The SARS-CoV-2 spike glycoprotein, mediating viral entry into human cells via ACE2 receptor binding, is a critical target for understanding viral evolution and pathogenesis. The D614G substitution, prevalent globally, enhances receptor binding by increasing the spike's open conformation. Emerging variants like Alpha and Beta have accumulated further spike substitutions impacting receptor binding, furin cleavage, and overall spike stability. This study aims to elucidate the structural and functional consequences of these mutations in Alpha (B.1.1.7) and Beta (B.1.351) variants, comparing them to the original Wuhan strain and the D614G variant. Using structure-fusion-stabilized spikes, the study directly compares the pre-fusion conformations, allowing for a detailed analysis of receptor-binding properties and their impact on viral infectivity. Understanding these evolutionary changes is paramount for developing effective antiviral strategies and predicting future variant emergence.

Previous research has highlighted the role of the D614G substitution in increasing SARS-CoV-2 infectivity by stabilizing the open conformation required for ACE2 binding and reducing S1 shedding. Studies on Alpha and Beta variants have shown increased RBD receptability and enhanced receptor binding. However, a detailed structural understanding of how these mutations affect spike conformation and stability was lacking. This study builds upon this existing knowledge by providing high-resolution structural data and functional assays to analyze the impact of specific mutations on receptor binding affinity and spike stability.

The study employed structure-fusion-stabilized spike proteins of Alpha and Beta variants, expressed in 293F cells. Cryo-electron microscopy (cryoEM) was used to determine the high-resolution structures of the spike proteins, both in the absence and presence of the ACE2 receptor. Biolayer interferometry was employed to quantitatively measure the binding affinity of the variant spikes to ACE2. Additionally, the impact of furin cleavage on spike stability was investigated by expressing the Alpha spike in the presence of a furin inhibitor. The structural analysis focused on the key mutations in the receptor-binding domain (RBD), the S1–S2 furin cleavage site, and the monomer–monomer interfaces. Data analysis included structural comparisons, quantitative binding affinity measurements, and assessment of spike trimer stability upon receptor binding. Specific methods included protein expression and purification, cryoEM data collection and processing, model building and refinement, and biolayer interferometry.

The study revealed several key structural and functional differences between the Alpha, Beta, and Wuhan spike proteins. Both Alpha and Beta variants shared the N501Y substitution in the RBD, leading to increased ACE2 binding affinity, particularly when coupled with D614G. The Alpha variant showed significantly increased stability as a trimer upon ACE2 binding compared to Wuhan and Beta variants. This increased stability was linked to the P681H substitution near the furin cleavage site, resulting in near-complete cleavage and a more stable, open conformation. In contrast, the Beta variant displayed a predominantly open conformation due to the K417N substitution combined with D614G. This open conformation, coupled with the N501Y mutation, contributes to increased receptor binding. Biolayer interferometry data showed a sixfold increase in binding strength for the Alpha spike and a twofold increase for the Beta spike compared to the Wuhan spike. The study further demonstrated that the D614G substitution was crucial for the enhanced ACE2 binding in both Alpha and Beta variants. The Y453F substitution in mink spike also showed increased binding affinity.

The findings highlight how SARS-CoV-2 has evolved to enhance its transmissibility in humans. The observed structural changes in Alpha and Beta spike proteins, specifically the increased stability and the predominantly open conformation, directly contribute to increased ACE2 binding affinity and enhanced viral entry into host cells. The increased stability of the Alpha spike in complex with ACE2 suggests a mechanism for improved infectivity. The predominance of the open conformation in Beta spike variants also contributes to heightened infectivity. These observations are consistent with the increased transmissibility observed in epidemiological studies of these variants. The study underscores the importance of continuous monitoring of SARS-CoV-2 evolution to understand the mechanisms of infectivity and to develop effective countermeasures.

This study provides high-resolution structural insights into the evolutionary adaptations of SARS-CoV-2 spike proteins in the Alpha and Beta variants. The key mutations identified contribute to increased ACE2 binding, enhanced spike stability, and a shift towards a more open conformation, collectively promoting higher infectivity. Future research should focus on exploring the interplay between these mutations and the immune response, further understanding the mechanisms of immune evasion, and predicting the emergence of future variants.

The study utilized stabilized spike proteins, which may not fully reflect the dynamics of the native spike protein on the virion surface. Further studies are needed to investigate the impact of these mutations on other aspects of viral pathogenesis, such as viral replication and immune evasion. While the study provides a comprehensive analysis of the Alpha and Beta variants, it is limited to these two variants, and the generalizability to other variants needs further investigation.