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1.
Proc Natl Acad Sci U S A ; 119(31): e2205412119, 2022 08 02.
Artículo en Inglés | MEDLINE | ID: mdl-35858383

RESUMEN

Camelid single-domain antibodies, also known as nanobodies, can be readily isolated from naïve libraries for specific targets but often bind too weakly to their targets to be immediately useful. Laboratory-based genetic engineering methods to enhance their affinity, termed maturation, can deliver useful reagents for different areas of biology and potentially medicine. Using the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and a naïve library, we generated closely related nanobodies with micromolar to nanomolar binding affinities. By analyzing the structure-activity relationship using X-ray crystallography, cryoelectron microscopy, and biophysical methods, we observed that higher conformational entropy losses in the formation of the spike protein-nanobody complex are associated with tighter binding. To investigate this, we generated structural ensembles of the different complexes from electron microscopy maps and correlated the conformational fluctuations with binding affinity. This insight guided the engineering of a nanobody with improved affinity for the spike protein.


Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , Afinidad de Anticuerpos , SARS-CoV-2 , Anticuerpos de Dominio Único , Glicoproteína de la Espiga del Coronavirus , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/genética , Anticuerpos Antivirales/química , Anticuerpos Antivirales/genética , Afinidad de Anticuerpos/genética , Microscopía por Crioelectrón , Entropía , Ingeniería Genética , Humanos , Unión Proteica , Dominios Proteicos , SARS-CoV-2/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/genética , Glicoproteína de la Espiga del Coronavirus/inmunología
2.
Proc Natl Acad Sci U S A ; 115(21): E4870-E4879, 2018 05 22.
Artículo en Inglés | MEDLINE | ID: mdl-29735649

RESUMEN

Capsules are surface layers of hydrated capsular polysaccharides (CPSs) produced by many bacteria. The human pathogen Salmonella enterica serovar Typhi produces "Vi antigen" CPS, which contributes to virulence. In a conserved strategy used by bacteria with diverse CPS structures, translocation of Vi antigen to the cell surface is driven by an ATP-binding cassette (ABC) transporter. These transporters are engaged in heterooligomeric complexes proposed to form an enclosed translocation conduit to the cell surface, allowing the transporter to power the entire process. We identified Vi antigen biosynthesis genetic loci in genera of the Burkholderiales, which are paradoxically distinguished from S. Typhi by encoding VexL, a predicted pectate lyase homolog. Biochemical analyses demonstrated that VexL is an unusual metal-independent endolyase with an acidic pH optimum that is specific for O-acetylated Vi antigen. A 1.22-Å crystal structure of the VexL-Vi antigen complex revealed features which distinguish common secreted catabolic pectate lyases from periplasmic VexL, which participates in cell-surface assembly. VexL possesses a right-handed parallel ß-superhelix, of which one face forms an electropositive glycan-binding groove with an extensive hydrogen bonding network that includes Vi antigen acetyl groups and confers substrate specificity. VexL provided a probe to interrogate conserved features of the ABC transporter-dependent export model. When introduced into S Typhi, VexL localized to the periplasm and degraded Vi antigen. In contrast, a cytosolic derivative had no effect unless export was disrupted. These data provide evidence that CPS assembled in ABC transporter-dependent systems is actually exposed to the periplasm during envelope translocation.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Burkholderia/enzimología , Liasas/metabolismo , Periplasma/enzimología , Polisacáridos Bacterianos/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Proteínas Bacterianas/química , Transporte Biológico , Liasas/química , Filogenia , Conformación Proteica
3.
Open Biol ; 14(6): 230252, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38835241

RESUMEN

The Omicron strains of SARS-CoV-2 pose a significant challenge to the development of effective antibody-based treatments as immune evasion has compromised most available immune therapeutics. Therefore, in the 'arms race' with the virus, there is a continuing need to identify new biologics for the prevention or treatment of SARS-CoV-2 infections. Here, we report the isolation of nanobodies that bind to the Omicron BA.1 spike protein by screening nanobody phage display libraries previously generated from llamas immunized with either the Wuhan or Beta spike proteins. The structure and binding properties of three of these nanobodies (A8, H6 and B5-5) have been characterized in detail providing insight into their binding epitopes on the Omicron spike protein. Trimeric versions of H6 and B5-5 neutralized the SARS-CoV-2 variant of concern BA.5 both in vitro and in the hamster model of COVID-19 following nasal administration. Thus, either alone or in combination could serve as starting points for the development of new anti-viral immunotherapeutics.


Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19 , SARS-CoV-2 , Anticuerpos de Dominio Único , Glicoproteína de la Espiga del Coronavirus , SARS-CoV-2/inmunología , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/farmacología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/química , COVID-19/inmunología , COVID-19/virología , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Humanos , Anticuerpos Antivirales/inmunología , Camélidos del Nuevo Mundo/inmunología , Epítopos/inmunología , Epítopos/química , Cricetinae , Unión Proteica , Modelos Moleculares
4.
Open Biol ; 13(3): 220373, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36944376

RESUMEN

The enterobacterial common antigen (ECA) is a carbohydrate polymer that is associated with the cell envelope in the Enterobacteriaceae. ECA contains a repeating trisaccharide which is polymerized by WzyE, a member of the Wzy membrane protein polymerase superfamily. WzyE activity is regulated by a membrane protein polysaccharide co-polymerase, WzzE. Förster resonance energy transfer experiments demonstrate that WzyE and WzzE from Pectobacterium atrosepticum form a complex in vivo, and immunoblotting and cryo-electron microscopy (cryo-EM) analysis confirm a defined stoichiometry of approximately eight WzzE to one WzyE. Low-resolution cryo-EM reconstructions of the complex, aided by an antibody recognizing the C-terminus of WzyE, reveals WzyE sits in the central membrane lumen formed by the octameric arrangement of the transmembrane helices of WzzE. The pairing of Wzy and Wzz is found in polymerization systems for other bacterial polymers, including lipopolysaccharide O-antigens and capsular polysaccharides. The data provide new structural insight into a conserved mechanism for regulating polysaccharide chain length in bacteria.


Asunto(s)
Bacterias , Polisacáridos , Microscopía por Crioelectrón , Bacterias/metabolismo , Oligosacáridos , Proteínas de la Membrana , Lípidos , Antígenos O/química , Antígenos O/metabolismo
5.
Science ; 377(6604): eabm3125, 2022 07 22.
Artículo en Inglés | MEDLINE | ID: mdl-35737812

RESUMEN

Many pathogens exploit host cell-surface glycans. However, precise analyses of glycan ligands binding with heavily modified pathogen proteins can be confounded by overlapping sugar signals and/or compounded with known experimental constraints. Universal saturation transfer analysis (uSTA) builds on existing nuclear magnetic resonance spectroscopy to provide an automated workflow for quantitating protein-ligand interactions. uSTA reveals that early-pandemic, B-origin-lineage severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike trimer binds sialoside sugars in an "end-on" manner. uSTA-guided modeling and a high-resolution cryo-electron microscopy structure implicate the spike N-terminal domain (NTD) and confirm end-on binding. This finding rationalizes the effect of NTD mutations that abolish sugar binding in SARS-CoV-2 variants of concern. Together with genetic variance analyses in early pandemic patient cohorts, this binding implicates a sialylated polylactosamine motif found on tetraantennary N-linked glycoproteins deep in the human lung as potentially relevant to virulence and/or zoonosis.


Asunto(s)
COVID-19 , Interacciones Huésped-Patógeno , SARS-CoV-2 , Ácidos Siálicos , Glicoproteína de la Espiga del Coronavirus , COVID-19/transmisión , Microscopía por Crioelectrón , Variación Genética , Humanos , Resonancia Magnética Nuclear Biomolecular , Polisacáridos/química , Unión Proteica , Dominios Proteicos , SARS-CoV-2/química , SARS-CoV-2/genética , Ácidos Siálicos/química , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética
6.
Nat Commun ; 12(1): 5469, 2021 09 22.
Artículo en Inglés | MEDLINE | ID: mdl-34552091

RESUMEN

SARS-CoV-2 remains a global threat to human health particularly as escape mutants emerge. There is an unmet need for effective treatments against COVID-19 for which neutralizing single domain antibodies (nanobodies) have significant potential. Their small size and stability mean that nanobodies are compatible with respiratory administration. We report four nanobodies (C5, H3, C1, F2) engineered as homotrimers with pmolar affinity for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Crystal structures show C5 and H3 overlap the ACE2 epitope, whilst C1 and F2 bind to a different epitope. Cryo Electron Microscopy shows C5 binding results in an all down arrangement of the Spike protein. C1, H3 and C5 all neutralize the Victoria strain, and the highly transmissible Alpha (B.1.1.7 first identified in Kent, UK) strain and C1 also neutralizes the Beta (B.1.35, first identified in South Africa). Administration of C5-trimer via the respiratory route showed potent therapeutic efficacy in the Syrian hamster model of COVID-19 and separately, effective prophylaxis. The molecule was similarly potent by intraperitoneal injection.


Asunto(s)
Anticuerpos Neutralizantes/farmacología , Tratamiento Farmacológico de COVID-19 , Anticuerpos de Dominio Único/farmacología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Administración Intranasal , Animales , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Microscopía por Crioelectrón , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Relación Dosis-Respuesta Inmunológica , Epítopos/química , Epítopos/metabolismo , Femenino , Masculino , Mesocricetus , Pruebas de Neutralización , SARS-CoV-2/efectos de los fármacos , Anticuerpos de Dominio Único/administración & dosificación , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/metabolismo , Glicoproteína de la Espiga del Coronavirus/química
8.
Nat Struct Mol Biol ; 27(9): 846-854, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32661423

RESUMEN

The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (KD of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Betacoronavirus/inmunología , Infecciones por Coronavirus , Pandemias , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral , Receptores Virales/metabolismo , Anticuerpos de Dominio Único/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Secuencia de Aminoácidos , Enzima Convertidora de Angiotensina 2 , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Neutralizantes/ultraestructura , Anticuerpos Antivirales/metabolismo , Anticuerpos Antivirales/ultraestructura , Afinidad de Anticuerpos , Reacciones Antígeno-Anticuerpo/inmunología , Betacoronavirus/metabolismo , Unión Competitiva , COVID-19 , Microscopía por Crioelectrón , Cristalografía por Rayos X , Epítopos/inmunología , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/inmunología , Modelos Moleculares , Biblioteca de Péptidos , Peptidil-Dipeptidasa A/ultraestructura , Unión Proteica , Conformación Proteica , Receptores Virales/ultraestructura , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , SARS-CoV-2 , Homología de Secuencia de Aminoácido , Anticuerpos de Dominio Único/metabolismo , Anticuerpos de Dominio Único/ultraestructura , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/ultraestructura
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