SARS-CoV-2 N439K mutation may be more infectious and resistant to antibodies than Wuhan strain

Researchers studied the effect of several SARS-CoV-2 mutations on its binding to the human angiotensin 2 converting enzyme using molecular dynamics simulations. They found that the N439K mutant binds more strongly than the original Wuhan strain, which could have implications for therapies such as monoclonal antibodies.

The 2019 coronavirus disease (COVID-19) pandemic raging worldwide is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The receptor binding domain of spike proteins present on the virus envelope binds to the human angiotensin 2 converting enzyme (ACE2), followed by fusion of the virus with the host cell membrane. The spike protein has two subunits: S1, which binds to host cells, and S2, which plays a role in membrane fusion.

The SARS-CoV-2 spike protein mediates viral entry into the host cell. 3d illustration. Image Credit: Design Cells / Shutterstock

Immunizing antibodies produced by the host immune system target RBD and disrupt the virus’s binding to ACE2. However, when there are mutations in the spike protein, it can affect the effectiveness of neutralizing antibodies. Currently, there are approximately 930 mutations reported around the world. According to reports, a mutation from ASP614 to GLY614 made the virus more contagious.

Most of the strategies developed to fight the virus in a similar way to antibodies are based on the spike protein sequence of the Wuhan reference strain. Missense mutations in earlier infectious coronaviruses such as MERS and SARS-CoV have been observed to become resistant to neutralizing antibodies to the original strain. Therefore, mutations in SARS-CoV-2 can also lead to strains resistant to antibody treatments in development. Therefore, mutations in circulating SARS-CoV-2 strains need to be monitored to develop better therapies.

Simulation of SARS-CoV-2 mutant binding with ACE2

Researchers from the Harbin Institute of Technology, China, modeled the complexes formed between human RBD and ACE2 and monoclonal antibodies and reported their findings in a paper published in bioRxiv * prepress server.

The authors compared the complexes formed by the wild-type virus and the mutated virus and performed molecular dynamics simulations together with the molecular mechanics / Poisson-Boltzmann surface area pattern. Mutations in SARS-CoV-2 were mainly observed in the open reading frame (ORF) regions, which encode non-structural proteins, nucleocapsid proteins, and spike proteins. The ORF3a protein, which can change the environment inside the infected cell and create holes in the membranes of the host cell, has a higher mutation rate in North America and Oceania and may allow the virus to spread better.

The authors found that the mutations were not on single sites. On the spike protein, the D614G variant was the majority mutation, followed by D936Y. The N439K variant, where asparagine at 439^th site is replaced by lysine, it is the most dominant in the RBD spike protein.

Molecular dynamics simulations showed greater flexibility changes in the N439K variant, which could lead to structural rearrangements in the SARS-CoV-2 RBD-ACE2 complex leading to stronger binding. Furthermore, the complex with the mutated virus forms more hydrogen bonds than the wild-type complex.

The binding energy of the N439K complex was also higher than that of the wild-type complex. This suggests that the mutant virus has a stronger association with human ACE2. The stronger bond could be due to the fact that replacing asparagine with lysine forms a new salt bridge in the complex with human ACE2, which could increase electrostatic interactions. In addition to this interaction, the complexes are also linked by van der Waals interactions and the free energy of polar solvation.

The N439K mutant may be resistant to some monoclonal antibodies

Although previous studies have suggested that mutant versions of the virus may be less infectious, the stronger link of human ACE2 to the N439K mutant suggests that this mutant strain may be more contagious. The N439K mutation is fully included in the D614G samples, which were observed to be more contagious than the original strain.

Structural and energetic details of both wild and mutant RBD-mAbs interactions (A) Crystal structures of the RDB-CB6 / REGN10987 complexes, the RBD is colored red, the CB6 heavy and light chains are represented as marine and green respectively, the chain heavy REGN10987 is colored yellow and the light is blue, and the 439 residues are described as the sphere. (B) Characteristic dynamic fluctuations of the RBD-REGN10987 and RBD (N439K) -REGN10987 complexes. The mutant type (100 ns) and the wild type (100 ns) are colored orange and blue, respectively. (C) The dynamic conformations are projected onto the main vectors (PC1 and PC2). Red and blue indicate mutant and wild-type 100 ns MD trajectories, respectively. (D) The RMSD of the receptor binding motif in four complexes during the MD 100-ns simulations. (E) The free binding energies for both REGN10987 mAb complexes (including heavy and light chains), color schemes are the same in Fig. 4A and Fig. 4C. (F) The binding free energies of 200 configurations at a 100ps interval from the simulations of the last 20ns. The t-test was conducted to verify the statistical significance of the difference between two binding free energy systems. A p value <0.05 indicates that the difference is statistically significant (95% confidence interval). The color scheme is the same as that in Fig. 4C

The authors also performed simulations of the N439K mutant of human ACE2 complexes with two neutralizing monoclonal antibodies, REGN10987 and CB6. REGN10987 binds to the CR2 and CR3 regions of RBD where N439K is found, while CB6 binds to CR1 and CR2. Analysis indicated that the N439K mutation reduced sensitivity to CB6 antibodies.

Although CB6 could neutralize the N439K mutant, the strain was quite resistant to REGN10987 antibodies. Therefore, as new antiviral strategies based on the Wuhan strain are being developed, given the possible mutations in SARS-CoV-2 that could become resistant to the antibodies developed for this strain, “it is necessary to consider the impact of several mutations on the efficacy of neutralizing antibodies. “, write the authors.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guide clinical practice / health-related behaviors, or treated as consolidated information.