Three highly pathogenic β-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 β-coronavirus spike glycoproteins, including all five human-infecting β-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 β-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-β-coronavirus vaccine.
How uromodulin helps flush out bacteria
Urinary tract infections (UTIs) are one of the most frequent bacterial infections in humans. The glycoprotein uromodulin is the most abundant urinary protein ...and can provide some protection from UTIs, but the precise mechanism has been unclear. Weiss
et al.
found that uromodulin forms stacked, fishbone-like filaments that act as a multivalent decoy for bacterial pathogens with adhesive pili that attach to the uromodulin glycans (see the Perspective by Kukulski). The resulting uromodulin-pathogen aggregates prevent bacterial adhesion to glycoproteins of the urinary epithelium and promote pathogen clearance as urine is excreted. This innate protection against UTIs is likely to be particularly important in infants and children.
Science
, this issue p.
1005
; see also p.
917
Uromodulin filaments in human urine associate with uropathogens and mediate bacterial aggregation and clearance.
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.
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.
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.
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.
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.
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The amino acid sequences of ACE2 decoy receptors have been extensively engineered for enhanced neutralization of SARS-CoV-2. Procko and colleagues find that in vivo properties of ACE2 decoy receptors are also highly dependent on how the protein is glycosylated, with long pharmacokinetics and high exposures requiring sialylated glycans.
The mechanisms underlying the biogenesis of the structurally unique, binuclear Cu
•Cu
redox center (Cu
) on subunit II (CoxB) of cytochrome oxidases have been a long-standing mystery. Here, we ...reconstituted the CoxB•Cu
center in vitro from
-CoxB and the
-forms of the copper transfer chaperones ScoI and PcuC. A previously unknown, highly stable ScoI•Cu
•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 Cu
and Cu
, and that only PcuC•Cu
•Cu
can release CoxB•Cu
from the ScoI•Cu
•CoxB complex. The CoxB•Cu
center was then formed quantitatively by transfer of Cu
from a second equivalent of PcuC•Cu
•Cu
to CoxB•Cu
. This metalation pathway is consistent with all available in vivo data and identifies the sources of the Cu ions required for Cu
center formation and the order of their delivery to CoxB.
We demonstrate how two copper atoms are brought together to build a metal center in an essential enzyme for cellular respiration.
The mechanisms underlying the biogenesis of the structurally unique, ...binuclear Cu
1.5+
•Cu
1.5+
redox center (Cu
A
) on subunit II (CoxB) of cytochrome oxidases have been a long-standing mystery. Here, we reconstituted the CoxB•Cu
A
center in vitro from
apo
-CoxB and the
holo
-forms of the copper transfer chaperones ScoI and PcuC. A previously unknown, highly stable ScoI•Cu
2+
•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 Cu
1+
and Cu
2+
, and that only PcuC•Cu
1+
•Cu
2+
can release CoxB•Cu
2+
from the ScoI•Cu
2+
•CoxB complex. The CoxB•Cu
A
center was then formed quantitatively by transfer of Cu
1+
from a second equivalent of PcuC•Cu
1+
•Cu
2+
to CoxB•Cu
2+
. This metalation pathway is consistent with all available in vivo data and identifies the sources of the Cu ions required for Cu
A
center formation and the order of their delivery to CoxB.
Targeting a range of betacoranaviruses In the past 20 years, three highly pathogenic β-coronaviruses have crossed from animals to humans, including the most recent: severe acute respiratory syndrome ...coronavirus 2 (SARS-CoV-2). A spike protein that decorates these viruses has an S1 domain that binds host cell receptors and an S2 domain that fuses the viral and cell membranes to allow cell entry. The S1 domain is the target of many neutralizing antibodies but is more genetically variable than S2, and antibodies can exert selective pressure, leading to resistant variants. Pintoet al. identified five monoclonal antibodies that interact with a helix in the S2 domain. The most broadly neutralizing antibody inhibited all β-coronavirus subgenera and reduced viral burden in hamsters infected with SARS-CoV-2. —VV
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