Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 21
Filtrar
1.
Cell ; 184(5): 1171-1187.e20, 2021 03 04.
Artículo en Inglés | MEDLINE | ID: mdl-33621484

RESUMEN

SARS-CoV-2 can mutate and evade immunity, with consequences for efficacy of emerging vaccines and antibody therapeutics. Here, we demonstrate that the immunodominant SARS-CoV-2 spike (S) receptor binding motif (RBM) is a highly variable region of S and provide epidemiological, clinical, and molecular characterization of a prevalent, sentinel RBM mutation, N439K. We demonstrate N439K S protein has enhanced binding affinity to the hACE2 receptor, and N439K viruses have similar in vitro replication fitness and cause infections with similar clinical outcomes as compared to wild type. We show the N439K mutation confers resistance against several neutralizing monoclonal antibodies, including one authorized for emergency use by the US Food and Drug Administration (FDA), and reduces the activity of some polyclonal sera from persons recovered from infection. Immune evasion mutations that maintain virulence and fitness such as N439K can emerge within SARS-CoV-2 S, highlighting the need for ongoing molecular surveillance to guide development and usage of vaccines and therapeutics.


Asunto(s)
COVID-19/inmunología , Aptitud Genética , Evasión Inmune , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Enzima Convertidora de Angiotensina 2/química , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/virología , Humanos , Mutación , Filogenia , SARS-CoV-2/química , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/química , Virulencia
2.
Nature ; 618(7965): 590-597, 2023 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-37258672

RESUMEN

Rapidly evolving influenza A viruses (IAVs) and influenza B viruses (IBVs) are major causes of recurrent lower respiratory tract infections. Current influenza vaccines elicit antibodies predominantly to the highly variable head region of haemagglutinin and their effectiveness is limited by viral drift1 and suboptimal immune responses2. Here we describe a neuraminidase-targeting monoclonal antibody, FNI9, that potently inhibits the enzymatic activity of all group 1 and group 2 IAVs, as well as Victoria/2/87-like, Yamagata/16/88-like and ancestral IBVs. FNI9 broadly neutralizes seasonal IAVs and IBVs, including the immune-evading H3N2 strains bearing an N-glycan at position 245, and shows synergistic activity when combined with anti-haemagglutinin stem-directed antibodies. Structural analysis reveals that D107 in the FNI9 heavy chain complementarity-determinant region 3 mimics the interaction of the sialic acid carboxyl group with the three highly conserved arginine residues (R118, R292 and R371) of the neuraminidase catalytic site. FNI9 demonstrates potent prophylactic activity against lethal IAV and IBV infections in mice. The unprecedented breadth and potency of the FNI9 monoclonal antibody supports its development for the prevention of influenza illness by seasonal and pandemic viruses.


Asunto(s)
Anticuerpos Antivirales , Especificidad de Anticuerpos , Virus de la Influenza A , Virus de la Influenza B , Vacunas contra la Influenza , Gripe Humana , Imitación Molecular , Neuraminidasa , Animales , Humanos , Ratones , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/uso terapéutico , Especificidad de Anticuerpos/inmunología , Arginina/química , Dominio Catalítico , Hemaglutininas Virales/inmunología , Virus de la Influenza A/clasificación , Virus de la Influenza A/enzimología , Virus de la Influenza A/inmunología , Subtipo H3N2 del Virus de la Influenza A/enzimología , Subtipo H3N2 del Virus de la Influenza A/inmunología , Virus de la Influenza B/clasificación , Virus de la Influenza B/enzimología , Virus de la Influenza B/inmunología , Vacunas contra la Influenza/química , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/uso terapéutico , Gripe Humana/inmunología , Gripe Humana/prevención & control , Neuraminidasa/antagonistas & inhibidores , Neuraminidasa/química , Neuraminidasa/inmunología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Estaciones del Año , Ácidos Siálicos/química
3.
Nature ; 598(7880): 342-347, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34464958

RESUMEN

SARS-CoV-2 infection-which involves both cell attachment and membrane fusion-relies on the angiotensin-converting enzyme 2 (ACE2) receptor, which is paradoxically found at low levels in the respiratory tract1-3, suggesting that there may be additional mechanisms facilitating infection. Here we show that C-type lectin receptors, DC-SIGN, L-SIGN and the sialic acid-binding immunoglobulin-like lectin 1 (SIGLEC1) function as attachment receptors by enhancing ACE2-mediated infection and modulating the neutralizing activity of different classes of spike-specific antibodies. Antibodies to the amino-terminal domain or to the conserved site at the base of the receptor-binding domain, while poorly neutralizing infection of ACE2-overexpressing cells, effectively block lectin-facilitated infection. Conversely, antibodies to the receptor binding motif, while potently neutralizing infection of ACE2-overexpressing cells, poorly neutralize infection of cells expressing DC-SIGN or L-SIGN and trigger fusogenic rearrangement of the spike, promoting cell-to-cell fusion. Collectively, these findings identify a lectin-dependent pathway that enhances ACE2-dependent infection by SARS-CoV-2 and reveal distinct mechanisms of neutralization by different classes of spike-specific antibodies.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Lectinas/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Moléculas de Adhesión Celular/metabolismo , Fusión Celular , Línea Celular , Cricetinae , Femenino , Humanos , Lectinas/inmunología , Lectinas Tipo C/metabolismo , Fusión de Membrana , Receptores de Superficie Celular/metabolismo , SARS-CoV-2/inmunología , Lectina 1 Similar a Ig de Unión al Ácido Siálico/metabolismo , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo
4.
Nature ; 597(7874): 97-102, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34261126

RESUMEN

An ideal therapeutic anti-SARS-CoV-2 antibody would resist viral escape1-3, have activity against diverse sarbecoviruses4-7, and be highly protective through viral neutralization8-11 and effector functions12,13. Understanding how these properties relate to each other and vary across epitopes would aid the development of therapeutic antibodies and guide vaccine design. Here we comprehensively characterize escape, breadth and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD). Despite a trade-off between in vitro neutralization potency and breadth of sarbecovirus binding, we identify neutralizing antibodies with exceptional sarbecovirus breadth and a corresponding resistance to SARS-CoV-2 escape. One of these antibodies, S2H97, binds with high affinity across all sarbecovirus clades to a cryptic epitope and prophylactically protects hamsters from viral challenge. Antibodies that target the angiotensin-converting enzyme 2 (ACE2) receptor-binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency. Nevertheless, we also characterize a potent RBM antibody (S2E128) with breadth across sarbecoviruses related to SARS-CoV-2 and a high barrier to viral escape. These data highlight principles underlying variation in escape, breadth and potency among antibodies that target the RBD, and identify epitopes and features to prioritize for therapeutic development against the current and potential future pandemics.


Asunto(s)
Anticuerpos ampliamente neutralizantes/inmunología , COVID-19/virología , Reacciones Cruzadas/inmunología , Evasión Inmune , SARS-CoV-2/clasificación , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Adulto , Anciano , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Afinidad de Anticuerpos , Anticuerpos ampliamente neutralizantes/química , COVID-19/inmunología , Vacunas contra la COVID-19/química , Vacunas contra la COVID-19/inmunología , Línea Celular , Cricetinae , Epítopos de Linfocito B/química , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/inmunología , Femenino , Humanos , Evasión Inmune/genética , Evasión Inmune/inmunología , Masculino , Mesocricetus , Persona de Mediana Edad , Modelos Moleculares , SARS-CoV-2/química , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Vacunología , Tratamiento Farmacológico de COVID-19
6.
J Am Chem Soc ; 141(17): 6906-6914, 2019 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-30943021

RESUMEN

Ergothioneine is an emergent factor in cellular redox biochemistry in humans and pathogenic bacteria. Broad consensus has formed around the idea that ergothioneine protects cells against reactive oxygen species. The recent discovery that anaerobic microorganisms make the same metabolite using oxygen-independent chemistry indicates that ergothioneine also plays physiological roles under anoxic conditions. In this report, we describe the crystal structure of the anaerobic ergothioneine biosynthetic enzyme EanB from green sulfur bacterium Chlorobium limicola. This enzyme catalyzes the oxidative sulfurization of N-α-trimethyl histidine. On the basis of structural and kinetic evidence, we describe the catalytic mechanism of this unusual C-S bond-forming reaction. Significant active-site conservation among distant EanB homologues suggests that the oxidative sulfurization of heterocyclic substrates may occur in a broad range of bacteria.


Asunto(s)
Biocatálisis , Ergotioneína/biosíntesis , Sulfurtransferasas/química , Dominio Catalítico/genética , Chlorobium/enzimología , Cristalografía por Rayos X , Cinética , Modelos Químicos , Mutagénesis Sitio-Dirigida , Sulfurtransferasas/genética , Sulfurtransferasas/metabolismo
7.
J Am Chem Soc ; 141(13): 5275-5285, 2019 04 03.
Artículo en Inglés | MEDLINE | ID: mdl-30883103

RESUMEN

Sulfoxide synthases are nonheme iron enzymes that catalyze oxidative carbon-sulfur bond formation between cysteine derivatives and N-α-trimethylhistidine as a key step in the biosynthesis of thiohistidines. The complex catalytic mechanism of this enzyme reaction has emerged as the controversial subject of several biochemical and computational studies. These studies all used the structure of the γ-glutamyl cysteine utilizing sulfoxide synthase, MthEgtB from Mycobacterium thermophilum (EC 1.14.99.50), as a structural basis. To provide an alternative model system, we have solved the crystal structure of CthEgtB from Chloracidobacterium thermophilum (EC 1.14.99.51) that utilizes cysteine as a sulfur donor. This structure reveals a completely different configuration of active site residues that are involved in oxygen binding and activation. Furthermore, comparison of the two EgtB structures enables a classification of all ergothioneine biosynthetic EgtBs into five subtypes, each characterized by unique active-site features. This active site diversity provides an excellent platform to examine the catalytic mechanism of sulfoxide synthases by comparative enzymology, but also raises the question as to why so many different solutions to the same biosynthetic problem have emerged.


Asunto(s)
Acidobacteria/enzimología , Ergotioneína/biosíntesis , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Oxígeno/metabolismo , Sitios de Unión , Biocatálisis , Ergotioneína/química , Estructura Molecular , Oxidación-Reducción , Oxígeno/química
8.
Proc Natl Acad Sci U S A ; 111(8): 2966-71, 2014 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-24516142

RESUMEN

Heteromeric amino acid transporters (HATs) are the unique example, known in all kingdoms of life, of solute transporters composed of two subunits linked by a conserved disulfide bridge. In metazoans, the heavy subunit is responsible for the trafficking of the heterodimer to the plasma membrane, and the light subunit is the transporter. HATs are involved in human pathologies such as amino acidurias, tumor growth and invasion, viral infection and cocaine addiction. However structural information about interactions between the heavy and light subunits of HATs is scarce. In this work, transmission electron microscopy and single-particle analysis of purified human 4F2hc/L-type amino acid transporter 2 (LAT2) heterodimers overexpressed in the yeast Pichia pastoris, together with docking analysis and crosslinking experiments, reveal that the extracellular domain of 4F2hc interacts with LAT2, almost completely covering the extracellular face of the transporter. 4F2hc increases the stability of the light subunit LAT2 in detergent-solubilized Pichia membranes, allowing functional reconstitution of the heterodimer into proteoliposomes. Moreover, the extracellular domain of 4F2hc suffices to stabilize solubilized LAT2. The interaction of 4F2hc with LAT2 gives insights into the structural bases for light subunit recognition and the stabilizing role of the ancillary protein in HATs.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Cadena Pesada de la Proteína-1 Reguladora de Fusión/química , Cadena Pesada de la Proteína-1 Reguladora de Fusión/metabolismo , Modelos Moleculares , Conformación Proteica , Western Blotting , Cromatografía de Afinidad , Cromatografía en Gel , Humanos , Microscopía Electrónica de Transmisión , Pichia , Unión Proteica
9.
Angew Chem Int Ed Engl ; 56(28): 8115-8119, 2017 07 03.
Artículo en Inglés | MEDLINE | ID: mdl-28544744

RESUMEN

The formylglycine-generating enzyme (FGE) is a unique copper protein that catalyzes oxygen-dependent C-H activation. We describe 1.66 Å- and 1.28 Å-resolution crystal structures of FGE from Thermomonospora curvata in complex with either AgI or CdII providing definitive evidence for a high-affinity metal-binding site in this enzyme. The structures reveal a bis-cysteine linear coordination of the monovalent metal, and tetrahedral coordination of the bivalent metal. Similar coordination changes may occur in the active enzyme as a result of CuI/II redox cycling. Complexation of copper atoms by two cysteine residues is common among copper-trafficking proteins, but is unprecedented for redox-active copper enzymes or synthetic copper catalysts.


Asunto(s)
Actinobacteria/enzimología , Cobre/química , Enzimas/química , Glicina/análogos & derivados , Metaloproteínas/química , Oxígeno/química , Sitios de Unión , Catálisis , Dominio Catalítico , Complejos de Coordinación/química , Cristalografía por Rayos X , Cisteína/química , Glicina/biosíntesis , Ligandos , Oxidación-Reducción , Conformación Proteica
10.
Angew Chem Int Ed Engl ; 56(41): 12508-12511, 2017 10 02.
Artículo en Inglés | MEDLINE | ID: mdl-28786519

RESUMEN

Ergothioneine is a sulfur metabolite that occurs in microorganisms, fungi, plants, and animals. The physiological function of ergothioneine is not clear. In recent years broad scientific consensus has formed around the idea that cellular ergothioneine primarily protects against reactive oxygen species. Herein we provide evidence that this focus on oxygen chemistry may be too narrow. We describe two enzymes from the strictly anaerobic green sulfur bacterium Chlorobium limicola that mediate oxygen-independent biosynthesis of ergothioneine. This anoxic origin suggests that ergothioneine is also important for oxygen-independent life. Furthermore, one of the discovered ergothioneine biosynthetic enzymes provides the first example of a rhodanese-like enzyme that transfers sulfur to non-activated carbon.


Asunto(s)
Proteínas Bacterianas/metabolismo , Chlorobium/metabolismo , Ergotioneína/metabolismo , Anaerobiosis , Vías Biosintéticas , Chlorobium/enzimología , Oxígeno/metabolismo
11.
Proc Natl Acad Sci U S A ; 110(42): E3978-86, 2013 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-24082128

RESUMEN

Peptide transporters (PTRs) of the large PTR family facilitate the uptake of di- and tripeptides to provide cells with amino acids for protein synthesis and for metabolic intermediates. Although several PTRs have been structurally and functionally characterized, how drugs modulate peptide transport remains unclear. To obtain insight into this mechanism, we characterize inhibitor binding to the Escherichia coli PTR dipeptide and tripeptide permease A (DtpA), which shows substrate specificities similar to its human homolog hPEPT1. After demonstrating that Lys[Z-NO2]-Val, the strongest inhibitor of hPEPT1, also acts as a high-affinity inhibitor for DtpA, we used single-molecule force spectroscopy to localize the structural segments stabilizing the peptide transporter and investigated which of these structural segments change stability upon inhibitor binding. This characterization was done with DtpA embedded in the lipid membrane and exposed to physiologically relevant conditions. In the unbound state, DtpA adopts two main alternate conformations in which transmembrane α-helix (TMH) 2 is either stabilized (in ∼43% of DtpA molecules) or not (in ∼57% of DtpA molecules). The two conformations are understood to represent the inward- and outward-facing conformational states of the transporter. With increasing inhibitor concentration, the conformation characterized by a stabilized TMH 2 becomes increasingly prevalent, reaching ∼92% at saturation. Our measurements further suggest that Lys[Z-NO2]-Val interacts with discrete residues in TMH 2 that are important for ligand binding and substrate affinity. These interactions in turn stabilize TMH 2, thereby promoting the inhibited conformation of DtpA.


Asunto(s)
Proteínas Bacterianas/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de Transporte de Membrana/química , Oligopéptidos/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Escherichia coli/genética , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/genética , Humanos , Proteínas de Transporte de Membrana/genética , Transportador de Péptidos 1 , Estabilidad Proteica , Estructura Secundaria de Proteína , Transporte de Proteínas/fisiología , Homología Estructural de Proteína , Simportadores/antagonistas & inhibidores , Simportadores/química , Simportadores/genética
12.
PLoS One ; 17(2): e0263328, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35143540

RESUMEN

Patients on dialysis are at risk of severe course of SARS-CoV-2 infection. Understanding the neutralizing activity and coverage of SARS-CoV-2 variants of vaccine-elicited antibodies is required to guide prophylactic and therapeutic COVID-19 interventions in this frail population. By analyzing plasma samples from 130 hemodialysis and 13 peritoneal dialysis patients after two doses of BNT162b2 or mRNA-1273 vaccines, we found that 35% of the patients had low-level or undetectable IgG antibodies to SARS-CoV-2 Spike (S). Neutralizing antibodies against the vaccine-matched SARS-CoV-2 and Delta variant were low or undetectable in 49% and 77% of patients, respectively, and were further reduced against other emerging variants. The fraction of non-responding patients was higher in SARS-CoV-2-naïve hemodialysis patients immunized with BNT162b2 (66%) than those immunized with mRNA-1273 (23%). The reduced neutralizing activity correlated with low antibody avidity. Patients followed up to 7 months after vaccination showed a rapid decay of the antibody response with an average 21- and 10-fold reduction of neutralizing antibodies to vaccine-matched SARS-CoV-2 and Delta variant, which increased the fraction of non-responders to 84% and 90%, respectively. These data indicate that dialysis patients should be prioritized for additional vaccination boosts. Nevertheless, their antibody response to SARS-CoV-2 must be continuously monitored to adopt the best prophylactic and therapeutic strategy.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Pruebas de Neutralización , Diálisis Renal , SARS-CoV-2/inmunología , Vacunación , Animales , Anticuerpos Neutralizantes/sangre , Afinidad de Anticuerpos , Células CHO , Vacunas contra la COVID-19/inmunología , Estudios de Casos y Controles , Cricetulus , Relación Dosis-Respuesta Inmunológica , Estudios de Seguimiento , Células HEK293 , Humanos , Inmunoglobulina G/sangre , Factores de Riesgo , Vacunas de ARNm/inmunología
13.
J Struct Biol ; 176(3): 395-403, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21996078

RESUMEN

The glucose transporter IICB of the Escherichia coli phosphotransferase system (PTS) consists of a polytopic membrane domain (IIC) responsible for substrate transport and a hydrophilic C-terminal domain (IIB) responsible for substrate phosphorylation. We have overexpressed and purified a triple mutant of IIC (mut-IIC), which had recently been shown to be suitable for crystallization purposes. Mut-IIC was homodimeric as determined by blue native-PAGE and gel-filtration, and had an eyeglasses-like structure as shown by negative-stain transmission electron microscopy (TEM) and single particle analysis. Glucose binding and transport by mut-IIC, mut-IICB and wildtype-IICB were compared with scintillation proximity and in vivo transport assays. Binding was reduced and transport was impaired by the triple mutation. The scintillation proximity assay allowed determination of substrate binding, affinity and specificity of wildtype-IICB by a direct method. 2D crystallization of mut-IIC yielded highly-ordered tubular crystals and made possible the calculation of a projection structure at 12Å resolution by negative-stain TEM. Immunogold labeling TEM revealed the sidedness of the tubular crystals, and high-resolution atomic force microscopy the surface structure of mut-IIC. This work presents the structure of a glucose PTS transporter at the highest resolution achieved so far and sets the basis for future structural studies.


Asunto(s)
Proteínas de Escherichia coli/química , Proteínas Facilitadoras del Transporte de la Glucosa/química , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/química , Cristalografía , Proteínas de Escherichia coli/genética , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Procesamiento de Imagen Asistido por Computador , Proteínas de la Membrana/química , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Estructura Terciaria de Proteína , Especificidad por Sustrato
14.
Biol Chem ; 392(1-2): 143-50, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21194358

RESUMEN

High-resolution microscopy techniques provide a plethora of information on biological structures from the cellular level down to the molecular level. In this review, we present the unique capabilities of transmission electron and atomic force microscopy to assess the structure, oligomeric state, function and dynamics of channel and transport proteins in their native environment, the lipid bilayer. Most importantly, membrane proteins can be visualized in the frozen-hydrated state and in buffer solution by cryo-transmission electron and atomic force microscopy, respectively. We also illustrate the potential of the scintillation proximity assay to study substrate binding of detergent-solubilized transporters prior to crystallization and structural characterization.


Asunto(s)
Proteínas de la Membrana/química , Microscopía de Fuerza Atómica , Transporte de Proteínas
15.
bioRxiv ; 2021 Apr 08.
Artículo en Inglés | MEDLINE | ID: mdl-33851154

RESUMEN

An ideal anti-SARS-CoV-2 antibody would resist viral escape 1-3 , have activity against diverse SARS-related coronaviruses 4-7 , and be highly protective through viral neutralization 8-11 and effector functions 12,13 . Understanding how these properties relate to each other and vary across epitopes would aid development of antibody therapeutics and guide vaccine design. Here, we comprehensively characterize escape, breadth, and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD), including S309 4 , the parental antibody of the late-stage clinical antibody VIR-7831. We observe a tradeoff between SARS-CoV-2 in vitro neutralization potency and breadth of binding across SARS-related coronaviruses. Nevertheless, we identify several neutralizing antibodies with exceptional breadth and resistance to escape, including a new antibody (S2H97) that binds with high affinity to all SARS-related coronavirus clades via a unique RBD epitope centered on residue E516. S2H97 and other escape-resistant antibodies have high binding affinity and target functionally constrained RBD residues. We find that antibodies targeting the ACE2 receptor binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency, but we identify one potent RBM antibody (S2E12) with breadth across sarbecoviruses closely related to SARS-CoV-2 and with a high barrier to viral escape. These data highlight functional diversity among antibodies targeting the RBD and identify epitopes and features to prioritize for antibody and vaccine development against the current and potential future pandemics.

16.
Science ; 370(6519): 950-957, 2020 11 20.
Artículo en Inglés | MEDLINE | ID: mdl-32972994

RESUMEN

Efficient therapeutic options are needed to control the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has caused more than 922,000 fatalities as of 13 September 2020. We report the isolation and characterization of two ultrapotent SARS-CoV-2 human neutralizing antibodies (S2E12 and S2M11) that protect hamsters against SARS-CoV-2 challenge. Cryo-electron microscopy structures show that S2E12 and S2M11 competitively block angiotensin-converting enzyme 2 (ACE2) attachment and that S2M11 also locks the spike in a closed conformation by recognition of a quaternary epitope spanning two adjacent receptor-binding domains. Antibody cocktails that include S2M11, S2E12, or the previously identified S309 antibody broadly neutralize a panel of circulating SARS-CoV-2 isolates and activate effector functions. Our results pave the way to implement antibody cocktails for prophylaxis or therapy, circumventing or limiting the emergence of viral escape mutants.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Betacoronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Pandemias/prevención & control , Peptidil-Dipeptidasa A/inmunología , Neumonía Viral/prevención & control , Glicoproteína de la Espiga del Coronavirus/antagonistas & inhibidores , Secuencias de Aminoácidos/inmunología , Enzima Convertidora de Angiotensina 2 , Animales , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Antivirales/administración & dosificación , Anticuerpos Antivirales/aislamiento & purificación , Células CHO , COVID-19 , Infecciones por Coronavirus/terapia , Cricetinae , Cricetulus , Microscopía por Crioelectrón , Células HEK293 , Humanos , Epítopos Inmunodominantes/química , Epítopos Inmunodominantes/inmunología , Microscopía Electrónica , Neumonía Viral/terapia , Dominios Proteicos/inmunología , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología
17.
Methods Mol Biol ; 1432: 223-42, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27485339

RESUMEN

This protocol describes the isolation of recombinant human and mammalian membrane proteins expressed in Xenopus laevis frog oocytes for structural studies. The cDNA-derived cRNA of the desired genes is injected into several hundreds of oocytes, which are incubated for several days to allow protein expression. Recombinant proteins are then purified via affinity chromatography. The novelty of this method comes from the design of a plasmid that produces multi-tagged proteins and, most importantly, the development of a protocol for efficiently discarding lipids, phospholipids, and lipoproteins from the oocyte egg yolk, which represent the major contaminants in protein purifications. Thus, the high protein purity and good yield obtained from this method allows protein structure determination by transmission electron microscopy of single detergent-solubilized protein particles and of 2D crystals of membrane protein embedded in lipid bilayers. Additionally, a radiotracer assay for functional analysis of the expressed target proteins in oocytes is described. Overall, this method is a valuable option for structural studies of mammalian and particularly human proteins, for which other expression systems often fail.


Asunto(s)
Mamíferos/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Xenopus laevis/genética , Animales , Cromatografía de Afinidad , Clonación Molecular , ADN Complementario/genética , Femenino , Vectores Genéticos/administración & dosificación , Humanos , Mamíferos/genética , Proteínas de la Membrana/genética , Oocitos/metabolismo , Plásmidos/genética , Conformación Proteica , ARN Complementario/genética , Proteínas Recombinantes/metabolismo , Xenopus laevis/metabolismo
18.
PLoS One ; 9(10): e109882, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25299125

RESUMEN

Human heteromeric amino acid transporters (HATs) are membrane protein complexes that facilitate the transport of specific amino acids across cell membranes. Loss of function or overexpression of these transporters is implicated in several human diseases such as renal aminoacidurias and cancer. HATs are composed of two subunits, a heavy and a light subunit, that are covalently connected by a disulphide bridge. Light subunits catalyse amino acid transport and consist of twelve transmembrane α-helix domains. Heavy subunits are type II membrane N-glycoproteins with a large extracellular domain and are involved in the trafficking of the complex to the plasma membrane. Structural information on HATs is scarce because of the difficulty in heterologous overexpression. Recently, we had a major breakthrough with the overexpression of a recombinant HAT, 4F2hc-LAT2, in the methylotrophic yeast Pichia pastoris. Microgram amounts of purified protein made possible the reconstruction of the first 3D map of a human HAT by negative-stain transmission electron microscopy. Here we report the important stabilization of purified human 4F2hc-LAT2 using a combination of two detergents, i.e., n-dodecyl-ß-D-maltopyranoside and lauryl maltose neopentyl glycol, and cholesteryl hemisuccinate. The superior quality and stability of purified 4F2hc-LAT2 allowed the measurement of substrate binding by scintillation proximity assay. In addition, an improved 3D map of this HAT could be obtained. The detergent-induced stabilization of the purified human 4F2hc-LAT2 complex presented here paves the way towards its crystallization and structure determination at high-resolution, and thus the elucidation of the working mechanism of this important protein complex at the molecular level.


Asunto(s)
Sistema de Transporte de Aminoácidos y+/aislamiento & purificación , Sistemas de Transporte de Aminoácidos/aislamiento & purificación , Cadena Pesada de la Proteína-1 Reguladora de Fusión/aislamiento & purificación , Cadenas Ligeras de la Proteína-1 Reguladora de Fusión/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Sistema de Transporte de Aminoácidos y+/química , Sistema de Transporte de Aminoácidos y+/metabolismo , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Detergentes/farmacología , Cadena Pesada de la Proteína-1 Reguladora de Fusión/química , Cadena Pesada de la Proteína-1 Reguladora de Fusión/metabolismo , Cadenas Ligeras de la Proteína-1 Reguladora de Fusión/química , Cadenas Ligeras de la Proteína-1 Reguladora de Fusión/metabolismo , Humanos , Maltosa/análogos & derivados , Maltosa/farmacología , Complejos Multiproteicos/química , Complejos Multiproteicos/aislamiento & purificación , Pichia , Estabilidad Proteica/efectos de los fármacos , Proteínas Recombinantes/efectos de los fármacos
19.
Protein Sci ; 20(3): 492-9, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21213248

RESUMEN

Numerous bacterial pathogens subvert cellular functions of eukaryotic host cells by the injection of effector proteins via dedicated secretion systems. The type IV secretion system (T4SS) effector protein BepA from Bartonella henselae is composed of an N-terminal Fic domain and a C-terminal Bartonella intracellular delivery domain, the latter being responsible for T4SS-mediated translocation into host cells. A proteolysis resistant fragment (residues 10-302) that includes the Fic domain shows autoadenylylation activity and adenylyl transfer onto Hela cell extract proteins as demonstrated by autoradiography on incubation with α-[(32)P]-ATP. Its crystal structure, determined to 2.9-Å resolution by the SeMet-SAD method, exhibits the canonical Fic fold including the HPFxxGNGRxxR signature motif with several elaborations in loop regions and an additional ß-rich domain at the C-terminus. On crystal soaking with ATP/Mg(2+), additional electron density indicated the presence of a PP(i) /Mg(2+) moiety, the side product of the adenylylation reaction, in the anion binding nest of the signature motif. On the basis of this information and that of the recent structure of IbpA(Fic2) in complex with the eukaryotic target protein Cdc42, we present a detailed model for the ternary complex of Fic with the two substrates, ATP/Mg(2+) and target tyrosine. The model is consistent with an in-line nucleophilic attack of the deprotonated side-chain hydroxyl group onto the α-phosphorus of the nucleotide to accomplish AMP transfer. Furthermore, a general, sequence-independent mechanism of target positioning through antiparallel ß-strand interactions between enzyme and target is suggested.


Asunto(s)
Proteínas Bacterianas/química , Sistemas de Secreción Bacterianos , Estructura Terciaria de Proteína , Secuencias de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bartonella henselae/química , Bartonella henselae/metabolismo , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Células HeLa , Humanos , Modelos Moleculares , Datos de Secuencia Molecular
20.
PLoS One ; 6(7): e21901, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21760919

RESUMEN

Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM) and single particle analysis (SPA) of two of these transporters, i.e., the potassium-chloride cotransporter 4 (KCC4) and the aquaporin-1 (AQP1) water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12) family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression.


Asunto(s)
Acuaporina 1/química , Acuaporina 1/metabolismo , Oocitos/metabolismo , Simportadores/química , Simportadores/metabolismo , Xenopus laevis/metabolismo , Animales , Acuaporina 1/aislamiento & purificación , Acuaporina 1/ultraestructura , Western Blotting , Membrana Celular/metabolismo , Cristalización , Yema de Huevo/metabolismo , Electroforesis en Gel de Poliacrilamida , Vectores Genéticos/genética , Humanos , Coloración Negativa , Oocitos/citología , Transporte de Proteínas , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Tinción con Nitrato de Plata , Simportadores/aislamiento & purificación , Simportadores/ultraestructura
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA