Current COVID-19 vaccines and many clinical diagnostics are based on the structure and function of the SARS-CoV-2 spike ectodomain. Using hydrogen-deuterium exchange monitored by mass spectrometry, we have uncovered that, in addition to the prefusion structure determined by cryo-electron microscopy, this protein adopts an alternative conformation that interconverts slowly with the canonical prefusion structure. This new conformation-an open trimer-contains easily accessible receptor-binding domains. It exposes the conserved trimer interface buried in the prefusion conformation, thus exposing potential epitopes for pan-coronavirus antibody and ligand recognition. The population of this state and kinetics of interconversion are modulated by temperature, receptor binding, antibody binding, and sequence variants observed in the natural population. Knowledge of the structure and populations of this conformation will help improve existing diagnostics, therapeutics, and vaccines.

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.

To investigate the evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the immune population, we coincupi bated the authentic virus with a highly neutralizing plasma from a COVID-19 convalescent patient. The plasma fully neutralized the virus for seven passages, but, after 45 d, the deletion of F140 in the spike N-terminal domain (NTD) N3 loop led to partial breakthrough. At day 73, an E484K substitution in the receptor-binding domain (RBD) occurred, followed, at day 80, by an insertion in the NTD N5 loop containing a new glycan sequon, which generated a variant completely resistant to plasma neutralization. Computational modeling predicts that the deletion and insertion in loops N3 and N5 prevent binding of neutralizing antibodies. The recent emergence in the United Kingdom, South Africa, Brazil, and Japan of natural variants with similar changes suggests that SARS-CoV-2 has the potential to escape an effective immune response and that vaccines and antibodies able to control emerging variants should be developed.

Persistent, high-risk human papillomavirus (HPV) infection is the primary cause of cervical cancer. Neutralizing antibodies elicited by L1-only virus-like particles (VLPs) can block HPV infection; however, the lack of high-resolution structures has limited our understanding of the mode of virus infection and the requirement for type specificity at the molecular level. Here, we describe two antibodies, A12A3 and 28F10, that specifically bind to and neutralize HPV58 and HPV59, respectively, through two distinct binding stoichiometries. We show that the epitopes of A12A3 are clustered in the DE loops of two adjacent HPV58 L1 monomers, whereas 28F10 recognizes the HPV59 FG loop of a single monomer. Via structure-based mutagenesis and analysis of antibody binding, we further identified the residues HPV58 D154, S168, and N170 and HPV59 M267, Q270, E273, Y276, K278, and R283, which play critical roles in virus infection. By substituting these strategic epitope residues into other HPV genotypes, we could then redirect the type-specific binding of the antibodies to these genotypes, thus highlighting the importance of these specific residues, HPV58 R161, S168, and N308 and HPV59 Q270, E273, and D281. Overall, our findings provide molecular insights into potential structural determinants of HPV required for infectivity and type specificity.IMPORTANCE High-risk human papillomaviruses (HPVs) are considered the major causative pathogens of cancers that affect epithelial mucosa, such as cervical cancer. However, because of the lack of high-resolution structural information on the sites of neutralization, we have yet to determine the precise mode of HPV infection and how different types of HPV cause infection. Our crystal structures in this study have uncovered discrete binding stoichiometries for two different antibodies. We show that one A12A3 Fab binds to the center of one HPV58 pentamer, whereas five 28F10 Fabs bind along the top fringe of one HPV59 pentamer. Furthermore, through targeted epitope analysis, we show that 6 to 7 discontinuous residues of the L1 major capsid protein of HPV are determinants, at least in part, for virus infection and type specificity. This knowledge will help us to unravel the process of HPV infection and can potentially be used to drive the development of therapeutics that target neutralization-sensitive sites.

The emergence of numerous variants of SARS-CoV-2, the causative agent of COVID-19, has presented new challenges to the global efforts to control the COVID-19 pandemic. Here, we obtain two cross-neutralizing antibodies (7D6 and 6D6) that target Sarbecoviruses' receptor-binding domain (RBD) with sub-picomolar affinities and potently neutralize authentic SARS-CoV-2. Crystal structures show that both antibodies bind a cryptic site different from that recognized by existing antibodies and highly conserved across Sarbecovirus isolates. Binding of these two antibodies to the RBD clashes with the adjacent N-terminal domain and disrupts the viral spike. Both antibodies confer good resistance to mutations in the currently circulating SARS-CoV-2 variants. Thus, our results have direct relevance to public health as options for passive antibody therapeutics and even active prophylactics. They can also inform the design of pan-sarbecovirus vaccines.

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.

Access to experimental X-ray diffraction image data is fundamental for validation and reproduction of macromolecular models and indispensable for development of structural biology processing methods. Here, we established a diffraction data publication and dissemination system, Structural Biology Data Grid (SBDG; data.sbgrid.org), to preserve primary experimental data sets that support scientific publications. Data sets are accessible to researchers through a community driven data grid, which facilitates global data access. Our analysis of a pilot collection of crystallographic data sets demonstrates that the information archived by SBDG is sufficient to reprocess data to statistics that meet or exceed the quality of the original published structures. SBDG has extended its services to the entire community and is used to develop support for other types of biomedical data sets. It is anticipated that access to the experimental data sets will enhance the paradigm shift in the community towards a much more dynamic body of continuously improving data analysis.