ABSTRACT
Previous studies on the structural relationship between human antibodies and SARS-CoV-2 have focused on generating static snapshots of antibody complexes with the Spike trimer. However, antibody-antigen interactions are dynamic, with significant binding-induced allosteric effects on conformations of antibody and its target antigen. In this study, we employ hydrogen-deuterium exchange mass spectrometry, in vitro assays, and molecular dynamics simulations to investigate the allosteric perturbations linked to binding events between a group of human antibodies with differential functional activities, and the Spike trimer from SARS-CoV-2. Our investigations have revealed key dynamic features that define weakly or moderately neutralizing antibodies versus those with strong neutralizing activity. These results provide mechanistic insights into the functional modes of human antibodies against COVID-19, and provide a rationale for effective antiviral strategies. TeaserDifferent neutralizing antibodies induce site-specific allosteric effects across SARS-CoV-2 Spike protein
ABSTRACT
Accumulating evidence indicates a potential role for bacterial lipopolysaccharide (LPS) in the overactivation of the immune response during SARS-CoV-2 infection. LPS is recognised by Toll-like receptor 4 (TLR4) in innate immunity. Here, we showed that LPS binds to multiple hydrophobic pockets spanning both the S1 and S2 subunits of the SARS-CoV-2 spike (S) protein. LPS binds to the S2 pocket with a lower affinity compared to S1, suggesting its possible role as an intermediate in the TLR4 cascade. Congruently, nuclear factor-kappa B (NF-{kappa}B) activation in vitro is strongly boosted by S2. In vivo, however, a boosting effect is observed for both S1 and S2, with the former potentially facilitated by proteolysis. Collectively, our study suggests the S protein may act as a delivery system for LPS in host innate immune pathways. The LPS binding pockets are highly conserved across different SARS-CoV-2 variants and therefore represent potential therapeutic targets.
ABSTRACT
Bats are the reservoir for numerous human pathogens including coronaviruses. The factors leading to the emergence and sustained transmission of coronaviruses in humans are poorly understood. An outstanding question is how coronaviruses can accomplish a host switch with a likely mismatch between the surface protein spike of a bat virus and the human cellular receptor at the time of zoonotic virus transmission. To identify potential novel evolutionary pathways for zoonotic virus emergence, we serially passaged six human 229E isolates in a newly established Rhinolophus lepidus (horseshoe bat) kidney cells and analyzed viral genetic changes. Here we observed extensive deletions within the spike and ORF4 genes of five 229E viruses after passaging in bat cells. As a result, spike protein expression and infectivity of human cells was lost in 5 of 6 viruses but the capability to infect bat cells was maintained. Only viruses that expressed the spike protein could be neutralized by 229E spike-specific antibodies in human cells, whereas there was no neutralizing effect on viruses that do not express the spike protein inoculated on bat cells. However, one isolate acquired an early stop codon abrogating spike expression but maintaining infection in bat cells. Upon passaging this isolate in human cells, spike expression was restored due to acquisition of nucleotide insertions amongst virus subpopulations. Spike-independent infection of coronaviruses provides an alternative mechanism for viral maintenance in bats that does not rely on the compatibility of viral surface proteins and cellular entry receptors.
ABSTRACT
Recent developments in the SARS-CoV-2 pandemic point to its inevitable transformation into an endemic disease, urging both diagnostics of emerging variants of concern (VOCs) and design of the variant-specific drugs in addition to vaccine adjustments. Exploring the structure and dynamics of the SARS-CoV-2 Spike protein, we argue that the high mutability characteristic of RNA viruses coupled with the remarkable flexibility and dynamics of viral proteins result in a substantial involvement of allosteric mechanisms. While allosteric effects of mutations should be considered in predictions and diagnostics of new VOCs, allosteric drugs advantageously avoid escaping mutations via non-competitive inhibition originating from many alternative distal locations. The exhaustive allosteric signalling and probing maps provide a comprehensive picture of allostery in the Spike protein, making it possible to locate sites of potential mutations that could work as new VOCs "drivers", and to determine binding patches that may be targeted by newly developed allosteric drugs.
ABSTRACT
The recent global COVID-19 pandemic has prompted a rapid response in terms of vaccine and drug development targeting the viral pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this work, we modelled a complete membrane-embedded SARS-CoV-2 spike (S) protein, the primary target of vaccine and therapeutics development, based on available structural data and known glycan content. We then used molecular dynamics (MD) simulations to study the system in the presence of benzene probes designed to enhance discovery of cryptic, potentially druggable pockets on the S protein surface. We uncovered a novel cryptic pocket with promising druggable properties located underneath the 617-628 loop, which was shown to be involved in the formation of S protein multimers on the viral surface. A marked multi-conformational behaviour of this loop in simulations was validated using hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments, supportive of opening and closing dynamics. Interestingly, the pocket is also the site of the D614G mutation, known to be important for SARS-CoV-2 fitness, and within close proximity to mutations in the novel SARS-CoV-2 strains B.1.1.7 and B.1.1.28, both of which are associated with increased transmissibility and severity of infection. The pocket was present in systems emulating both immature and mature glycosylation states, suggesting its druggability may not be dependent upon the stage of virus maturation. Overall, the predominantly hydrophobic nature of the cryptic pocket, its well conserved surface, and proximity to regions of functional relevance in viral assembly and fitness are all promising indicators of its potential for therapeutic targeting. Our method also successfully recapitulated hydrophobic pockets in the receptor binding domain and N-terminal domain associated with detergent or lipid binding in prior cryo-electron microscopy (cryo-EM) studies. Collectively, this work highlights the utility of the benzene mapping approach in uncovering potential druggable sites on the surface of SARS-CoV-2 targets.
ABSTRACT
A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity between the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against infectious virus S protein. We find patterns which are conserved across all samples and this can be associated with site-specific stalling of glycan maturation which act as a highly sensitive reporter of protein structure. Molecular dynamics (MD) simulations of a fully glycosylated spike support s a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.