The severity of the COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, calls for the urgent development of a vaccine. The primary immunological target is the SARS-CoV-2 spike (S) protein. S is exposed on the viral surface to mediate viral entry into the host cell. To identify possible antibody binding sites not shielded by glycans, we performed multi-microsecond molecular dynamics simulations of a 4.1 million atom system containing a patch of viral membrane with four full-length, fully glycosylated and palmitoylated S proteins. By mapping steric accessibility, structural rigidity, sequence conservation and generic antibody binding signatures, we recover known epitopes on S and reveal promising epitope candidates for vaccine development. We find that the extensive and inherently flexible glycan coat shields a surface area larger than expected from static structures, highlighting the importance of structural dynamics in epitope mapping.
The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required for cell entry and is the primary focus for vaccine development. In this study, we combined cryo–electron tomography, subtomogram averaging, and molecular dynamics simulations to structurally analyze S in situ. Compared with the recombinant S, the viral S was more heavily glycosylated and occurred mostly in the closed prefusion conformation. We show that the stalk domain of S contains three hinges, giving the head unexpected orientational freedom. We propose that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat. The structure of native S contributes to our understanding of SARS-CoV-2 infection and potentially to the development of safe vaccines.
The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required for cell entry and is the major focus for vaccine development. We combine cryo electron tomography, subtomogram averaging and molecular dynamics simulations to structurally analyze S in situ. Compared to recombinant S, the viral S is more heavily glycosylated and occurs predominantly in a closed pre-fusion conformation. We show that the stalk domain of S contains three hinges that give the globular domain unexpected orientational freedom. We propose that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat. The structure of native S contributes to our understanding of SARS-CoV-2 infection and the development of safe vaccines. The large scale tomography data set of SARS-CoV-2 used for this study is therefore sufficient to resolve structural features to below 5 Ångstrom, and is publicly available at EMPIAR-10453.
More than 75% of surface and secreted proteins are modified by covalent addition of complex sugars through N- and O-glycosylation. Unlike proteins, glycans do not typically adopt specific secondary structures and remain very mobile, influencing protein dynamics and interactions with other molecules. Glycan conformational freedom impairs complete structural elucidation of glycoproteins. Computer simulations may be used to model glycan structure and dynamics. However, such simulations typically require thousands of computing hours on specialized supercomputers, thus limiting routine use. Here, we describe a reductionist method that can be implemented on personal computers to graft ensembles of realistic glycan conformers onto static protein structures in a matter of minutes. Using this open-source pipeline, we reconstructed the full glycan cover of SARS-CoV-2 Spike protein (S-protein) and a human GABAA receptor. Focusing on S-protein, we show that GlycoSHIELD recapitulates key features of extended simulations of the glycosylated protein, including epitope masking, and provides new mechanistic insights on N-glycan impact on protein structural dynamics.
The spike protein of SARS-CoV-2 is a highly flexible membrane receptor that triggers the translocation of the virus into cells by attaching to the human receptors. Like other type I membrane receptors, this protein has several extracellular domains connected by flexible hinges. The presence of these hinges results in high flexibility, which consequently results in challenges in defining the conformation of the protein. Here, We developed a new method to define the conformational space based on a few variables inspired by the robotic field’s methods to determine a robotic arm’s forward kinematics. Using newly performed atomistic molecular dynamics (MD) simulations and publicly available data, we found that the Denavit-Hartenberg (DH) parameters can reliably show the changes in the local conformation. Furthermore, the rotational and translational components of the homogenous transformation matrix constructed based on the DH parameters can identify the changes in the global conformation of the spike and also differentiate between the conformation with a similar position of the spike head, which other types of parameters, such as spherical coordinates, fail to distinguish between such conformations. Finally, the new method will be beneficial for looking at the conformational heterogeneity in all other type I membrane receptors.
Abstract
The primary immunological target of COVID-19 vaccines is the SARS-CoV-2 spike (S) protein. S is exposed on the viral surface and mediates viral entry into the host cell. To identify possible antibody binding sites, we performed multi-microsecond molecular dynamics simulations of a 4.1 million atom system containing a patch of viral membrane with four full-length, fully glycosylated and palmitoylated S proteins. By mapping steric accessibility, structural rigidity, sequence conservation, and generic antibody binding signatures, we recover known epitopes on S and reveal promising epitope candidates for structure-based vaccine design. We find that the extensive and inherently flexible glycan coat shields a surface area larger than expected from static structures, highlighting the importance of structural dynamics. The protective glycan shield and the high flexibility of its hinges give the stalk overall low epitope scores. Our computational epitope-mapping procedure is general and should thus prove useful for other viral envelope proteins whose structures have been characterized.
Author summary
The SARS-CoV-2 virus has caused a global health crisis. The spike protein exposed at its surface is key for infection and the primary antibody target. However, spike is covered by highly mobile glycan molecules that could impair antibody binding. To identify accessible epitopes, we performed molecular dynamics simulations of an atomistic model of glycosylated spike embedded in a membrane. By combining extensive simulations with bioinformatics analyses, we recovered known antibody binding sites and identified several epitope candidates as targets for further vaccine development.
