Beyond Shielding: The Roles of Glycans in the SARS-CoV‑2 Spike Protein

The ongoing COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 28,000,000 infections and 900,000 deaths worldwide to date. Antibody development efforts mainly revolve around the extensively glycosylated SARS-CoV-2 spike (S) protein, which mediates host cell entry by binding to the angiotensin-converting enzyme 2 (ACE2). Similar to many other viral fusion proteins, the SARS-CoV-2 spike utilizes a glycan shield to thwart the host immune response. Here, we built a full-length model of the glycosylated SARS-CoV-2 S protein, both in the open and closed states, augmenting the available structural and biological data. Multiple microsecond-long, all-atom molecular dynamics simulations were used to provide an atomistic perspective on the roles of glycans and on the protein structure and dynamics. We reveal an essential structural role of N-glycans at sites N165 and N234 in modulating the conformational dynamics of the spike’s receptor binding domain (RBD), which is responsible for ACE2 recognition. This finding is corroborated by biolayer interferometry experiments, which show that deletion of these glycans through N165A and N234A mutations significantly reduces binding to ACE2 as a result of the RBD conformational shift toward the “down” state. Additionally, end-to-end accessibility analyses outline a complete overview of the vulnerabilities of the glycan shield of the SARS-CoV-2 S protein, which may be exploited in the therapeutic efforts targeting this molecular machine. Overall, this work presents hitherto unseen functional and structural insights into the SARS-CoV-2 S protein and its glycan coat, providing a strategy to control the conformational plasticity of the RBD that could be harnessed for vaccine development

Stabilized Coronavirus Spike Stem Elicits a Broadly Protective Antibody

Current coronavirus vaccines primarily target immunodominant epitopes in the S1 subunit, which are poorly conserved and susceptible to escape mutations, thus threatening vaccine efficacy. Here, we use structure-guided protein engineering to remove the S1 subunit from the MERS-CoV spike (S) glycoprotein and develop stabilized stem (SS) antigens. Vaccination with MERS SS elicits cross-reactive β-coronavirus antibody responses and protects mice against lethal MERS-CoV challenge. High-throughput screening of antibody secreting cells from MERS SS-immunized mice leads to discovery of a panel of cross-reactive monoclonal antibodies. Among them, antibody IgG22 binds with high affinity to both MERS-CoV and SARS-CoV-2 S proteins, and a combination of electron microscopy and crystal structures localizes the epitope to a conserved coiled-coil region in the S2 subunit. Passive transfer of IgG22 protects mice against both MERS-CoV and SARS-CoV-2 challenge. Collectively, these results provide proof-of-principle for cross-reactive coronavirus antibodies and inform the development of pan-coronavirus vaccines and therapeutic antibodies

Structural basis for receptor binding and antibody-mediated neutralization by Bordetella adenylate cyclase toxin

Bordetella pertussis, the gram-negative pathogen that causes whooping cough, caused 89,000 deaths in 2018 globally. In the United States, introduction of killed whole-cell B. pertussis (wP) vaccines in the 1940s reduced the number of yearly cases from over 100,000 to less than 10,000 in 1965. However, public fears about the reactogenicity of wP vaccine led many countries to switch to less effective acellular B. pertussis (aP) vaccines, containing specific purified antigens. An improved version of aP vaccine is desired, which could be achieved by including better or more optimized antigens. Notably, the adenylate cyclase toxin (ACT) of B. pertussis has been hypothesized to be an effective antigen to include in the aP vaccine as it is essential for lung colonization in mouse intranasal models and has elicited protective immunity in mice. However, ACT was never included in human vaccines as it was too difficult to produce. ACT is a virulence factor secreted by B. pertussis that inserts into host leukocytes and translocates an adenylate cyclase enzyme into the target cell cytosol. The rapid formation of cAMP in host leukocytes inhibits their bactericidal activities, thus promoting survival of B. pertussis in the respiratory epithelium. As a ‘pore-forming RTX toxin’, ACT has a C-terminal calcium-binding ‘RTX’ domain that mediates secretion. However, biochemical data had previously shown that the RTX domain of ACT contains the binding site for the ACT receptor, integrin αMβ₂. The RTX domain was also shown to harbor the epitopes for neutralizing antibodies that prevent αMβ₂ binding, suggesting that this interaction could be targeted with an RTX domain immunogen. However, the structural determinants of the ACT RTX domain’s interaction with αMβ₂ were not well understood prior to this work. Specifically, it was not known how the ACT RTX domain had been adapted to engage in protein-protein interactions, which regions of the RTX domain contacted the αMβ₂ receptor as well as neutralizing antibodies, and how binding of ACT to αMβ₂ favors membrane insertion of ACT. In addition, ACT preferentially binds to the inactive conformation of αMβ₂, and the structural basis for this preference is not understood. Therefore, this work set out to structurally define the interaction of ACT with αMβ₂ and with neutralizing antibodies, to understand key epitopes for mediating protection against B. pertussis. Chapter 1 provides an outline of the epidemiology of B. pertussis and the known details of the ACT intoxication and receptor-binding mechanism. In Chapter 2, a crystal structure of an engineered RTX domain fragment harboring the αMβ₂-binding site bound to neutralizing antibodies is described. This structure showed neutralizing antibodies bound to the variable “linker” regions of the RTX domain, which had a previously unknown fold and connect the segments of the RTX domain containing consensus 9-residue repeats known to form a helical calcium-binding structure. This suggested that the RTX linkers were involved in binding αMβ₂ as these antibodies prevent αMβ₂ binding to the ACT RTX domain. In Chapter 3, a cryo-electron microscopy structure of the ACT RTX domain in complex with the αMβ₂ ectodomain is reported. This structure showed that the RTX linkers, shown in Chapter 2 to harbor the neutralizing epitopes bound by antibodies M2B10 and M1H5, each form a separate binding site on αMβ₂, and can only contact both of these sites when αMβ₂ is in the bent, inactive conformation. This structure also showed that αMβ₂ binding would position lysine residues that require post-translational acylation for ACT activity directly at the host cell membrane, coupling receptor binding to membrane insertion. This work defined RTX linkers as key protein-protein interaction modules of the ACT RTX domain and provides a framework for the design of RTX domain vaccines.Cellular and Molecular Biolog

Beyond Shielding: The Roles of Glycans in the SARS-CoV‑2 Spike Protein

The ongoing COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 28,000,000 infections and 900,000 deaths worldwide to date. Antibody development efforts mainly revolve around the extensively glycosylated SARS-CoV-2 spike (S) protein, which mediates host cell entry by binding to the angiotensin-converting enzyme 2 (ACE2). Similar to many other viral fusion proteins, the SARS-CoV-2 spike utilizes a glycan shield to thwart the host immune response. Here, we built a full-length model of the glycosylated SARS-CoV-2 S protein, both in the open and closed states, augmenting the available structural and biological data. Multiple microsecond-long, all-atom molecular dynamics simulations were used to provide an atomistic perspective on the roles of glycans and on the protein structure and dynamics. We reveal an essential structural role of N-glycans at sites N165 and N234 in modulating the conformational dynamics of the spike's receptor binding domain (RBD), which is responsible for ACE2 recognition. This finding is corroborated by biolayer interferometry experiments, which show that deletion of these glycans through N165A and N234A mutations significantly reduces binding to ACE2 as a result of the RBD conformational shift toward the "down" state. Additionally, end-to-end accessibility analyses outline a complete overview of the vulnerabilities of the glycan shield of the SARS-CoV-2 S protein, which may be exploited in the therapeutic efforts targeting this molecular machine. Overall, this work presents hitherto unseen functional and structural insights into the SARS-CoV-2 S protein and its glycan coat, providing a strategy to control the conformational plasticity of the RBD that could be harnessed for vaccine development

A glycan gate controls opening of the SARS-CoV-2 spike protein.

SARS-CoV-2 infection is controlled by the opening of the spike protein receptor binding domain (RBD), which transitions from a glycan-shielded 'down' to an exposed 'up' state to bind the human angiotensin-converting enzyme 2 receptor and infect cells. While snapshots of the 'up' and 'down' states have been obtained by cryo-electron microscopy and cryo-electron tomagraphy, details of the RBD-opening transition evade experimental characterization. Here over 130 µs of weighted ensemble simulations of the fully glycosylated spike ectodomain allow us to characterize more than 300 continuous, kinetically unbiased RBD-opening pathways. Together with ManifoldEM analysis of cryo-electron microscopy data and biolayer interferometry experiments, we reveal a gating role for the N-glycan at position N343, which facilitates RBD opening. Residues D405, R408 and D427 also participate. The atomic-level characterization of the glycosylated spike activation mechanism provided herein represents a landmark study for ensemble pathway simulations and offers a foundation for understanding the fundamental mechanisms of SARS-CoV-2 viral entry and infection

Mapping the co-localization of the circadian proteins PER2 and BMAL1 with enkephalin and substance P throughout the rodent forebrain

Despite rhythmic expression of clock genes being found throughout the central nervous system, very little is known about their function outside of the suprachiasmatic nucleus. Determining the pattern of clock gene expression across neuronal subpopulations is a key step in understanding their regulation and how they may influence the functions of various brain structures. Using immunofluorescence and confocal microscopy, we quantified the co-expression of the clock proteins BMAL1 and PER2 with two neuropeptides, Substance P (SubP) and Enkephalin (Enk), expressed in distinct neuronal populations throughout the forebrain. Regions examined included the limbic forebrain (dorsal striatum, nucleus accumbens, amygdala, stria terminalis), thalamus medial habenula of the thalamus, paraventricular nucleus and arcuate nucleus of the hypothalamus and the olfactory bulb. In most regions examined, BMAL1 was homogeneously expressed in nearly all neurons (~90%), and PER2 was expressed in a slightly lower proportion of cells. There was no specific correlation to SubP- or Enk- expressing subpopulations. The olfactory bulb was unique in that PER2 and BMAL1 were expressed in a much smaller percentage of cells, and Enk was rarely found in the same cells that expressed the clock proteins (SubP was undetectable). These results indicate that clock genes are not unique to specific cell types, and further studies will be required to determine the factors that contribute to the regulation of clock gene expression throughout the brain