Fig. 4. Antigenicity of the 2019-nCoV RBD. (A) SARS-CoV RBD shown as a white molecular surface (PDB ID: 2AJF), with residues that vary in the 2019-nCoV RBD colored red. The ACE2-binding site is outlined with a black dashed line. (B) Biolayer interferometry sensorgram showing binding to ACE2 by the 2019-nCoV RBD-SD1. Binding data are shown as a black line, and the best fit of the data to a 1:1 binding model is shown in red.(C) Biolayer interferometry to measure cross-reactivity of the SARS-CoV RBD-directed antibodies S230, m396, and 80R. Sensor tips with immobilized antibodies were dipped into wells containing 2019-nCoV RBD-SD1, and the resulting data are shown as a black line.

1The outbreak of a novel betacoronavirus (2019-nCov) represents a pandemic threat that has been 2 declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a 3 key target for urgently needed vaccines, therapeutic antibodies, and diagnostics. To facilitate 4 medical countermeasure (MCM) development we determined a 3.5 Å-resolution cryo-EM 5 structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the 6 trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible 7 conformation. We also show biophysical and structural evidence that the 2019-nCoV S binds 8 ACE2 with higher affinity than SARS-CoV S. Additionally we tested several published SARS-9CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to 10 nCoV-2019 S, suggesting antibody cross-reactivity may be limited between the two virus RBDs. 11The atomic-resolution structure of 2019-nCoV S should enable rapid development and evaluation

The COVID-19 pandemic has led to accelerated efforts to develop therapeutics and vaccines. A key target of these efforts is the spike (S) protein, which is metastable and difficult to produce recombinantly. Here, we characterized 100 structure-guided spike designs and identified 26 individual substitutions that increased protein yields and stability. Testing combinations of beneficial substitutions resulted in the identification of HexaPro, a variant with six beneficial proline substitutions exhibiting ~10-fold higher expression than its parental construct and the ability to withstand heat stress, storage at room temperature, and three freeze-thaw cycles. A 3.2 Å-resolution cryo-EM structure of HexaPro confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for SARS-CoV-2.

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.

The ongoing COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 7,000,000 infections and 400,000 deaths worldwide to date. Antibody development efforts mainly revolve around the extensively glycosylated SARS-CoV-2 spike (S) protein, which mediates the host cell entry by binding to the angiotensinconverting enzyme 2 (ACE2). In the context of vaccine design, similar to many other viruses, the SARS-CoV-2 spike utilizes a glycan shield to thwart the host immune response. Here, we built a full-length model of 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 glycan shield and the protein structure, stability, and dynamics. End-to-end accessibility analyses outline a complete overview of the vulnerabilities of the glycan shield of SARS-CoV-2 S protein, which can be harnessed for vaccine development. In addition, a dynamic analysis of the main antibody epitopes is provided. Finally, beyond shielding, a possible structural role of N-glycans at N165 and N234 is hypothesized to modulate and stabilize the conformational dynamics of the spike's receptor binding domain, which is responsible for ACE2 recognition. Overall, this work presents hitherto unseen functional and structural insights into the SARS-CoV-2 S protein and its glycan coat, which may be exploited by therapeutic efforts targeting this essential molecular machine. Glycan Shield of the Receptor Binding DomainAs discussed in the previous section, the glycan shield plays a critical role in hiding the S protein surface from molecular recognition. However, to effectively function, the spike needs to recognize and bind to ACE2 receptors as the primary host cell infection route. For this reason, the RBM must become fully exposed and accessible. 48 In this scenario, the glycan shield works in concert with a large conformational change that allows the RBD to emerge above the N-glycan coverage. Here, we quantify the ASA of the RBM within RBD-A, corresponding to the RBD/ACE2-interacting region (residues 400-508), at various probe radii in both the Open and Closed systems (Figures 3A and 3D, full data in Tables S4-S6.). As expected, the ASA plots show a significant difference between the "down" (Closed) and "up" (Open) RBD conformations, with the RBM area covered by glycans being remarkably larger in the former. When RBD-A is in the "up" conformation, its RBM shows an average (across all radii) of only ~9% surface area covered by glycans, compared with ~35% in the Closed system (Figures 3A and 3D). This difference is further amplified when considering a larger probe radius of 15 Å, with a maximum of 11% and 46% for Open and Closed, respectively. Interestingly, for smaller probes (1.4-3 Å) the shielding becomes weak in both systems, with an average of 6% and 16% for Open and Closed, respectively.Note that the RBD regio...