Exploring Nanobody Inhibitory Mechanism against SARS-CoV-2 Spike Glycoprotein Using Molecular Dynamics Simulations

SARS-CoV-2 consists of a 30 kb single-stranded RNA genome encapsulated by a lipid bilayer and three distinct structural proteins embedded within the lipid membrane: envelope (E), membrane (M), and spike (S). Host cell entry is primarily mediated by homotrimeric S glycoproteins located on the viral membrane. Each S protomer consists of S1 and S2 subunits that mediate binding to the host cell receptor and fusion of the viral envelope, respectively. The receptor-binding domain (RBD) of S1 undergoes a large rigid body motion to bind to ACE2. In the closed state, all RBDs of the S trimer are in the down position, and the binding surface is inaccessible to ACE2. It had been proposed that the S protein needs to transition into a fully open state before it can bind ACE2 bind. In our recent MD simulation study [1], which was performed with the support of COVID-19 HPC Consortium, we showed that switching of one of the RBDs into a semi-open intermediate state is sufficient to expose the ACE2 binding surface and stabilize the RBD in its up position. With the resources provided by the COVID-19 HPC Consortium, we also performed an extensive set of all-atom MD simulations to study the S protein-ACE2 binding interface [2]. We identified an extended network of salt bridges, hydrophobic and electrostatic interactions, and hydrogen bonding between the S protein and ACE2. In silico mutagenesis of a single or a pair of these residues on RBD was not sufficient to destabilize its binding, but reduced the average work to unbind it from ACE2 under force. \n \nBecause RBD makes multiple contacts with ACE2 through an extended surface, small molecules or peptides that target a specific region in the RBD-ACE2 interaction surface may not be sufficient to prevent binding of the S protein to ACE2. Instead, blocking of a larger surface of the CR1 region with a neutralizing antibody or nanobody is more likely to prevent the S protein-ACE2 interactions. Consistent with this prediction, recent studies identified 14 antibodies and 3 nanobodies that have a neutralizing effect against the S protein and block its interactions with ACE2. The mechanism by which these antibodies and nanobodies target RBD and prevent its binding to ACE2 remains to be determined. \n \nBecause nanobodies are smaller than antibodies in size, it is computationally less expensive to study their interactions with RBD using all-atom MD simulations. The most promising nanobodies identified so far are H11-D4 and H11-D4 from llama and Ty1 from alpaca. Because crystal structures of these nanobodies in complex with RBD are available, we have the required starting structural data for our simulations. Our previous simulations showed that the nanobody H11-D4 can bind the RBD in its closed state without showing a steric clash with the remaining S protein structure. Interestingly, docking of the RBD-bound structure of the nanobodies to RBD-ACE2 structure revealed that the nanobodies do not overlap with ACE2 (Fig.1). Yet, these nanobodies interact with RBD residues critical for ACE2 binding. We propose that there could be two alternative mechanisms by which the nanobodies prevent S-ACE2 interactions without overlapping with the ACE2 binding site. First, these nanobodies bind to RBD in its closed conformation and prevent its transition to the semi-intermediate or open-state, thereby blocking access of ACE2. To test this model, we will position the nanobodies near their RBD binding site of the S protein in its closed conformation and determine if it prevents opening of RBD. Second, the nanobodies may more strongly interact with the residues critical for ACE2 binding in the open conformation of RBD, thereby prevent ACE2 binding. We will test this possibility by forming the RBD-nanobody complex in the open conformation and test whether RBD is capable of binding ACE2 while in complex with the nanobody. \n \nIt is also possible that nanobodies strongly interact with RBD and dissociate it from ACE2 after binding. Starting from the crystal structure of RBD-ACE2, the effect of the nanobodies will be investigated by placing them close to their binding pose and subsequently performing MD simulations. We will test whether this disrupts the critical interactions between RBD and ACE2, and points the critical RBD residues to interact with the nanobody. The results of these simulations will reveal the molecular mechanism for the inhibitory effect of the nanobody for ACE2 binding. \n \nAs a second part of the project, we will perform an extensive set of in silico mutagenesis analysis to identify the critical nanobody residues that facilitate the binding of the nanobodies to the S protein. Mutagenesis of these residues and pulling the RBD away from ACE2 will enable us to estimate the free energy of binding and the order of events that result in the unbinding of nanobodies from SARS-CoV-2 S protein. In silico, RBD will be pulled away from the nanobody at velocities comparable to AFM pulling speeds to generate experimentally testable predictions. These low velocities will also enable us to more accurately estimate the binding free energy of native and mutant nanobodies. \n \n References \n \n [1] Gur, M., Taka, E., Yilmaz, S. Z., Kilinc, C., Aktas, U., & Golcuk, M. (2020). Conformational transition of SARS-CoV-2 spike glycoprotein between its closed and open states. The Journal of Chemical Physics, 153(7), 075101. \n \n [2] Taka, E., Yilmaz, S. Z., Golcuk, M., Kilinc, C., Aktas, U., Yildiz, A., & Gur, M. (2020). Critical Interactions Between the SARS-CoV-2 Spike Glycoprotein and the Human ACE2 Receptor. bioRxiv.