RESUMEN
Epstein-Barr virus (EBV) is the major cause of infectious mononucleosis and is associated with several human cancers and, more recently, multiple sclerosis. Despite its prevalence and health impact, there are currently no vaccines or treatments. Four viral glycoproteins (gp), gp350 and gH/gL/gp42, mediate entry into the major sites of viral replication, B cells, and epithelial cells. Here, we designed a nanoparticle vaccine displaying these proteins and showed that it elicits potent neutralizing antibodies that protect against infection in vivo. We designed single-chain gH/gL and gH/gL/gp42 proteins that were each fused to bacterial ferritin to form a self-assembling nanoparticle. Structural analysis revealed that single-chain gH/gL and gH/gL/gp42 adopted a similar conformation to the wild-type proteins, and the protein spikes were observed by electron microscopy. Single-chain gH/gL or gH/gL/gp42 nanoparticle vaccines were constructed to ensure product homogeneity needed for clinical development. These vaccines elicited neutralizing antibodies in mice, ferrets, and nonhuman primates that inhibited EBV entry into both B cells and epithelial cells. When mixed with a previously reported gp350 nanoparticle vaccine, gp350D123, no immune competition was observed. To confirm its efficacy in vivo, humanized mice were challenged with EBV after passive transfer of IgG from mice vaccinated with control, gH/gL/gp42+gp350D123, or gH/gL+gp350D123 nanoparticles. Although all control animals were infected, only one mouse in each vaccine group that received immune IgG had detectable transient viremia. Furthermore, no EBV lymphomas were detected in immune animals. This bivalent EBV nanoparticle vaccine represents a promising candidate to prevent EBV infection and EBV-related malignancies in humans.
Asunto(s)
Infecciones por Virus de Epstein-Barr , Vacunas , Animales , Anticuerpos Neutralizantes , Infecciones por Virus de Epstein-Barr/prevención & control , Hurones , Herpesvirus Humano 4 , Inmunoglobulina G , Ratones , Vacunas CombinadasRESUMEN
Interaction of factor VIII (FVIII) with von Willebrand factor (VWF) is mediated by the VWF D'D3 domains and thrombin-mediated release is essential for hemostasis after vascular injury. VWF-D'D3 mutations resulting in loss of FVIII binding are the underlying cause of von Willebrand disease (VWD) type 2N. Furthermore, the FVIII-VWF interaction has significant implications for the development of therapeutics for bleeding disorders, particularly hemophilia A, in which endogenous VWF clearance imposes a half-life ceiling on replacement FVIII therapy. To understand the structural basis of FVIII engagement by VWF, we solved the structure of BIVV001 by cryo-electron microscopy to 2.9 Å resolution. BIVV001 is a bioengineered clinical-stage FVIII molecule for the treatment of hemophilia A. In BIVV001, VWF-D'D3 is covalently linked to an Fc domain of a B domain-deleted recombinant FVIII (rFVIII) Fc fusion protein, resulting in a stabilized rFVIII/VWF-D'D3 complex. Our rFVIII/VWF structure resolves BIVV001 architecture and provides a detailed spatial understanding of previous biochemical and clinical observations related to FVIII-VWF engagement. Notably, the FVIII acidic a3 peptide region (FVIII-a3), established as a critical determinant of FVIII/VWF complex formation, inserts into a basic groove formed at the VWF-D'/rFVIII interface. Our structure shows direct interaction of sulfated Y1680 in FVIII-a3 and VWF-R816 that, when mutated, leads to severe hemophilia A or VWD type 2N, respectively. These results provide insight on this key coagulation complex, explain the structural basis of many hemophilia A and VWD type 2N mutations, and inform studies to further elucidate how VWF dissociates rapidly from FVIII upon activation.
Asunto(s)
Microscopía por Crioelectrón/métodos , Factor VIII/química , Proteínas Recombinantes de Fusión/química , Factor de von Willebrand/química , Combinación de Medicamentos , Humanos , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Mapeo de Interacción de Proteínas , Proteínas Recombinantes de Fusión/ultraestructuraRESUMEN
N-substituted azaindoles were discovered as potent pan-PIM inhibitors. Lead optimization, guided by structure and focused on physico-chemical properties allowed us to solve inherent hERG and permeability liabilities, and provided compound 27, which subsequently impacted KG-1 tumor growth in a mouse model.
Asunto(s)
Antineoplásicos/farmacología , Compuestos Aza/farmacología , Indoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Compuestos Aza/síntesis química , Compuestos Aza/metabolismo , Línea Celular Tumoral , Cristalografía por Rayos X , Humanos , Indoles/síntesis química , Indoles/metabolismo , Ratones , Piperidinas/síntesis química , Piperidinas/metabolismo , Piperidinas/farmacología , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Pirrolidinas/síntesis química , Pirrolidinas/metabolismo , Pirrolidinas/farmacología , Ratas , Estereoisomerismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
This work describes the rational amelioration of Cytochrome P450 4/5 (CYP3A4/5) induction through the Pregnane-X Receptor (PXR) pathway in a series of compounds that modulate the metabotropic glutamate Receptor 2 (mGluR2) via an allosteric mechanism. The compounds were initially shown to induce CYP3A4/5 via the gold-standard induction assay measured in primary human hepatocytes. This was followed up by testing the compounds in a PXR assay which correlated well with the assay in primary cells. Further, one of the compounds was crystallized with PXR (pdb code 6DUP). Analysis of this co-crystal structure, together with previously published PXR co-crystal structures, lead to modification ideas. The compounds synthesized based on these ideas were shown not to be CYP3A4/5 inducers. The mGluR2 activity of the resulting compounds was maintained.
Asunto(s)
Citocromo P-450 CYP3A/biosíntesis , Receptor X de Pregnano/fisiología , Receptores de Glutamato Metabotrópico/efectos de los fármacos , Regulación Alostérica , Animales , Cristalografía por Rayos X , Inducción Enzimática/fisiología , Humanos , Receptor X de Pregnano/química , RatasRESUMEN
Plasmodium parasites use specialized ligands which bind to red blood cell (RBC) receptors during invasion. Defining the mechanism of receptor recognition is essential for the design of interventions against malaria. Here, we present the structural basis for Duffy antigen (DARC) engagement by P. vivax Duffy binding protein (DBP). We used NMR to map the core region of the DARC ectodomain contacted by the receptor binding domain of DBP (DBP-RII) and solved two distinct crystal structures of DBP-RII bound to this core region of DARC. Isothermal titration calorimetry studies show these structures are part of a multi-step binding pathway, and individual point mutations of residues contacting DARC result in a complete loss of RBC binding by DBP-RII. Two DBP-RII molecules sandwich either one or two DARC ectodomains, creating distinct heterotrimeric and heterotetrameric architectures. The DARC N-terminus forms an amphipathic helix upon DBP-RII binding. The studies reveal a receptor binding pocket in DBP and critical contacts in DARC, reveal novel targets for intervention, and suggest that targeting the critical DARC binding sites will lead to potent disruption of RBC engagement as complex assembly is dependent on DARC binding. These results allow for models to examine inter-species infection barriers, Plasmodium immune evasion mechanisms, P. knowlesi receptor-ligand specificity, and mechanisms of naturally acquired P. vivax immunity. The step-wise binding model identifies a possible mechanism by which signaling pathways could be activated during invasion. It is anticipated that the structural basis of DBP host-cell engagement will enable development of rational therapeutics targeting this interaction.
Asunto(s)
Antígenos de Protozoos/química , Sistema del Grupo Sanguíneo Duffy/química , Eritrocitos/química , Plasmodium vivax/química , Proteínas Protozoarias/química , Receptores de Superficie Celular/química , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Línea Celular , Sistema del Grupo Sanguíneo Duffy/genética , Sistema del Grupo Sanguíneo Duffy/inmunología , Eritrocitos/inmunología , Eritrocitos/parasitología , Humanos , Evasión Inmune , Malaria Vivax/genética , Malaria Vivax/inmunología , Plasmodium vivax/inmunología , Plasmodium vivax/metabolismo , Mutación Puntual , Unión Proteica , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Relación Estructura-ActividadRESUMEN
We compare the structure, activity, and linkage of DNA-binding domains (DBDs) from σ(54) transcriptional activators and discuss how the properties of the DBDs and the linker to the neighboring domain are affected by the overall properties and requirements of the full proteins. These transcriptional activators bind upstream of specific promoters that utilize σ(54)-polymerase. Upon receiving a signal the activators assemble into hexamers, which then, through adenosine triphosphate (ATP) hydrolysis, drive a conformational change in polymerase that enables transcription initiation. We present structures of the DBDs of activators nitrogen regulatory protein C 1 (NtrC1) and Nif-like homolog 2 (Nlh2) from the thermophile Aquifex aeolicus. The structures of these domains and their relationship to other parts of the activators are discussed. These structures are compared with previously determined structures of the DBDs of NtrC4, NtrC, ZraR, and factor for inversion stimulation. The N-terminal linkers that connect the DBDs to the central domains in NtrC1 and Nlh2 were studied and found to be unstructured. Additionally, a crystal structure of full-length NtrC1 was solved, but density of the DBDs was extremely weak, further indicating that the linker between ATPase and DBDs functions as a flexible tether. Flexible linking of ATPase and DBDs is likely necessary to allow assembly of the active hexameric ATPase ring. The comparison of this set of activators also shows clearly that strong dimerization of the DBD only occurs when other domains do not dimerize strongly.
Asunto(s)
Estructura Terciaria de Proteína , ARN Polimerasa Sigma 54 , Secuencias de Aminoácidos , Proteínas Bacterianas/metabolismo , ADN , Proteínas de Unión al ADN/química , Transactivadores/química , Factores de TranscripciónRESUMEN
The σ subunits of bacterial RNA polymerase occur in many variant forms and confer promoter specificity to the holopolymerase. Members of the σ(54) family of σ subunits require the action of a 'transcriptional activator' protein to open the promoter and initiate transcription. The activator proteins undergo regulated assembly from inactive dimers to hexamers that are active ATPases. These contact σ(54) directly and, through ATP hydrolysis, drive a conformational change that enables promoter opening. σ(54) activators use several different kinds of regulatory domains to respond to a wide variety of intracellular signals. One common regulatory module, the GAF domain, is used by σ(54) activators to sense small-molecule ligands. The structural basis for GAF domain regulation in σ(54) activators has not previously been reported. Here, we present crystal structures of GAF regulatory domains for Aquifex aeolicus σ(54) activators NifA-like homolog (Nlh)2 and Nlh1 in three functional states-an 'open', ATPase-inactive state; a 'closed', ATPase-inactive state; and a 'closed', ligand-bound, ATPase-active state. We also present small-angle X-ray scattering data for Nlh2-linked GAF-ATPase domains in the inactive state. These GAF domain dimers regulate σ(54) activator proteins by holding the ATPase domains in an inactive dimer conformation. Ligand binding of Nlh1 dramatically remodels the GAF domain dimer interface, disrupting the contacts with the ATPase domains. This mechanism has strong parallels to the response to phosphorylation in some two-component regulated σ(54) activators. We describe a structural mechanism of GAF-mediated enzyme regulation that appears to be conserved among humans, plants, and bacteria.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Bacterias Gramnegativas/química , ARN Polimerasa Sigma 54/química , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Dimerización , Regulación Bacteriana de la Expresión Génica , Bacterias Gramnegativas/enzimología , Bacterias Gramnegativas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , ARN Polimerasa Sigma 54/metabolismo , Alineación de Secuencia , Transducción de Señal , Transactivadores/química , Transactivadores/metabolismoRESUMEN
Erythrocyte-binding antigen 140 (PfEBA-140) is a critical Plasmodium falciparum erythrocyte invasion ligand that engages glycophorin C on host erythrocytes during malaria infection. The minimal receptor-binding region of PfEBA-140 contains two conserved Duffy binding-like (DBL) domains, a fold unique to Plasmodium species. Here, we present the crystal structure of the receptor-binding region of PfEBA-140 at 2.4 Å resolution. The two-domain binding region is present as a monomer in the asymmetric unit, and the structure reveals novel features in PfEBA-140 that are likely determinants of receptor specificity. Analysis by small-angle x-ray scattering demonstrated that the minimal binding region is monomeric in solution, consistent with the crystal structure. Erythrocyte binding assays showed that the full-length binding region containing the tandem DBL domains is required for erythrocyte engagement, suggesting that both domains contain critical receptor contact sites. The electrostatic surface of PfEBA-140 elucidates a basic patch that constitutes a putative high-affinity binding interface spanning both DBL domains. Mutation of residues within this interface results in severely diminished erythrocyte binding. This study provides insight into the structural basis and mechanism of PfEBA-140 receptor engagement and forms a basis for future studies of this critical interaction. In addition, the solution and crystal structures allow the first identification of likely determinants of erythrocyte receptor specificity for P. falciparum invasion ligands. A complete understanding of the PfEBA-140 erythrocyte invasion pathway will aid in the design of invasion inhibitory therapeutics and vaccines.
Asunto(s)
Antígenos de Protozoos/química , Eritrocitos/parasitología , Interacciones Huésped-Parásitos , Plasmodium falciparum/fisiología , Proteínas Protozoarias/química , Receptores de Superficie Celular/química , Cristalografía por Rayos X , Eritrocitos/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Superficie Celular/metabolismo , Dispersión del Ángulo PequeñoRESUMEN
Plasmodium vivax and Plasmodium knowlesi invasion depends on the parasite Duffy-binding protein DBL domain (RII-PvDBP or RII-PkDBP) engaging the Duffy antigen receptor for chemokines (DARC) on red blood cells. Inhibition of this key interaction provides an excellent opportunity for parasite control. There are competing models for whether Plasmodium ligands engage receptors as monomers or dimers, a question whose resolution has profound implications for parasite biology and control. We report crystallographic, solution and functional studies of RII-PvDBP showing that dimerization is required for and driven by receptor engagement. This work provides a unifying framework for prior studies and accounts for the action of naturally acquired blocking antibodies and the mechanism of immune evasion. We show that dimerization is conserved in DBL-domain receptor engagement and propose that receptor-mediated ligand dimerization drives receptor affinity and specificity. Because dimerization is prevalent in signaling, our studies raise the possibility that induced dimerization may activate pathways for invasion.
Asunto(s)
Antígenos de Protozoos/metabolismo , Sistema del Grupo Sanguíneo Duffy/metabolismo , Plasmodium vivax/metabolismo , Proteínas Protozoarias/metabolismo , Receptores de Superficie Celular/metabolismo , Antígenos de Protozoos/química , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Sistema del Grupo Sanguíneo Duffy/química , Humanos , Ligandos , Modelos Moleculares , Plasmodium falciparum/metabolismo , Estructura Terciaria de Proteína , Proteínas Protozoarias/química , Receptores de Superficie Celular/química , Transducción de SeñalRESUMEN
Electrospray ionization (ESI) mass spectrometry (MS) is a powerful method for analyzing the active forms of macromolecular complexes of biomolecules. However, these solutions often contain high concentrations of salts and/or detergents that adversely effect ESI performance by making ion formation less reproducible, causing severe adduction or ion suppression. Many methods for separating complexes from nonvolatile additives are routinely used with ESI-MS, but these methods may not be appropriate for complexes that require such stabilizers for activity. Here, the effects of buffer loading using concentrations of ammonium acetate ranging from 0.22 to 1.41 M on the ESI mass spectra of a solution containing a domain truncation mutant of a sigma(54) activator from Aquifex aeolicus were studied. This 44.9 kDa protein requires the presence of millimolar concentrations of Mg(2+), BeF(3)(-), and ADP, (at approximately 60 degrees C) to assemble into an active homo-hexamer. Addition of ammonium acetate can improve signal stability and reproducibility, and can significantly lower adduction and background signals. However, at higher concentrations, the relative ion abundance of the hexamer is diminished, while that of the constituent monomer is enhanced. These results are consistent with loss of enzymatic activity as measured by ATP hydrolysis and indicate that the high concentration of ammonium acetate interferes with assembly of the hexamer. This shows that buffer loading with ammonium acetate is effective for obtaining ESI signal for complexes that require high concentrations of essential salts, but can interfere with formation of, and/or destabilize complexes by disrupting crucial electrostatic interactions at high concentration.
Asunto(s)
Acetatos/química , Complejos Multiproteicos/química , ARN Polimerasa Sigma 54/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Bacterias/química , Tampones (Química) , Peso Molecular , Estructura Cuaternaria de Proteína , Reproducibilidad de los ResultadosRESUMEN
A common challenge with studies of proteins in vitro is determining which constructs and conditions are most physiologically relevant. sigma(54) activators are proteins that undergo regulated assembly to form an active ATPase ring that enables transcription by sigma(54)-polymerase. Previous studies of AAA(+) ATPase domains from sigma(54) activators have shown that some are heptamers, while others are hexamers. Because active oligomers assemble from off-state dimers, it was thought that even-numbered oligomers should dominate, and that heptamer formation would occur when individual domains of the activators, rather than the intact proteins, were studied. Here we present results from electrospray ionization mass spectrometry experiments characterizing the assembly states of intact NtrC4 (a sigma(54) activator from Aquifex aeolicus, an extreme thermophile), as well as its ATPase domain alone, and regulatory-ATPase and ATPase-DNA binding domain combinations. We show that the full-length and activated regulatory-ATPase proteins form hexamers, whereas the isolated ATPase domain, unactivated regulatory-ATPase, and ATPase-DNA binding domain form heptamers. Activation of the N-terminal regulatory domain is the key factor stabilizing the hexamer form of the ATPase, relative to the heptamer.
Asunto(s)
Archaea/química , Proteínas Arqueales/química , ARN Polimerasa Sigma 54/química , Espectrometría de Masa por Ionización de Electrospray , Peso Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Espectrometría de Masas en TándemRESUMEN
Genetic changes lead gradually to altered protein function, making deduction of the molecular basis for activity from a sequence difficult. Comparative studies provide insights into the functional consequences of specific changes. Here we present structural and biochemical studies of NtrC4, a sigma-54 activator from Aquifex aeolicus, and compare it with NtrC1 (a paralog) and NtrC (a homolog from Salmonella enterica) to provide insight into how a substantial change in regulatory mechanism may have occurred. Activity assays show that assembly of NtrC4's active oligomer is repressed by the N-terminal receiver domain, and that BeF3- addition (mimicking phosphorylation) removes this repression. Observation of assembly without activation for NtrC4 indicates that it is much less strongly repressed than NtrC1. The crystal structure of the unactivated receiver-ATPase domain combination shows a partially disrupted interface. NMR structures of the regulatory domain show that its activation mechanism is very similar to that of NtrC1. The crystal structure of the NtrC4 DNA-binding domain shows that it is dimeric and more similar in structure to NtrC than NtrC1. Electron microscope images of the ATPase-DNA-binding domain combination show formation of oligomeric rings. Sequence alignments provide insights into the distribution of activation mechanisms in this family of proteins.
Asunto(s)
Bacterias/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Evolución Molecular , Regulación Bacteriana de la Expresión Génica , Transcripción Genética , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/ultraestructura , Biología Computacional , Cristalografía por Rayos X , ADN Bacteriano/metabolismo , Dimerización , Hidrólisis , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Soluciones , Transactivadores/químicaRESUMEN
The hepatitis delta virus (HDV) ribozyme catalyzes viral RNA self-cleavage through general acid-base chemistry in which an active-site cytidine and at least one metal ion are involved. Monovalent metal ions support slow catalysis and were proposed to substitute for structural, but not catalytic, divalent metal ions in the RNA. To investigate the role of monovalent cations in ribozyme structure and function, we determined the crystal structure of the precursor HDV ribozyme in the presence of thallium ions (Tl(+)). Two Tl(+) ions can occupy a previously observed divalent metal ion hexahydrate-binding site located near the scissile phosphate, but are easily competed away by cobalt hexammine, a magnesium hexahydrate mimic and potent reaction inhibitor. Intriguingly, a third Tl(+) ion forms direct inner-sphere contacts with the ribose 2'-OH nucleophile and the pro-S(p) scissile phosphate oxygen. We discuss possible structural and catalytic implications of monovalent cation binding for the HDV ribozyme mechanism.
Asunto(s)
Virus de la Hepatitis Delta/genética , ARN Catalítico/química , Dominio Catalítico/fisiología , Cationes Monovalentes , Cristalografía por Rayos X , Virus de la Hepatitis Delta/química , Virus de la Hepatitis Delta/fisiología , Unión Proteica , ARN Catalítico/fisiologíaRESUMEN
It is of great interest to determine how solutes such as urea, sugars, guanidinium salts, and trimethylamine N-oxide affect the stability, solubility, and solvation of globular proteins. A key hypothesis in this field states that solutes affect protein stability indirectly by making or breaking water structure. We used a new technique, pressure perturbation calorimetry, to measure the temperature dependence of a solute's partial compressibility. Using fundamental thermodynamic relations, we converted these data to the pressure dependence of the partial heat capacity to examine the impact of protein stabilizing and denaturing solutes on water structure by applying the classic two-state mixture model for water. Contrary to widely held expectations, we found no correlation between a solute's impact on water structure and its effect on protein stability. Our results indicate that efforts to explain solute effects should focus on other hypotheses, including those based on preferential interaction and excluded volume.