RESUMEN
The atomic-level mechanisms by which G protein-coupled receptors (GPCRs) transmit extracellular ligand binding events through their transmembrane helices to activate intracellular G proteins remain unclear. Using a comprehensive library of mutations covering all 352 residues of the GPCR CXC chemokine receptor 4 (CXCR4), we identified 41 amino acids that are required for signaling induced by the chemokine ligand CXCL12 (stromal cell-derived factor 1). CXCR4 variants with each of these mutations do not signal properly but remain folded, based on receptor surface trafficking, reactivity to conformationally sensitive monoclonal antibodies, and ligand binding. When visualized on the structure of CXCR4, the majority of these residues form a continuous intramolecular signaling chain through the transmembrane helices; this chain connects chemokine binding residues on the extracellular side of CXCR4 to G protein-coupling residues on its intracellular side. Integrated into a cohesive model of signal transmission, these CXCR4 residues cluster into five functional groups that mediate (i) chemokine engagement, (ii) signal initiation, (iii) signal propagation, (iv) microswitch activation, and (v) G protein coupling. Propagation of the signal passes through a "hydrophobic bridge" on helix VI that coordinates with nearly every known GPCR signaling motif. Our results agree with known conserved mechanisms of GPCR activation and significantly expand on understanding the structural principles of CXCR4 signaling.
Asunto(s)
Conformación Proteica , Receptores CXCR4/química , Receptores CXCR4/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Sitios de Unión/genética , Quimiocina CXCL12/química , Quimiocina CXCL12/metabolismo , Células HEK293 , Humanos , Ligandos , Modelos Moleculares , Mutación , Unión Proteica , Multimerización de Proteína , Receptores CXCR4/genética , Homología de Secuencia de AminoácidoRESUMEN
A number of structures have been solved for the Envelope (E) protein from dengue virus and closely related flaviviruses, providing detailed pictures of the conformational states of the protein at different stages of infectivity. However, the key functional residues responsible for mediating the dynamic changes between these structures remain largely unknown. Using a comprehensive library of functional point mutations covering all 390 residues of the dengue virus E protein ectodomain, we identified residues that are critical for virus infectivity, but that do not affect E protein expression, folding, virion assembly, or budding. The locations and atomic interactions of these critical residues within different structures representing distinct fusogenic conformations help to explain how E protein (i) regulates fusion-loop exposure by shielding, tethering, and triggering its release; (ii) enables hinge movements between E domain interfaces during triggered structural transformations; and (iii) drives membrane fusion through late-stage zipper contacts with stem. These results provide structural targets for drug and vaccine development and integrate the findings from structural studies and isolated mutagenesis efforts into a cohesive model that explains how specific residues in this class II viral fusion protein enable virus infectivity.
Asunto(s)
Virus del Dengue/genética , Dengue/metabolismo , Modelos Moleculares , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus , Virus del Dengue/metabolismo , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Células HEK293 , Humanos , Luciferasas de Renilla , Proteínas del Envoltorio Viral/genética , Virión/metabolismoRESUMEN
UNLABELLED: Chikungunya virus (CHIKV) is a reemerging alphavirus that causes a debilitating arthritic disease and infects millions of people and for which no specific treatment is available. Like many alphaviruses, the structural targets on CHIKV that elicit a protective humoral immune response in humans are poorly defined. Here we used phage display against virus-like particles (VLPs) to isolate seven human monoclonal antibodies (MAbs) against the CHIKV envelope glycoproteins E2 and E1. One MAb, IM-CKV063, was highly neutralizing (50% inhibitory concentration, 7.4 ng/ml), demonstrated high-affinity binding (320 pM), and was capable of therapeutic and prophylactic protection in multiple animal models up to 24 h postexposure. Epitope mapping using a comprehensive shotgun mutagenesis library of 910 mutants with E2/E1 alanine mutations demonstrated that IM-CKV063 binds to an intersubunit conformational epitope on domain A, a functionally important region of E2. MAbs against the highly conserved fusion loop have not previously been reported but were also isolated in our studies. Fusion loop MAbs were broadly cross-reactive against diverse alphaviruses but were nonneutralizing. Fusion loop MAb reactivity was affected by temperature and reactivity conditions, suggesting that the fusion loop is hidden in infectious virions. Visualization of the binding sites of 15 different MAbs on the structure of E2/E1 revealed that all epitopes are located at the membrane-distal region of the E2/E1 spike. Interestingly, epitopes on the exposed topmost and outer surfaces of the E2/E1 trimer structure were neutralizing, whereas epitopes facing the interior of the trimer were not, providing a rationale for vaccine design and therapeutic MAb development using the intact CHIKV E2/E1 trimer. IMPORTANCE: CHIKV is the most important alphavirus affecting humans, resulting in a chronic arthritic condition that can persist for months or years. In recent years, millions of people have been infected globally, and the spread of CHIKV to the Americas is now beginning, with over 100,000 cases occurring in the Caribbean within 6 months of its arrival. Our study reports on seven human MAbs against the CHIKV envelope, including a highly protective MAb and rarely isolated fusion loop MAbs. Epitope mapping of these MAbs demonstrates how some E2/E1 epitopes are exposed or hidden from the human immune system and suggests a structural mechanism by which these MAbs protect (or fail to protect) against CHIKV infection. Our results suggest that the membrane-distal end of CHIKV E2/E1 is the primary target for the humoral immune response to CHIKV, and antibodies targeting the exposed topmost and outer surfaces of the E2/E1 trimer determine the neutralizing efficacy of this response.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Virus Chikungunya/inmunología , Epítopos/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Antivirales/aislamiento & purificación , Sitios de Unión , Técnicas de Visualización de Superficie Celular , Fiebre Chikungunya/prevención & control , Modelos Animales de Enfermedad , Mapeo Epitopo , Femenino , Humanos , Inmunización Pasiva , Ratones Endogámicos C57BL , Modelos Moleculares , Conformación Proteica , Análisis de SupervivenciaRESUMEN
The influenza virus M2 protein is a well-validated yet underexploited proton-selective ion channel essential for influenza virus infectivity. Because M2 is a toxic viral ion channel, existing M2 inhibitors have been discovered through live virus inhibition or medicinal chemistry rather than M2-targeted high-throughput screening (HTS), and direct measurement of its activity has been limited to live cells or reconstituted lipid bilayers. Here, we describe a cell-free ion channel assay in which M2 ion channels are incorporated into virus-like particles (VLPs) and proton conductance is measured directly across the viral lipid bilayer, detecting changes in membrane potential, ion permeability, and ion channel function. Using this approach in high-throughput screening of over 100,000 compounds, we identified 19 M2-specific inhibitors, including two novel chemical scaffolds that inhibit both M2 function and influenza virus infectivity. Counterscreening for nonspecific disruption of viral bilayer ion permeability also identified a broad-spectrum antiviral compound that acts by disrupting the integrity of the viral membrane. In addition to its application to M2 and potentially other ion channels, this technology enables direct measurement of the electrochemical and biophysical characteristics of viral membranes.
Asunto(s)
Antivirales/farmacología , Membrana Celular/virología , Virus de la Influenza A/fisiología , Gripe Humana/virología , Canales Iónicos/efectos de los fármacos , Protones , Proteínas de la Matriz Viral/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Membrana Celular/metabolismo , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Concentración de Iones de Hidrógeno , Gripe Humana/tratamiento farmacológico , Gripe Humana/patología , Membrana Dobles de Lípidos/metabolismo , Bibliotecas de Moléculas Pequeñas , Proteínas de la Matriz Viral/metabolismo , ViriónRESUMEN
Epitopes that define the immunodominant regions of conformationally complex integral membrane proteins have been difficult to reliably delineate. Here, a high-throughput approach termed shotgun mutagenesis was used to map the binding epitopes of five different monoclonal antibodies targeting the GPCR CCR5. The amino acids, and in some cases the atoms, that comprise the critical contact points of each epitope were identified, defining the immunodominant structures of this GPCR and their physicochemistry.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Mapeo Epitopo/métodos , Epítopos Inmunodominantes/análisis , Receptores CCR5/inmunología , Anticuerpos Monoclonales/química , Técnica del Anticuerpo Fluorescente/métodos , Modelos Moleculares , Mutagénesis , Reacción en Cadena de la Polimerasa/métodos , Receptores CCR5/genéticaRESUMEN
Although bitter taste receptors (TAS2Rs) are important for human health, little is known of the determinants of ligand specificity. TAS2Rs such as TAS2R16 help define gustatory perception and dietary preferences that ultimately influence human health and disease. Each TAS2R must accommodate a broad diversity of chemical structures while simultaneously achieving high specificity so that diverse bitter toxins can be detected without all foods tasting bitter. However, how these G protein-coupled receptors achieve this balance is poorly understood. Here we used a comprehensive mutation library of human TAS2R16 to map its interactions with existing and novel agonists. We identified 13 TAS2R16 residues that contribute to ligand specificity and 38 residues whose mutation eliminated signal transduction by all ligands, providing a comprehensive assessment of how this GPCR binds and signals. Our data suggest a model in which hydrophobic residues on TM3 and TM7 form a broad ligand-binding pocket that can accommodate the diverse structural features of ß-glycoside ligands while still achieving high specificity.