , Adenosina Trifosfato/metabolismo , Cristalografía por Rayos X , ADN Helicasas/química , ADN Helicasas/metabolismo , Ácido Glutámico/metabolismo , Modelos Moleculares , ARN/metabolismo , ARN Helicasas/metabolismo , Rec A Recombinasas/química , Factor Rho/metabolismoRESUMEN
The circuitry of the brain is characterized by cell heterogeneity, sprawling cellular anatomy, and astonishingly complex patterns of connectivity. Determining how complex neural circuits control behavior is a major challenge that is often approached using surgical, chemical, or transgenic approaches to ablate neurons. However, all these approaches suffer from a lack of precise spatial and temporal control. This drawback would be overcome if cellular ablation could be controlled with light. Cells are naturally and cleanly ablated through apoptosis due to the terminal activation of caspases. Here, we describe the engineering of a light-activated human caspase-3 (Caspase-LOV) by exploiting its natural spring-loaded activation mechanism through rational insertion of the light-sensitive LOV2 domain that expands upon illumination. We apply the light-activated caspase (Caspase-LOV) to study neurodegeneration in larval and adult Drosophila Using the tissue-specific expression system (UAS)-GAL4, we express Caspase-LOV specifically in three neuronal cell types: retinal, sensory, and motor neurons. Illumination of whole flies or specific tissues containing Caspase-LOV-induced cell death and allowed us to follow the time course and sequence of neurodegenerative events. For example, we find that global synchronous activation of caspase-3 drives degeneration with a different time-course and extent in sensory versus motor neurons. We believe the Caspase-LOV tool we engineered will have many other uses for neurobiologists and others for specific temporal and spatial ablation of cells in complex organisms.
Asunto(s)
Apoptosis/fisiología , Caspasa 3/genética , Drosophila melanogaster/metabolismo , Activación Enzimática/genética , Luz , Neuronas Motoras/metabolismo , Células Receptoras Sensoriales/metabolismo , Técnicas de Ablación , Animales , Animales Modificados Genéticamente , Encéfalo/fisiología , Caspasa 3/metabolismo , Caspasas/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Conducción Nerviosa/fisiología , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas Virales/metabolismoRESUMEN
Ring-shaped hexameric helicases and translocases support essential DNA-, RNA-, and protein-dependent transactions in all cells and many viruses. How such systems coordinate ATPase activity between multiple subunits to power conformational changes that drive the engagement and movement of client substrates is a fundamental question. Using the Escherichia coli Rho transcription termination factor as a model system, we have used solution and crystallographic structural methods to delineate the range of conformational changes that accompany distinct substrate and nucleotide cofactor binding events. Small-angle X-ray scattering data show that Rho preferentially adopts an open-ring state in solution and that RNA and ATP are both required to cooperatively promote ring closure. Multiple closed-ring structures with different RNA substrates and nucleotide occupancies capture distinct catalytic intermediates accessed during translocation. Our data reveal how RNA-induced ring closure templates a sequential ATP-hydrolysis mechanism, provide a molecular rationale for how the Rho ATPase domains distinguishes between distinct RNA sequences, and establish structural snapshots of substepping events in a hexameric helicase/translocase.
Asunto(s)
ADN Helicasas/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Proteínas Portadoras de Nucleobases, Nucleósidos, Nucleótidos y Ácidos Nucleicos/química , Adenosina Trifosfato/química , Dominio Catalítico , Hidrólisis , Cinética , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Estructura Cuaternaria de Proteína , ARN Bacteriano/químicaRESUMEN
Superfamily 1 and superfamily 2 RNA helicases are ubiquitous messenger-RNA-protein complex (mRNP) remodelling enzymes that have critical roles in all aspects of RNA metabolism. The superfamily 2 DEAD-box ATPase Dbp5 (human DDX19) functions in mRNA export and is thought to remodel mRNPs at the nuclear pore complex (NPC). Dbp5 is localized to the NPC via an interaction with Nup159 (NUP214 in vertebrates) and is locally activated there by Gle1 together with the small-molecule inositol hexakisphosphate (InsP(6)). Local activation of Dbp5 at the NPC by Gle1 is essential for mRNA export in vivo; however, the mechanistic role of Dbp5 in mRNP export is poorly understood and it is not known how Gle1(InsP6) and Nup159 regulate the activity of Dbp5. Here we report, from yeast, structures of Dbp5 in complex with Gle1(InsP6), Nup159/Gle1(InsP6) and RNA. These structures reveal that InsP(6) functions as a small-molecule tether for the Gle1-Dbp5 interaction. Surprisingly, the Gle1(InsP6)-Dbp5 complex is structurally similar to another DEAD-box ATPase complex essential for translation initiation, eIF4G-eIF4A, and we demonstrate that Gle1(InsP6) and eIF4G both activate their DEAD-box partner by stimulating RNA release. Furthermore, Gle1(InsP6) relieves Dbp5 autoregulation and cooperates with Nup159 in stabilizing an open Dbp5 intermediate that precludes RNA binding. These findings explain how Gle1(InsP6), Nup159 and Dbp5 collaborate in mRNA export and provide a general mechanism for DEAD-box ATPase regulation by Gle1/eIF4G-like activators.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , ARN Helicasas DEAD-box/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Ácido Fítico/metabolismo , Transporte de ARN , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/química , ARN Helicasas DEAD-box/química , Activación Enzimática , Factor 4A Eucariótico de Iniciación/química , Factor 4A Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/química , Factor 4G Eucariótico de Iniciación/metabolismo , Modelos Biológicos , Modelos Moleculares , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Conformación Proteica , ARN de Hongos/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
There is great interest in developing selective protein kinase inhibitors by targeting allosteric sites, but these sites often involve protein-protein or protein-peptide interfaces that are very challenging to target with small molecules. Here we present a systematic approach to targeting a functionally conserved allosteric site on the protein kinase PDK1 called the PDK1-interacting fragment (PIF)tide-binding site, or PIF pocket. More than two dozen prosurvival and progrowth kinases dock a conserved peptide tail into this binding site, which recruits them to PDK1 to become activated. Using a site-directed chemical screen, we identified and chemically optimized ligand-efficient, selective, and cell-penetrant small molecules (molecular weight â¼ 380 Da) that compete with the peptide docking motif for binding to PDK1. We solved the first high-resolution structure of a peptide docking motif (PIFtide) bound to PDK1 and mapped binding energy hot spots using mutational analysis. We then solved structures of PDK1 bound to the allosteric small molecules, which revealed a binding mode that remarkably mimics three of five hot-spot residues in PIFtide. These allosteric small molecules are substrate-selective PDK1 inhibitors when used as single agents, but when combined with an ATP-competitive inhibitor, they completely suppress the activation of the downstream kinases. This work provides a promising new scaffold for the development of high-affinity PIF pocket ligands, which may be used to enhance the anticancer activity of existing PDK1 inhibitors. Moreover, our results provide further impetus for exploring the helix αC patches of other protein kinases as potential therapeutic targets even though they involve protein-protein interfaces.
Asunto(s)
Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Simulación del Acoplamiento Molecular , Péptidos , Inhibidores de Proteínas Quinasas , Proteínas Quinasas Dependientes de 3-Fosfoinosítido/antagonistas & inhibidores , Proteínas Quinasas Dependientes de 3-Fosfoinosítido/química , Proteínas Quinasas Dependientes de 3-Fosfoinosítido/genética , Proteínas Quinasas Dependientes de 3-Fosfoinosítido/metabolismo , Regulación Alostérica/efectos de los fármacos , Sitio Alostérico , Secuencias de Aminoácidos , Antineoplásicos/química , Antineoplásicos/farmacología , Cristalografía por Rayos X , Células HEK293 , Humanos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/genética , Neoplasias/patología , Péptidos/química , Péptidos/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Estructura Secundaria de ProteínaRESUMEN
Procaspase-3 (P3) and procaspase-7 (P7) are activated through proteolytic maturation to form caspase-3 (C3) and caspase-7 (C7), respectively, which serve overlapping but nonredundant roles as the executioners of apoptosis in humans. However, it is unclear if differences in P3 and P7 maturation mechanisms underlie their unique biological functions, as the structure of P3 remains unknown. Here, we report structures of P3 in a catalytically inactive conformation, structures of P3 and P7 bound to covalent peptide inhibitors that reveal the active conformation of the zymogens, and the structure of a partially matured C7:P7 heterodimer. Along with a biochemical analysis, we show that P3 is catalytically inactive and matures through a symmetric all-or-nothing process. In contrast, P7 contains latent catalytic activity and matures through an asymmetric and tiered mechanism, suggesting a lower threshold for activation. Finally, we use our structures to design a selection strategy for conformation specific antibody fragments that stimulate procaspase activity, showing that executioner procaspase conformational equilibrium can be rationally modulated. Our studies provide a structural framework that may help to explain the unique roles of these important proapoptotic enzymes, and suggest general strategies for the discovery of proenzyme activators.
Asunto(s)
Caspasa 3/química , Caspasa 7/química , Precursores Enzimáticos/química , Multimerización de Proteína/fisiología , Apoptosis/fisiología , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 7/genética , Caspasa 7/metabolismo , Activación Enzimática/fisiología , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Humanos , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
BACKGROUND: HIV envelope (env) diversity represents a significant challenge for the use of broadly neutralizing antibodies (bNAbs) in HIV treatment and cure studies. Screening for viral sensitivity to bNAbs to select eligible trial participants will be important to improve clinical efficacy; however, no universal approach has been established. METHODS: Pre-antiretroviral therapy plasma virus from participants in the Zurich Primary HIV Infection (ZPHI) study was genotyped and phenotyped for sensitivity to the bNAbs elipovimab (EVM, formerly GS-9722) and 3BNC117. The genotyping and phenotyping assessments were performed following the Clinical Laboratory Improvement Amendments of 1988 guidelines as required for entry into clinical trials. The genotypic-based prediction of bNAb sensitivity was based on HIV env amino acid signatures identified from a genotypic-phenotypic correlation algorithm using a subtype B database. RESULTS: Genotyping the plasma virus and applying env sensitivity signatures, ZPHI study participants with viral sensitivity to EVM and 3BNC117 were identified. ZPHI study participants with virus sensitive to EVM and 3BNC117 were also identified by phenotyping the plasma virus. Comparison of the genotypic and phenotypic sensitivity assessments showed strong agreement between the 2 methodologies. CONCLUSIONS: The genotypic assessment was found to be as predictive as the direct measurement of bNAb sensitivity by phenotyping and may, therefore, be preferred because of more rapid turnaround time and assay simplicity. A significant number of the participants were predicted to have virus sensitive to EVM and 3BNC117 and could, thus, be potential participants for clinical trials involving these bNAbs.
Asunto(s)
Terapia Antirretroviral Altamente Activa , Anticuerpos ampliamente neutralizantes/genética , Anticuerpos Anti-VIH/inmunología , Anticuerpos Anti-VIH/metabolismo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , VIH-1/efectos de los fármacos , Anticuerpos Monoclonales Humanizados , Anticuerpos Neutralizantes , Farmacorresistencia Viral , Genotipo , Infecciones por VIH/virología , VIH-1/genética , VIH-1/inmunología , Humanos , FenotipoRESUMEN
Many fundamental cellular processes depend on enzymes that utilize chemical energy to catalyse unfavourable reactions. Certain classes of ATPases provide a particularly vivid example of the process of energy conversion, employing cycles of nucleotide turnover to move and/or rearrange biological polymers such as proteins and nucleic acids. Four well-characterized classes of ATP-dependent protein/nucleic acid translocases and remodelling factors are found in all three domains of life (bacteria, archaea and eukarya): additional strand catalytic 'E' (ASCE) P-loop NTPases, GHL proteins, actin-fold enzymes and chaperonins. These unrelated protein superfamilies have each evolved the ability to couple ATP binding and hydrolysis to the generation of motion and force along or within their substrates. The past several years have witnessed the emergence of a wealth of structural data that help explain how such molecular engines link nucleotide turnover to conformational change. In this review, we highlight several recent advances to illustrate some of the mechanisms by which each family of ATP-dependent motors facilitates the rearrangement and movement of proteins, protein complexes and nucleic acids.
Asunto(s)
Adenosina Trifosfatasas/química , Proteínas Arqueales/metabolismo , Proteínas Bacterianas/química , Proteínas Fúngicas/química , Ácidos Nucleicos/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Proteínas Arqueales/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Catálisis , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hidrólisis , Ácidos Nucleicos/química , Conformación ProteicaRESUMEN
Most bacteria possess two type IIA topoisomerases, DNA gyrase and topo IV, that together help manage chromosome integrity and topology. Gyrase primarily introduces negative supercoils into DNA, an activity mediated by the C-terminal domain of its DNA binding subunit (GyrA). Although closely related to gyrase, topo IV preferentially decatenates DNA and relaxes positive supercoils. Here we report the structure of the full-length Escherichia coli ParC dimer at 3.0 A resolution. The N-terminal DNA binding region of ParC is highly similar to that of GyrA, but the ParC dimer adopts a markedly different conformation. The C-terminal domain (CTD) of ParC is revealed to be a degenerate form of the homologous GyrA CTD, and is anchored to the top of the N-terminal domains in a configuration different from that thought to occur in gyrase. Biochemical assays show that the ParC CTD controls the substrate specificity of topo IV, likely by capturing DNA segments of certain crossover geometries. This work delineates strong mechanistic parallels between topo IV and gyrase, while explaining how structural differences between the two enzyme families have led to distinct activity profiles. These findings in turn explain how the structures and functions of bacterial type IIA topoisomerases have evolved to meet specific needs of different bacterial families for the control of chromosome superstructure.
Asunto(s)
Topoisomerasa de ADN IV/metabolismo , Topoisomerasa de ADN IV/química , Modelos Moleculares , Conformación Proteica , Especificidad por SustratoRESUMEN
Aberrant changes in post-translational modifications (PTMs) such as phosphate groups underlie a majority of human diseases. However, detection and quantification of PTMs for diagnostic or biomarker applications often require PTM-specific monoclonal antibodies (mAbs), which are challenging to generate using traditional antibody-selection methods. Here we outline a general strategy for producing synthetic, PTM-specific mAbs by engineering a motif-specific 'hot spot' into an antibody scaffold. Inspired by a natural phosphate-binding motif, we designed and selected mAb scaffolds with hot spots specific for phosphoserine, phosphothreonine or phosphotyrosine. Crystal structures of the phospho-specific mAbs revealed two distinct modes of phosphoresidue recognition. Our data suggest that each hot spot functions independently of the surrounding scaffold, as phage display antibody libraries using these scaffolds yielded >50 phospho- and target-specific mAbs against 70% of target peptides. Our motif-specific scaffold strategy may provide a general solution for rapid, robust development of anti-PTM mAbs for signaling, diagnostic and therapeutic applications.
Asunto(s)
Anticuerpos Fosfo-Específicos/biosíntesis , Especificidad de Anticuerpos/inmunología , Ingeniería de Proteínas , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Anticuerpos Fosfo-Específicos/química , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Fosfopéptidos/química , Fosfopéptidos/inmunología , Anticuerpos de Cadena Única/inmunologíaRESUMEN
Hexameric helicases couple the energy of ATP hydrolysis to processive movement along nucleic acids and are critical components of cells and many viruses. Molecular motion derives from ATP hydrolysis at up to six distinct catalytic centers, which is coupled to the coordinated action of translocation loops in the center of the hexamer. Due to the structural dynamics and catalytic complexity of hexameric helicases, few have been crystallized with a full complement of bound substrates, and instead tend to form crystals belonging to high-symmetry space groups that obscure the differences among catalytic subunits. We were able to overcome these difficulties and solve an asymmetric structure of the Rho transcription termination factor from Escherichia coli bound to ATP mimics and RNA. Here, we present some considerations used for crystallization of this hexameric helicase, discuss the utility of substrate-centric crystal-screening strategies, and outline a crystal-aging screen that allowed us to overcome the adverse effects of nonmerohedral twinning.
Asunto(s)
Cristalografía por Rayos X/métodos , ARN Helicasas/química , ARN Helicasas/metabolismo , Adenosina Trifosfatasas/metabolismo , Rec A Recombinasas/metabolismoRESUMEN
The essential Mycobacterium tuberculosis Ser/Thr protein kinase (STPK), PknB, plays a key role in regulating growth and division, but the structural basis of activation has not been defined. Here, we provide biochemical and structural evidence that dimerization through the kinase-domain (KD) N-lobe activates PknB by an allosteric mechanism. Promoting KD pairing using a small-molecule dimerizer stimulates the unphosphorylated kinase, and substitutions that disrupt N-lobe pairing decrease phosphorylation activity in vitro and in vivo. Multiple crystal structures of two monomeric PknB KD mutants in complex with nucleotide reveal diverse inactive conformations that contain large active-site distortions that propagate > 30 Å from the mutation site. These results define flexible, inactive structures of a monomeric bacterial receptor KD and show how "back-to-back" N-lobe dimerization stabilizes the active KD conformation. This general mechanism of bacterial receptor STPK activation affords insights into the regulation of homologous eukaryotic kinases that form structurally similar dimers.