RESUMO
Multivalent carbohydrate-lectin interactions at host-pathogen interfaces play a crucial role in the establishment of infections. Although competitive antagonists that prevent pathogen adhesion are promising antimicrobial drugs, the molecular mechanisms underlying these complex adhesion processes are still poorly understood. Here, we characterize the interactions between the fimbrial adhesin FimH from uropathogenic Escherichia coli strains and its natural high-mannose type N-glycan binding epitopes on uroepithelial glycoproteins. Crystal structures and a detailed kinetic characterization of ligand-binding and dissociation revealed that the binding pocket of FimH evolved such that it recognizes the terminal α(1-2)-, α(1-3)-, and α(1-6)-linked mannosides of natural high-mannose type N-glycans with similar affinity. We demonstrate that the 2000-fold higher affinity of the domain-separated state of FimH compared to its domain-associated state is ligand-independent and consistent with a thermodynamic cycle in which ligand-binding shifts the association equilibrium between the FimH lectin and the FimH pilin domain. Moreover, we show that a single N-glycan can bind up to three molecules of FimH, albeit with negative cooperativity, so that a molar excess of accessible N-glycans over FimH on the cell surface favors monovalent FimH binding. Our data provide pivotal insights into the adhesion properties of uropathogenic Escherichia coli strains to their target receptors and a solid basis for the development of effective FimH antagonists.
Assuntos
Adesinas de Escherichia coli/metabolismo , Proteínas de Fímbrias/metabolismo , Mananas/metabolismo , Manosídeos/metabolismo , Adesinas de Escherichia coli/química , Sítios de Ligação , Escherichia coli/química , Proteínas de Fímbrias/química , Cinética , Ligantes , Mananas/química , Manosídeos/química , Ligação Proteica , Conformação Proteica , TermodinâmicaRESUMO
The Spike of SARS-CoV-2 recognizes a transmembrane protease, angiotensin-converting enzyme 2 (ACE2), on host cells to initiate infection. Soluble derivatives of ACE2, in which Spike affinity is enhanced and the protein is fused to Fc of an immunoglobulin, are potent decoy receptors that reduce disease in animal models of COVID-19. Mutations were introduced into an ACE2 decoy receptor, including adding custom N-glycosylation sites and a cavity-filling substitution together with Fc modifications, which increased the decoy's catalytic activity and provided small to moderate enhancements of pharmacokinetics following intravenous and subcutaneous administration in humanized FcRn mice. Most prominently, sialylation of native glycans increases exposures by orders of magnitude, and the optimized decoy is therapeutically efficacious in a mouse COVID-19 model. Ultimately, an engineered and highly sialylated decoy receptor produced using methods suitable for manufacture of representative drug substance has high exposure with a 5- to 9-day half-life. Finally, peptide epitopes at mutated sites in the decoys generally have low binding to common HLA class II alleles and the predicted immunogenicity risk is low. Overall, glycosylation is a critical molecular attribute of ACE2 decoy receptors and modifications that combine tighter blocking of Spike with enhanced pharmacokinetics elevate this class of molecules as viable drug candidates.
RESUMO
Three highly pathogenic ß-coronaviruses have crossed the animal-to-human species barrier in the past two decades: SARS-CoV, MERS-CoV and SARS-CoV-2. To evaluate the possibility of identifying antibodies with broad neutralizing activity, we isolated a monoclonal antibody, termed B6, that cross-reacts with eight ß-coronavirus spike glycoproteins, including all five human-infecting ß-coronaviruses. B6 broadly neutralizes entry of pseudotyped viruses from lineages A and C, but not from lineage B, and the latter includes SARS-CoV and SARS-CoV-2. Cryo-EM, X-ray crystallography and membrane fusion assays reveal that B6 binds to a conserved cryptic epitope located in the fusion machinery. The data indicate that antibody binding sterically interferes with the spike conformational changes leading to membrane fusion. Our data provide a structural framework explaining B6 cross-reactivity with ß-coronaviruses from three lineages, along with a proof of concept for antibody-mediated broad coronavirus neutralization elicited through vaccination. This study unveils an unexpected target for next-generation structure-guided design of a pan-ß-coronavirus vaccine.
Assuntos
Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Glicoproteína da Espícula de Coronavírus , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Infecções por Coronavirus/prevenção & controle , Reações Cruzadas , Epitopos , Feminino , Células HEK293 , Humanos , Camundongos , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Three highly pathogenic ß-coronaviruses crossed the animal-to-human species barrier in the past two decades: SARS-CoV, MERS-CoV and SARS-CoV-2. SARS-CoV-2 has infected more than 64 million people worldwide, claimed over 1.4 million lives and is responsible for the ongoing COVID-19 pandemic. We isolated a monoclonal antibody, termed B6, cross-reacting with eight ß-coronavirus spike glycoproteins, including all five human-infecting ß-coronaviruses, and broadly inhibiting entry of pseudotyped viruses from two coronavirus lineages. Cryo-electron microscopy and X-ray crystallography characterization reveal that B6 binds to a conserved cryptic epitope located in the fusion machinery and indicate that antibody binding sterically interferes with spike conformational changes leading to membrane fusion. Our data provide a structural framework explaining B6 cross-reactivity with ß-coronaviruses from three lineages along with proof-of-concept for antibody-mediated broad coronavirus neutralization elicited through vaccination. This study unveils an unexpected target for next-generation structure-guided design of a pan-coronavirus vaccine.
RESUMO
The spillovers of betacoronaviruses in humans and the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants highlight the need for broad coronavirus countermeasures. We describe five monoclonal antibodies (mAbs) cross-reacting with the stem helix of multiple betacoronavirus spike glycoproteins isolated from COVID-19 convalescent individuals. Using structural and functional studies, we show that the mAb with the greatest breadth (S2P6) neutralizes pseudotyped viruses from three different subgenera through the inhibition of membrane fusion, and we delineate the molecular basis for its cross-reactivity. S2P6 reduces viral burden in hamsters challenged with SARS-CoV-2 through viral neutralization and Fc-mediated effector functions. Stem helix antibodies are rare, oftentimes of narrow specificity, and can acquire neutralization breadth through somatic mutations. These data provide a framework for structure-guided design of pan-betacoronavirus vaccines eliciting broad protection.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Betacoronavirus/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Internalização do Vírus , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/isolamento & purificação , Convalescença , Cricetinae , Reações Cruzadas , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Células Jurkat , Pulmão/imunologia , Fusão de Membrana/imunologia , Testes de Neutralização , Mapeamento de Peptídeos , Conformação Proteica em alfa-Hélice , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Carga Viral/imunologiaRESUMO
Uromodulin is the most abundant protein in human urine, and it forms filaments that antagonize the adhesion of uropathogens; however, the filament structure and mechanism of protection remain poorly understood. We used cryo-electron tomography to show that the uromodulin filament consists of a zigzag-shaped backbone with laterally protruding arms. N-glycosylation mapping and biophysical assays revealed that uromodulin acts as a multivalent ligand for the bacterial type 1 pilus adhesin, presenting specific epitopes on the regularly spaced arms. Imaging of uromodulin-uropathogen interactions in vitro and in patient urine showed that uromodulin filaments associate with uropathogens and mediate bacterial aggregation, which likely prevents adhesion and allows clearance by micturition. These results provide a framework for understanding uromodulin in urinary tract infections and in its more enigmatic roles in physiology and disease.
Assuntos
Infecções Urinárias/metabolismo , Uromodulina/química , Uromodulina/fisiologia , Adesinas Bacterianas/química , Microscopia Crioeletrônica , Glicosilação , Humanos , LigantesRESUMO
The mechanisms underlying the biogenesis of the structurally unique, binuclear Cu1.5+â¢Cu1.5+ redox center (CuA) on subunit II (CoxB) of cytochrome oxidases have been a long-standing mystery. Here, we reconstituted the CoxBâ¢CuA center in vitro from apo-CoxB and the holo-forms of the copper transfer chaperones ScoI and PcuC. A previously unknown, highly stable ScoIâ¢Cu2+â¢CoxB complex was shown to be rapidly formed as the first intermediate in the pathway. Moreover, our structural data revealed that PcuC has two copper-binding sites, one each for Cu1+ and Cu2+, and that only PcuCâ¢Cu1+â¢Cu2+ can release CoxBâ¢Cu2+ from the ScoIâ¢Cu2+â¢CoxB complex. The CoxBâ¢CuA center was then formed quantitatively by transfer of Cu1+ from a second equivalent of PcuCâ¢Cu1+â¢Cu2+ to CoxBâ¢Cu2+. This metalation pathway is consistent with all available in vivo data and identifies the sources of the Cu ions required for CuA center formation and the order of their delivery to CoxB.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cobre/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Metalochaperonas/química , Metalochaperonas/metabolismo , Apoproteínas/metabolismo , Sítios de Ligação , Bradyrhizobium/metabolismo , Cristalografia por Raios X , Modelos Biológicos , Oxirredução , Domínios Proteicos , Relação Estrutura-AtividadeRESUMO
Ligand-receptor interactions that are reinforced by mechanical stress, so-called catch-bonds, play a major role in cell-cell adhesion. They critically contribute to widespread urinary tract infections by pathogenic Escherichia coli strains. These pathogens attach to host epithelia via the adhesin FimH, a two-domain protein at the tip of type I pili recognizing terminal mannoses on epithelial glycoproteins. Here we establish peptide-complemented FimH as a model system for fimbrial FimH function. We reveal a three-state mechanism of FimH catch-bond formation based on crystal structures of all states, kinetic analysis of ligand interaction and molecular dynamics simulations. In the absence of tensile force, the FimH pilin domain allosterically accelerates spontaneous ligand dissociation from the FimH lectin domain by 100,000-fold, resulting in weak affinity. Separation of the FimH domains under stress abolishes allosteric interplay and increases the affinity of the lectin domain. Cell tracking demonstrates that rapid ligand dissociation from FimH supports motility of piliated E. coli on mannosylated surfaces in the absence of shear force.