O-glycosylation on SARS-CoV-2: Emergence of Model D

Saptaparna Dasgupta, Bennett University

SARS-CoV-2, a strain from the Coronavirus family, is responsible for the ongoing global pandemic of COVID-19, taking the lives of millions of people via an infectious respiratory illness. Several studies have been conducted on this virus to find target proteins responsible for the infection as a therapeutic. Thus, spike (S) protein emerged as a promising target. Glycosylation of the spike (S) protein present on the virus and the Angiotensin-Converting Enzyme 2 (ACE2) receptor present on the host cell are crucial components for the virus to infect humans. Spike proteins contain a domain for the ACE2 receptor to bind, known as the Receptor Binding Domain (RBD). A novel site, mutated on the RBD, known as the O-glycosylation site (D494S) on the RBD was revealed as a crucial mediator of the interaction between the virus and the host.

Mutation of D494S:

As stated in the study by Rahnama et al., the interaction in between the RBD and ACE2 is conducted via the four ββ strands (β4-β7) along with the two αα helices (H1, H2) on the binding interface of ACE2. Mutation in the RBD domain of the SARS-CoV-2 results in attenuated binding affinity with the potential inhibitors of the virus. Mutations at the intersection point of the S1 and S2 proteins on the virus led to the emergence of a cleavage site, polybasic in nature. The site is positioned adjacent to the O-glycosylation site, which is a unique characteristic of SARS-CoV-2. An enzyme named Furin helps in the recognition of the polybasic cleavage sites at time of O-glycosylation. It has also been found that patients with high blood glucose levels are in higher chances of getting infected.

Model D:

The crystallized complex of the SARS-CoV-2 RBD with ACE2 as an extracellular domain was taken as the initial structure. Ions, namely Zn²⁺ and Cl⁻ were used to stabilize the structures of ACE2 and S1 and S2 subunits. The Asparagine residues, located with N-glycosylation were glycosylated and GLYCAM online builder was used to attach oligosaccharide N-glycan to each of the sites. Similarly, the Serine494 residue was O-glycosylated using the GLYCAM online builder. These modified O-glycan models were attached to the RBD complex, and on the other hand, the RBD-ACE2 complex was N-glycosylated. Serine494 was substituted with Aspartic acid residues and was referred to as model D (in Fig 1). The molecular dynamic simulation was performed at 310K temperature and 1 bar pressure, respectively, followed by which, the binding free energies were calculated.

Result and Discussion:

The RMSD plot data reveals the fluctuations of RMSD within the limited timescale at the time of the molecular simulation. Amongst the three models of O-glycosylated systems, model III, and model II show similar flexibility with S protein. Model D was observed to depict high fluctuations in the RMSD values. Thus, it could be concluded that the mutation in D494S stabilizes the interactions within RBD-ACE2 in SARS-CoV-2. Model D was observed to have the highest RMSF value, and thus, it was concluded as the most flexible complex. Also, it was observed that mutation in D494S influences the stabilization of the interaction between RBD and ACE2. The relative difference between the model, represented by the fluctuations which are determined using the formula:

Wherein,

RMSF_i is per residue RMSF of each model

D_RMSF is lower relative to RMSF

As revealed by the calculated binding free energy, the interaction between RBD and ACE2 is more favorable upon O-glycosylation in models II and III. The binding free energy calculation is also influenced by the glycan groups, where larger groups are more favorable.

Significance of the study:

The site unique to SARS-CoV-2 is the S494 is located on the binding interface of RBD-ACE2, which is enhanced by O-glycans. The results from the molecular dynamic simulation state that O-glycosylation of S494 helps in the build-up of strong interaction between RBD-ACE2. This interaction could potentially increase the infectivity potential of the virus.

Attachment of elongated O-glycans was observed to lead to less flexible ACE2-RBD dynamics. Also, a decreased distance was observed between RBD and the αα helices of ACE2. The MD simulation of un-glycosylated S494 and substituting the S residue to D residue was used to confirm the results that occur in SARS-CoV-2. This research adds to our understanding of SARS-CoV-2-ACE2 glycosylation and its involvement in the rate of infection of the virus. This hypothesis is a good candidate for experimental validation, and if it is found to be essential, it should be considered in future treatment designs.

Also read: Xylans – an undervalued biorefinery gem

Reference:

1. Rahnama, S., Azimzadeh Irani, M., Amininasab, M., & Ejtehadi, M. R. (2021). S494 O-glycosylation site on the SARS-CoV-2 RBD affects the virus affinity to ACE2 and its infectivity; a molecular dynamics study. Scientific Reports, 11(1), 15162. https://doi.org/10.1038/s41598-021-94602-w

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Author info:

Saptaparna Dasgupta, currently a B. Tech 3^rd year student, pursuing Biotechnology, is a diligent student and determined in terms of her career goals. Being a budding biotechnologist, she is open to all research fields of her course and passionate about knowledge. She is focused and constantly tries to improve her writing skills, also a project enthusiast and is fond of gaining hands-on experience in laboratories. She believes that all hard work and efforts pays off eventually and follows this as the motto of her life.