RESUMEN
While cryogenic electron microscopy (cryo-EM) is fruitfully used for harvesting high-resolution structures of sizable macromolecules, its application to small or flexible proteins composed of small domains like immunoglobulin (IgG) remain challenging. Here, we applied single particle cryo-EM to Rituximab, a therapeutic IgG mediating anti-tumor toxicity, to explore its solution conformations. We found Rituximab molecules exhibited aggregates in cryo-EM specimens contrary to its solution behavior, and utilized a non-ionic detergent to successfully disperse them as isolated particles amenable to single particle analysis. As the detergent adversely reduced the protein-to-solvent contrast, we employed phase plate contrast to mitigate the impaired protein visibility. Assisted by phase plate imaging, we obtained a canonical three-arm IgG structure with other structures displaying variable arm densities co-existing in solution, affirming high flexibility of arm-connecting linkers. Furthermore, we showed phase plate imaging enables reliable structure determination of Fab to sub-nanometer resolution from ab initio, yielding a characteristic two-lobe structure that could be unambiguously docked with crystal structure. Our findings revealed conformation diversity of IgG and demonstrated phase plate was viable for cryo-EM analysis of small proteins without symmetry. This work helps extend cryo-EM boundaries, providing a valuable imaging and structural analysis framework for macromolecules with similar challenging features.
Asunto(s)
Microscopía por Crioelectrón , Fragmentos Fab de Inmunoglobulinas , Inmunoglobulina G , Conformación Proteica , Microscopía por Crioelectrón/métodos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/ultraestructura , Inmunoglobulina G/química , Rituximab/química , Humanos , Modelos MolecularesRESUMEN
Rotavirus live-attenuated vaccines, both mono- and pentavalent, generate broadly heterotypic protection. B-cells isolated from adults encode neutralizing antibodies, some with affinity for VP5*, that afford broad protection in mice. We have mapped the epitope of one such antibody by determining the high-resolution cryo-EM structure of its antigen-binding fragment (Fab) bound to the virion of a candidate vaccine strain, CDC-9. The Fab contacts both the distal end of a VP5* ß-barrel domain and the two VP8* lectin-like domains at the tip of a projecting spike. Its interactions with VP8* do not impinge on the likely receptor-binding site, suggesting that the mechanism of neutralization is at a step subsequent to initial attachment. We also examined structures of CDC-9 virions from two different stages of serial passaging. Nearly all the VP4 (cleaved to VP8*/VP5*) spikes on particles from the earlier passage (wild-type isolate) had transitioned from the "upright" conformation present on fully infectious virions to the "reversed" conformation that is probably the end state of membrane insertion, unable to mediate penetration, consistent with the very low in vitro infectivity of the wild-type isolate. About half the VP4 spikes were upright on particles from the later passage, which had recovered substantial in vitro infectivity but had acquired an attenuated phenotype in neonatal rats. A mutation in VP4 that occurred during passaging appears to stabilize the interface at the apex of the spike and could account for the greater stability of the upright spikes on the late-passage, attenuated isolate. IMPORTANCE Rotavirus live-attenuated vaccines generate broadly heterotypic protection, and B-cells isolated from adults encode antibodies that are broadly protective in mice. Determining the structural and mechanistic basis of broad protection can contribute to understanding the current limitations of vaccine efficacy in developing countries. The structure of an attenuated human rotavirus isolate (CDC-9) bound with the Fab fragment of a broadly heterotypic protective antibody shows that protection is probably due to inhibition of the conformational transition in the viral spike protein (VP4) critical for viral penetration, rather than to inhibition of receptor binding. A comparison of structures of CDC-9 virus particles at two stages of serial passaging supports a proposed mechanism for initial steps in rotavirus membrane penetration.
Asunto(s)
Anticuerpos ampliamente neutralizantes , Proteínas de la Cápside , Epítopos de Linfocito B , Rotavirus , Vacunas Atenuadas , Virión , Animales , Anticuerpos ampliamente neutralizantes/inmunología , Anticuerpos ampliamente neutralizantes/ultraestructura , Proteínas de la Cápside/química , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/ultraestructura , Microscopía por Crioelectrón , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/ultraestructura , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/ultraestructura , Ratones , Conformación Proteica , Ratas , Rotavirus/química , Rotavirus/clasificación , Rotavirus/inmunología , Rotavirus/fisiología , Pase Seriado , Vacunas Atenuadas/química , Vacunas Atenuadas/inmunología , Vacunas Atenuadas/metabolismo , Virión/inmunología , Virión/metabolismo , Virión/ultraestructuraRESUMEN
N-methyl-D-aspartate receptors (NMDARs) are critically involved in basic brain functions and neurodegeneration as well as tumor invasiveness. Targeting specific subtypes of NMDARs with distinct activities has been considered an effective therapeutic strategy for neurological disorders and diseases. However, complete elimination of off-target effects of small chemical compounds has been challenging and thus, there is a need to explore alternative strategies for targeting NMDAR subtypes. Here we report identification of a functional antibody that specifically targets the GluN1-GluN2B NMDAR subtype and allosterically down-regulates ion channel activity as assessed by electrophysiology. Through biochemical analysis, x-ray crystallography, single-particle electron cryomicroscopy, and molecular dynamics simulations, we show that this inhibitory antibody recognizes the amino terminal domain of the GluN2B subunit and increases the population of the non-active conformational state. The current study demonstrates that antibodies may serve as specific reagents to regulate NMDAR functions for basic research and therapeutic objectives.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/ultraestructura , Clonación Molecular , Microscopía por Crioelectrón , Cristalografía por Rayos X , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Fragmentos Fab de Inmunoglobulinas/farmacología , Fragmentos Fab de Inmunoglobulinas/ultraestructura , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/aislamiento & purificación , Región Variable de Inmunoglobulina/farmacología , Región Variable de Inmunoglobulina/ultraestructura , Simulación de Dinámica Molecular , Oocitos , Ratas , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/ultraestructura , Células Sf9 , Spodoptera , Xenopus laevisRESUMEN
Specific antibody interactions with short peptides have made epitope tagging systems a vital tool employed in virtually all fields of biological research. Here, we present a novel epitope tagging system comprised of a monoclonal antibody named GD-26, which recognises the TD peptide (GTGATPADD) derived from Haloarcula marismortui bacteriorhodopsin I (HmBRI) D94N mutant. The crystal structure of the antigen-binding fragment (Fab) of GD-26 complexed with the TD peptide was determined to a resolution of 1.45 Å. The TD peptide was found to adopt a 310 helix conformation within the binding cleft, providing a characteristic peptide structure for recognition by GD-26 Fab. Based on the structure information, polar and nonpolar forces collectively contribute to the strong binding. Attempts to engineer the TD peptide show that the proline residue is crucial for the formation of the 310 helix in order to fit into the binding cleft. Isothermal calorimetry (ITC) reported a dissociation constant KD of 12 ± 2.8 nm, indicating a strong interaction between the TD peptide and GD-26 Fab. High specificity of GD-26 IgG to the TD peptide was demonstrated by western blotting, ELISA and immunofluorescence as only TD-tagged proteins were detected, suggesting the effectiveness of the GD-26/TD peptide tagging system. In addition to already-existing epitope tags such as the FLAG tag and the ALFA tag adopting either extended or α-helix conformations, the unique 310 helix conformation of the TD peptide together with the corresponding monoclonal antibody GD-26 offers a novel tagging option for research.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Bacteriorodopsinas/inmunología , Epítopos/inmunología , Péptidos/inmunología , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/ultraestructura , Especificidad de Anticuerpos/genética , Bacteriorodopsinas/genética , Bacteriorodopsinas/ultraestructura , Cristalografía por Rayos X , Ensayo de Inmunoadsorción Enzimática , Epítopos/genética , Epítopos/ultraestructura , Haloarcula marismortui/inmunología , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/ultraestructura , Péptidos/genéticaRESUMEN
Respiratory syncytial virus (RSV) is the most common cause of acute lower respiratory tract infections resulting in medical intervention and hospitalizations during infancy and early childhood, and vaccination against RSV remains a public health priority. The RSV F glycoprotein is a major target of neutralizing antibodies, and the prefusion stabilized form of F (DS-Cav1) is under investigation as a vaccine antigen. AM14 is a human monoclonal antibody with the exclusive capacity of binding an epitope on prefusion F (PreF), which spans two F protomers. The quality of recognizing a trimer-specific epitope makes AM14 valuable for probing PreF-based immunogen conformation and functionality during vaccine production. Currently, only a low-resolution (5.5 Å) X-ray structure is available of the PreF-AM14 complex, revealing few reliable details of the interface. Here, we perform complementary structural studies using X-ray crystallography and cryo-electron microscopy (cryo-EM) to provide improved resolution structures at 3.6 Å and 3.4 Å resolutions, respectively. Both X-ray and cryo-EM structures provide clear side-chain densities, which allow for accurate mapping of the AM14 epitope on DS-Cav1. The structures help rationalize the molecular basis for AM14 loss of binding to RSV F monoclonal antibody-resistant mutants and reveal flexibility for the side chain of a key antigenic residue on PreF. This work provides the basis for a comprehensive understanding of RSV F trimer specificity with implications in vaccine design and quality assessment of PreF-based immunogens.
Asunto(s)
Anticuerpos Monoclonales/ultraestructura , Fragmentos Fab de Inmunoglobulinas/ultraestructura , Infecciones por Virus Sincitial Respiratorio/prevención & control , Vacunas contra Virus Sincitial Respiratorio/química , Virus Sincitial Respiratorio Humano/inmunología , Proteínas Virales de Fusión/inmunología , Proteínas Virales de Fusión/ultraestructura , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Sitios de Unión de Anticuerpos , Células CHO , Cricetulus , Microscopía por Crioelectrón , Cristalografía por Rayos X , Epítopos , Fragmentos Fab de Inmunoglobulinas/inmunología , Modelos Moleculares , Mutación , Conformación Proteica , Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/virología , Vacunas contra Virus Sincitial Respiratorio/genética , Vacunas contra Virus Sincitial Respiratorio/inmunología , Virus Sincitial Respiratorio Humano/genética , Virus Sincitial Respiratorio Humano/patogenicidad , Relación Estructura-Actividad , Desarrollo de Vacunas , Proteínas Virales de Fusión/genéticaRESUMEN
We report the development of a platform of dual targeting Fab (DutaFab) molecules, which comprise two spatially separated and independent binding sites within the human antibody CDR loops: the so-called H-side paratope encompassing HCDR1, HCDR3 and LCDR2, and the L-side paratope encompassing LCDR1, LCDR3 and HCDR2. Both paratopes can be independently selected and combined into the desired bispecific DutaFabs in a modular manner. X-ray crystal structures illustrate that DutaFabs are able to bind two target molecules simultaneously at the same Fv region comprising a VH-VL heterodimer. In the present study, this platform is applied to generate DutaFabs specific for VEGFA and PDGF-BB, which show high affinities, physico-chemical stability and solubility, as well as superior efficacy over anti-VEGF monotherapy in vivo. These molecules exemplify the usefulness of DutaFabs as a distinct class of antibody therapeutics, which is currently being evaluated in patients.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Neovascularización Coroidal/tratamiento farmacológico , Desarrollo de Medicamentos/métodos , Fragmentos Fab de Inmunoglobulinas/farmacología , Ingeniería de Proteínas , Secuencia de Aminoácidos/genética , Animales , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Biespecíficos/ultraestructura , Becaplermina/antagonistas & inhibidores , Sitios de Unión de Anticuerpos/genética , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Fragmentos Fab de Inmunoglobulinas/ultraestructura , Concentración 50 Inhibidora , Inyecciones Intravítreas , Masculino , Modelos Moleculares , Prueba de Estudio Conceptual , Conformación Proteica , Ratas , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic continues, with devasting consequences for human lives and the global economy1,2. The discovery and development of virus-neutralizing monoclonal antibodies could be one approach to treat or prevent infection by this coronavirus. Here we report the isolation of sixty-one SARS-CoV-2-neutralizing monoclonal antibodies from five patients infected with SARS-CoV-2 and admitted to hospital with severe coronavirus disease 2019 (COVID-19). Among these are nineteen antibodies that potently neutralized authentic SARS-CoV-2 in vitro, nine of which exhibited very high potency, with 50% virus-inhibitory concentrations of 0.7 to 9 ng ml-1. Epitope mapping showed that this collection of nineteen antibodies was about equally divided between those directed against the receptor-binding domain (RBD) and those directed against the N-terminal domain (NTD), indicating that both of these regions at the top of the viral spike are immunogenic. In addition, two other powerful neutralizing antibodies recognized quaternary epitopes that overlap with the domains at the top of the spike. Cryo-electron microscopy reconstructions of one antibody that targets the RBD, a second that targets the NTD, and a third that bridges two separate RBDs showed that the antibodies recognize the closed, 'all RBD-down' conformation of the spike. Several of these monoclonal antibodies are promising candidates for clinical development as potential therapeutic and/or prophylactic agents against SARS-CoV-2.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Betacoronavirus/inmunología , Infecciones por Coronavirus/inmunología , Epítopos de Linfocito B/inmunología , Neumonía Viral/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/ultraestructura , Anticuerpos Neutralizantes/análisis , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/ultraestructura , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/química , Anticuerpos Antivirales/ultraestructura , Betacoronavirus/química , Betacoronavirus/ultraestructura , COVID-19 , Infecciones por Coronavirus/prevención & control , Microscopía por Crioelectrón , Modelos Animales de Enfermedad , Mapeo Epitopo , Epítopos de Linfocito B/química , Epítopos de Linfocito B/ultraestructura , Femenino , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/ultraestructura , Pulmón/patología , Pulmón/virología , Masculino , Mesocricetus , Modelos Moleculares , Pruebas de Neutralización , Pandemias/prevención & control , Neumonía Viral/prevención & control , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/ultraestructuraRESUMEN
Neutralizing antibody responses to coronaviruses mainly target the receptor-binding domain (RBD) of the trimeric spike. Here, we characterized polyclonal immunoglobulin Gs (IgGs) and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their focus on RBD epitopes, recognition of alpha- and beta-coronaviruses, and contributions of avidity to increased binding/neutralization of IgGs over Fabs. Using electron microscopy, we examined specificities of polyclonal plasma Fabs, revealing recognition of both S1A and RBD epitopes on SARS-CoV-2 spike. Moreover, a 3.4 Å cryo-electron microscopy (cryo-EM) structure of a neutralizing monoclonal Fab-spike complex revealed an epitope that blocks ACE2 receptor binding. Modeling based on these structures suggested different potentials for inter-spike crosslinking by IgGs on viruses, and characterized IgGs would not be affected by identified SARS-CoV-2 spike mutations. Overall, our studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.
Asunto(s)
Anticuerpos Neutralizantes/química , Betacoronavirus/química , Infecciones por Coronavirus/inmunología , Fragmentos Fab de Inmunoglobulinas/química , Inmunoglobulina G/química , Neumonía Viral/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/aislamiento & purificación , Betacoronavirus/inmunología , COVID-19 , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/terapia , Reacciones Cruzadas , Microscopía por Crioelectrón , Mapeo Epitopo , Epítopos , Humanos , Inmunización Pasiva , Fragmentos Fab de Inmunoglobulinas/sangre , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Fragmentos Fab de Inmunoglobulinas/ultraestructura , Inmunoglobulina G/sangre , Inmunoglobulina G/aislamiento & purificación , Inmunoglobulina G/ultraestructura , Coronavirus del Síndrome Respiratorio de Oriente Medio/química , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología , Modelos Moleculares , Pandemias , Neumonía Viral/sangre , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/química , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/inmunología , Sueroterapia para COVID-19RESUMEN
Dipeptidase 3 (DPEP3) is one of three glycosylphosphatidylinositol-anchored metallopeptidases potentially involved in the hydrolytic metabolism of dipeptides. While its exact biological function is not clear, DPEP3 expression is normally limited to testis, but can be elevated in ovarian cancer. Antibody drug conjugates targeting DPEP3 have shown efficacy in preclinical models with a pyrrolobenzodiazepine conjugate, SC-003, dosed in a phase I clinical trial (NCT02539719). Here we reveal the novel atomic structure of DPEP3 alone and in complex with the SC-003 Fab fragment at 1.8 and 2.8 Å, respectively. The structure of DPEP3/SC-003 Fab complex reveals an eighteen-residue epitope across the DPEP3 dimerization interface distinct from the enzymatic active site. DPEP1 and DPEP3 extracellular domains share a conserved, dimeric TIM (ß/α)8-barrel fold, consistent with 49% sequence identity. However, DPEP3 diverges from DPEP1 and DPEP2 in key positions of its active site: a histidine to tyrosine variation at position 269 reduces affinity for the ß zinc and may cause substrate steric hindrance, whereas an aspartate to asparagine change at position 359 abolishes activation of the nucleophilic water/hydroxide, resulting in no in vitro activity against a variety of dipeptides and biological substrates (imipenem, leukotriene D4 and cystinyl-bis-glycine). Hence DPEP3, unlike DPEP1 and DPEP2, may require an activating co-factor in vivo or may remain an inactive, degenerate enzyme. This report sheds light on the structural discriminants between active and inactive membrane dipeptidases and provides a benchmark to characterize current and future DPEP3-targeted therapeutic approaches.
Asunto(s)
Dipeptidasas/ultraestructura , Epítopos/ultraestructura , Inmunoconjugados/ultraestructura , Anticuerpos/química , Anticuerpos/inmunología , Anticuerpos/ultraestructura , Dipeptidasas/química , Dipeptidasas/genética , Dipeptidasas/inmunología , Epítopos/genética , Epítopos/inmunología , Humanos , Inmunoconjugados/genética , Inmunoconjugados/inmunología , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/ultraestructura , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/ultraestructura , ProteolisisRESUMEN
The oncoprotein Smoothened (SMO), a G-protein-coupled receptor (GPCR) of the Frizzled-class (class-F), transduces the Hedgehog signal from the tumour suppressor Patched-1 (PTCH1) to the glioma-associated-oncogene (GLI) transcription factors, which activates the Hedgehog signalling pathway1,2. It has remained unknown how PTCH1 modulates SMO, how SMO is stimulated to form a complex with heterotrimeric G proteins and whether G-protein coupling contributes to the activation of GLI proteins3. Here we show that 24,25-epoxycholesterol, which we identify as an endogenous ligand of PTCH1, can stimulate Hedgehog signalling in cells and can trigger G-protein signalling via human SMO in vitro. We present a cryo-electron microscopy structure of human SMO bound to 24(S),25-epoxycholesterol and coupled to a heterotrimeric Gi protein. The structure reveals a ligand-binding site for 24(S),25-epoxycholesterol in the 7-transmembrane region, as well as a Gi-coupled activation mechanism of human SMO. Notably, the Gi protein presents a different arrangement from that of class-A GPCR-Gi complexes. Our work provides molecular insights into Hedgehog signal transduction and the activation of a class-F GPCR.
Asunto(s)
Microscopía por Crioelectrón , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/ultraestructura , Oxiesteroles/química , Receptor Smoothened/química , Receptor Smoothened/ultraestructura , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/ultraestructura , Ligandos , Modelos Moleculares , Oxiesteroles/metabolismo , Receptor Patched-1/metabolismo , Conformación Proteica , Transducción de Señal , Receptor Smoothened/metabolismo , Alcaloides de Veratrum/químicaRESUMEN
Human parechovirus 3 (HPeV3) infection is associated with sepsis characterized by significant immune activation and subsequent tissue damage in neonates. Strategies to limit infection have been unsuccessful due to inadequate molecular diagnostic tools for early detection and the lack of a vaccine or specific antiviral therapy. Toward the latter, we present a 2.8-Å-resolution structure of HPeV3 in complex with fragments from a neutralizing human monoclonal antibody, AT12-015, using cryo-electron microscopy (cryo-EM) and image reconstruction. Modeling revealed that the epitope extends across neighboring asymmetric units with contributions from capsid proteins VP0, VP1, and VP3. Antibody decoration was found to block binding of HPeV3 to cultured cells. Additionally, at high resolution, it was possible to model a stretch of RNA inside the virion and, from this, identify the key features that drive and stabilize protein-RNA association during assembly.IMPORTANCE Human parechovirus 3 (HPeV3) is receiving increasing attention as a prevalent cause of sepsis-like symptoms in neonates, for which, despite the severity of disease, there are no effective treatments available. Structural and molecular insights into virus neutralization are urgently needed, especially as clinical cases are on the rise. Toward this goal, we present the first structure of HPeV3 in complex with fragments from a neutralizing monoclonal antibody. At high resolution, it was possible to precisely define the epitope that, when targeted, prevents virions from binding to cells. Such an atomic-level description is useful for understanding host-pathogen interactions and viral pathogenesis mechanisms and for finding potential cures for infection and disease.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Parechovirus/inmunología , Parechovirus/ultraestructura , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Cápside/metabolismo , Proteínas de la Cápside/inmunología , Línea Celular Tumoral , Microscopía por Crioelectrón/métodos , Epítopos/metabolismo , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/ultraestructuraRESUMEN
The olfactory system must recognize and discriminate amongst an enormous variety of chemicals in the environment. To contend with such diversity, insects have evolved a family of odorant-gated ion channels comprised of a highly conserved co-receptor (Orco) and a divergent odorant receptor (OR) that confers chemical specificity. Here, we present the single-particle cryo-electron microscopy structure of an Orco homomer from the parasitic fig wasp Apocrypta bakeri at 3.5 Å resolution, providing structural insight into this receptor family. Orco possesses a novel channel architecture, with four subunits symmetrically arranged around a central pore that diverges into four lateral conduits that open to the cytosol. The Orco tetramer has few inter-subunit interactions within the membrane and is bound together by a small cytoplasmic anchor domain. The minimal sequence conservation among ORs maps largely to the pore and anchor domain, shedding light on how the architecture of this receptor family accommodates its remarkable sequence diversity and facilitates the evolution of odour tuning.
Asunto(s)
Microscopía por Crioelectrón , Insectos/ultraestructura , Receptores Odorantes/química , Receptores Odorantes/ultraestructura , Secuencias de Aminoácidos , Animales , Sitios de Unión , Secuencia Conservada , Interacciones Hidrofóbicas e Hidrofílicas , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/ultraestructura , Insectos/química , Insectos/clasificación , Activación del Canal Iónico , Modelos Moleculares , Filogenia , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Receptores Odorantes/metabolismo , Alineación de SecuenciaRESUMEN
Antibody Fab fragments have been exploited with significant success to facilitate the structure determination of challenging macromolecules as crystallization chaperones and as molecular fiducial marks for single particle cryo-electron microscopy approaches. However, the inherent flexibility of the "elbow" regions, which link the constant and variable domains of the Fab, can introduce disorder and thus diminish their effectiveness. We have developed a phage display engineering strategy to generate synthetic Fab variants that significantly reduces elbow flexibility, while maintaining their high affinity and stability. This strategy was validated using previously recalcitrant Fab-antigen complexes where introduction of an engineered elbow region enhanced crystallization and diffraction resolution. Furthermore, incorporation of the mutations appears to be generally portable to other synthetic antibodies and may serve as a universal strategy to enhance the success rates of Fabs as structure determination chaperones.
Asunto(s)
Antígenos/química , Microscopía por Crioelectrón/métodos , Fragmentos Fab de Inmunoglobulinas/química , Antígenos/ultraestructura , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/ultraestructura , Cristalización/métodos , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/ultraestructura , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/ultraestructura , Biblioteca de Péptidos , Conformación Proteica , Ingeniería de Proteínas , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestructuraRESUMEN
For many enveloped viruses, binding to a receptor(s) on a host cell acts as the first step in a series of events culminating in fusion with the host cell membrane and transfer of genetic material for replication. The envelope glycoprotein (Env) trimer on the surface of HIV is responsible for receptor binding and fusion. Although Env can tolerate a high degree of mutation in five variable regions (V1-V5), and also at N-linked glycosylation sites that contribute roughly half the mass of Env, the functional sites for recognition of receptor CD4 and co-receptor CXCR4/CCR5 are conserved and essential for viral fitness. Soluble SOSIP Env trimers are structural and antigenic mimics of the pre-fusion native, surface-presented Env, and are targets of broadly neutralizing antibodies. Thus, they are attractive immunogens for vaccine development. Here we present high-resolution cryo-electron microscopy structures of subtype B B41 SOSIP Env trimers in complex with CD4 and antibody 17b, or with antibody b12, at resolutions of 3.7 Å and 3.6 Å, respectively. We compare these to cryo-electron microscopy reconstructions of B41 SOSIP Env trimers with no ligand or in complex with either CD4 or the CD4-binding-site antibody PGV04 at 5.6 Å, 5.2 Å and 7.4 Å resolution, respectively. Consequently, we present the most complete description yet, to our knowledge, of the CD4-17b-induced intermediate and provide the molecular basis of the receptor-binding-induced conformational change required for HIV-1 entry into host cells. Both CD4 and b12 induce large, previously uncharacterized conformational rearrangements in the gp41 subunits, and the fusion peptide becomes buried in a newly formed pocket. These structures provide key details on the biological function of the type I viral fusion machine from HIV-1 as well as new templates for inhibitor design.
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Regulación Alostérica , Microscopía por Crioelectrón , VIH-1/química , VIH-1/ultraestructura , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen env del Virus de la Inmunodeficiencia Humana/ultraestructura , Regulación Alostérica/efectos de los fármacos , Secuencia de Aminoácidos , Anticuerpos/química , Anticuerpos/inmunología , Anticuerpos/farmacología , Anticuerpos/ultraestructura , Sitios de Unión/efectos de los fármacos , Antígenos CD4/química , Antígenos CD4/metabolismo , Antígenos CD4/ultraestructura , Proteína gp41 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/ultraestructura , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/farmacología , Fragmentos Fab de Inmunoglobulinas/ultraestructura , Ligandos , Modelos Moleculares , Receptores CCR5/química , Receptores CCR5/metabolismo , Receptores del VIH/química , Receptores del VIH/metabolismo , Receptores del VIH/ultraestructura , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
ABCG2 is a constitutively expressed ATP-binding cassette (ABC) transporter that protects many tissues against xenobiotic molecules. Its activity affects the pharmacokinetics of commonly used drugs and limits the delivery of therapeutics into tumour cells, thus contributing to multidrug resistance. Here we present the structure of human ABCG2 determined by cryo-electron microscopy, providing the first high-resolution insight into a human multidrug transporter. We visualize ABCG2 in complex with two antigen-binding fragments of the human-specific, inhibitory antibody 5D3 that recognizes extracellular loops of the transporter. We observe two cholesterol molecules bound in the multidrug-binding pocket that is located in a central, hydrophobic, inward-facing translocation pathway between the transmembrane domains. Combined with functional in vitro analyses, our results suggest a multidrug recognition and transport mechanism of ABCG2, rationalize disease-causing single nucleotide polymorphisms and the allosteric inhibition by the 5D3 antibody, and provide the structural basis of cholesterol recognition by other G-subfamily ABC transporters.
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Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/química , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/ultraestructura , Microscopía por Crioelectrón , Proteínas de Neoplasias/química , Proteínas de Neoplasias/ultraestructura , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/antagonistas & inhibidores , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/ultraestructura , Secuencia de Aminoácidos , Anticuerpos/química , Anticuerpos/inmunología , Anticuerpos/ultraestructura , Sitios de Unión , Transporte Biológico , Colesterol/química , Colesterol/metabolismo , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/ultraestructura , Modelos Moleculares , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Polimorfismo de Nucleótido Simple/genética , Dominios ProteicosRESUMEN
While neutralizing antibodies are highly effective against ebolavirus infections, current experimental ebolavirus vaccines primarily elicit species-specific antibody responses. Here, we describe an immunization-elicited macaque antibody (CA45) that clamps the internal fusion loop with the N terminus of the ebolavirus glycoproteins (GPs) and potently neutralizes Ebola, Sudan, Bundibugyo, and Reston viruses. CA45, alone or in combination with an antibody that blocks receptor binding, provided full protection against all pathogenic ebolaviruses in mice, guinea pigs, and ferrets. Analysis of memory B cells from the immunized macaque suggests that elicitation of broadly neutralizing antibodies (bNAbs) for ebolaviruses is possible but difficult, potentially due to the rarity of bNAb clones and their precursors. Unexpectedly, germline-reverted CA45, while exhibiting negligible binding to full-length GP, bound a proteolytically remodeled GP with picomolar affinity, suggesting that engineered ebolavirus vaccines could trigger rare bNAb precursors more robustly. These findings have important implications for developing pan-ebolavirus vaccine and immunotherapeutic cocktails.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/aislamiento & purificación , Vacunas contra el Virus del Ébola/inmunología , Fiebre Hemorrágica Ebola/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/química , Regiones Determinantes de Complementariedad , Reacciones Cruzadas , Ebolavirus/inmunología , Mapeo Epitopo , Epítopos de Linfocito B/inmunología , Femenino , Hurones , Cobayas , Fragmentos Fab de Inmunoglobulinas/ultraestructura , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos MolecularesRESUMEN
Structure-based vaccine design depends on extensive structural analyses of antigen-antibody complexes.Single-particle electron cryomicroscopy (cryoEM) can circumvent some of the problems of x-ray crystallography as a pipeline for obtaining the required structures. We have examined the potential of single-particle cryoEM for determining the structure of influenza-virus hemagglutinin (HA):single-chain variable-domain fragment complexes, by studying a complex we failed to crystallize in pursuing an extended project on the human immune response to influenza vaccines.The result shows that a combination of cryoEM and molecular modeling can yield details of the antigen-antibody interface, although small variation in the twist of the rod-likeHA trimer limited the overall resolution to about 4.5Å.Comparison of principal 3D classes suggests ways to modify the HA trimer to overcome this limitation. A closely related antibody from the same donor did yield crystals when bound with the same HA, giving us an independent validation of the cryoEM results.The two structures also augment our understanding of receptor-binding site recognition by antibodies that neutralize a wide range of influenza-virus variants.
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Anticuerpos Antivirales/ultraestructura , Antígenos Virales/ultraestructura , Glicoproteínas Hemaglutininas del Virus de la Influenza/ultraestructura , Anticuerpos Antivirales/química , Antígenos Virales/química , Sitios de Unión , Microscopía por Crioelectrón , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/ultraestructura , Modelos Moleculares , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/ultraestructuraRESUMEN
Antibodies represent a highly successful class of molecules that bind a wide-range of targets in therapeutic-, diagnostic- and research-based applications. The antibody repertoire is composed of the building blocks required to develop an effective adaptive immune response against foreign insults. A number of species have developed novel genetic and structural mechanisms from which they derive these antibody repertoires, however, traditionally antibodies are isolated from human, and rodent sources. Due to their high-value therapeutic, diagnostic, biotechnological and research applications, much innovation has resulted in techniques and approaches to isolate novel antibodies. These approaches are bolstered by advances in our understanding of species immune repertoires, next generation sequencing capacity, combinatorial antibody discovery and high-throughput screening. Structural determination of antibodies and antibody-antigen complexes has proven to be pivotal to our current understanding of the immune repertoire for a range of species leading to advances in man-made libraries and fine tuning approaches to develop antibodies from immune-repertoires. Furthermore, the isolation of antibodies directed against antigens of importance in health, disease and developmental processes, has yielded a plethora of structural and functional insights. This review highlights the significant contribution of antibody-based crystallography to our understanding of adaptive immunity and its application to providing critical information on a range of human-health related indications.
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Inmunización Pasiva/métodos , Fragmentos Fab de Inmunoglobulinas/ultraestructura , Inmunoglobulina G/ultraestructura , Anticuerpos de Cadena Única/ultraestructura , Inmunidad Adaptativa , Animales , Antígenos/inmunología , Cristalografía por Rayos X , Humanos , Fragmentos Fab de Inmunoglobulinas/biosíntesis , Fragmentos Fab de Inmunoglobulinas/química , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/química , Modelos Moleculares , Chaperonas Moleculares/biosíntesis , Chaperonas Moleculares/química , Chaperonas Moleculares/ultraestructura , Conformación Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Anticuerpos de Cadena Única/biosíntesis , Anticuerpos de Cadena Única/química , Especificidad de la EspecieRESUMEN
We screened a panel of mouse and human monoclonal antibodies (MAbs) against chikungunya virus and identified several with inhibitory activity against multiple alphaviruses. Passive transfer of broadly neutralizing MAbs protected mice against infection by chikungunya, Mayaro, and O'nyong'nyong alphaviruses. Using alanine-scanning mutagenesis, loss-of-function recombinant proteins and viruses, and multiple functional assays, we determined that broadly neutralizing MAbs block multiple steps in the viral lifecycle, including entry and egress, and bind to a conserved epitope on the B domain of the E2 glycoprotein. A 16 Å resolution cryo-electron microscopy structure of a Fab fragment bound to CHIKV E2 B domain provided an explanation for its neutralizing activity. Binding to the B domain was associated with repositioning of the A domain of E2 that enabled cross-linking of neighboring spikes. Our results suggest that B domain antigenic determinants could be targeted for vaccine or antibody therapeutic development against multiple alphaviruses of global concern.
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Infecciones por Alphavirus/inmunología , Alphavirus/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Epítopos , Proteínas del Envoltorio Viral/inmunología , Alphavirus/clasificación , Alphavirus/metabolismo , Infecciones por Alphavirus/prevención & control , Infecciones por Alphavirus/terapia , Secuencia de Aminoácidos , Animales , Virus Chikungunya/química , Virus Chikungunya/inmunología , Microscopía por Crioelectrón , Glicoproteínas/química , Glicoproteínas/inmunología , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/ultraestructura , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Proteínas del Envoltorio Viral/química , Vacunas Virales/inmunología , Internalización del VirusRESUMEN
Human cytomegalovirus (HCMV) poses a significant threat to immunocompromised individuals and neonates infected in utero. Glycoprotein B (gB), the herpesvirus fusion protein, is a target for neutralizing antibodies and a vaccine candidate due to its indispensable role in infection. Here we show the crystal structure of the HCMV gB ectodomain bound to the Fab fragment of 1G2, a neutralizing human monoclonal antibody isolated from a seropositive subject. The gB/1G2 interaction is dominated by aromatic residues in the 1G2 heavy chain CDR3 protruding into a hydrophobic cleft in the gB antigenic domain 5 (AD-5). Structural analysis and comparison with HSV gB suggest the location of additional neutralizing antibody binding sites on HCMV gB. Finally, immunoprecipitation experiments reveal that 1G2 can bind to HCMV virion gB suggesting that its epitope is exposed and accessible on the virus surface. Our data will support the development of vaccines and therapeutic antibodies against HCMV infection.