The coronavirus (CoV) spike protein (S), involved in host cell-virus fusion, is the primary immunogenic target for virus neutralization and the current focus of many vaccine design efforts. The highly flexible protein S, with its mobile domains, presents a moving target for the immune system.

Here, to better understand protein S mobility, we implement a vector analysis based on the structure of available protein S β-CoV structures. Despite a general similarity in domain organization, we found that S proteins from different β-CoV show different configurations.

Based on this analysis, we developed two soluble ectodomain constructs for the SARS-CoV-2 protein S, in which the highly immunogenic and mobile receptor-binding domain (RBD) is blocked in the “down” position of all RBDs. . or it adopts “rising” state conformations more readily than wild-type protein S. These results demonstrate that protein S conformation can be controlled by rational design and can provide a framework for the development of S CoV proteins designed for vaccine applications.

The wealth of structural information for the S β-CoV proteins, including the recently determined cryo-MS structures of the SARS-CoV-21 peak,2,3,4,5,6,7,8,9,10,11, has provided a rich source of detailed geometric information from which to begin an accurate examination of the macromolecular transitions underlying the activation of this fusion machine.

Several soluble ectodomain construct structures have been determined that retain the entire S1 subunit and the surface exposed S2 subunit. These include SARS-CoV-21,3, SARS4,5,6,7,8, MERS4,9, and other human1,10 and murine11 S β-CoV proteins. These structures revealed remarkable conformational heterogeneity in protein Speaks, especially in the RBD region.

Within a single protomer, the RBD could adopt a closed state in which the RBD covers the apical region of the S2 protein near the C-terminus of the first heptad repeat (HR1) or an open state in which the RBD dissociates from the apical central axis of S2 and the NTD (Fig. 1a). Furthermore, cryo-EM structures strongly suggest a high degree of domain flexibility in both the down and up states in the NTD and RBD.

Although these structures have provided essential information for identifying the relative arrangement of these domains, it remains to be determined the degree to which conformational heterogeneity can be altered by mutation during the natural evolution of the virus and in a vaccine immunogen design context.

In this study, we quantify the variability in the S1 and S2 geometric arrangements to reveal important regions of flexibility to consider and target for structure-based immunogen design. Based on these analyzes, we design mutations that alter the conformational distribution of domains in protein S.

We visualize the effect of our designs using a structural determination pipeline that relies first on single-particle analysis by negative staining electron microscopy. (NSEM) for rapid and low-cost assessment of low-resolution peak ectodomains, followed by cryo-EM for high-resolution information on the changes introduced by these mutations.

Our results reveal a heterogeneous conformational landscape of the SARS-CoV-2 peak that is highly susceptible to modification by the introduction of mutations at the contact sites between the S1 and S2 subunits. We also present data on stabilized modified SARS-CoV-2 ectodomain constructs in conformations that have not yet been seen in currently available frameworks, with great interest and direct application in vaccine design.