Omicron Flap Structure and Dynamics.
The stability of the isolated RBD associated with its ½ β barrel motif creates an isolated stable structure that has led to its use as highly effective COVID-19 vaccines12. Here the flap dynamics of an isolated RBD are studied beginning with the RBD, Up-Chain conformation (residues C336-L518) of the trimeric spike protein parsed from published experimental structure files in a “One-Up” conformation [PDB ID: 7THK13 (Omicron, BA.1), 7WK314 (Omicron, BA.1), 6Z9715 (WT), 6VSB16 (WT)]. In addition to its use as vaccine, studying the isolated Up-chain RBD allows for a more focused study on dynamic behavior and also correlation of the RBD binding to ACE2 using experimental structure files, which often consider the RBD in isolation. These starting structures are studied over a relatively long 0.3 to 0.6 microsecond time period abstracting static and dynamic information to ascertain key differences between WT and Omicron RBD. For the sake of completeness, we note that the “Down State” RBD and its interactions with its remaining S1 and S2 domains as well as neighboring chains have been studied previously17 and are not considered here.
Figure 2 and Supplementary 1 (S1) show the RMSF values across four different structure files (2 Omicron, 2 WT) after a 0.3 microsecond simulation. The enhanced stability of the Omicron flap domain over WT is evident.
Similar RMSF results were obtained using PDB ID’s: 6VSB16 and 7WK318 (Supplementary 1), although parts of the loop or flap in 6VSB are missing residues that were added prior to simulation, as discussed in the Methods Section. Figure 3 shows the corresponding C-alpha positions from Fig. 2 across the entire RBD over the last 50 nsec. of MD simulation for both WT and Omicron RBD. The more diffuse vector positions in the flap domain are clearly shown for WT compared to Omicron. The open flap conformation of Omicron is maintained throughout the entire 0.3 microsecond simulation. (Supplement 2)
To further illustrate the orientational stability, the order parameter, which is the plateau value of the orientational correlation function for the backbone residue NH vectors, is shown in Fig. 4. The order parameter is similar in both cases with the important exception of the turn domains with mutations N440K and N501Y demonstrating enhanced rotational stability for Omicron over WT.
Intra-loop and loop-RBD interactions
We conducted dominant energetic mappings, as described earlier, in order to pinpoint the precise new residue interactions that lead to the more stable internal translational and orientational state of Omicron RBD (Figs. 5 and 6). The stability of Omicron’s RBD is shown to be associated with increased interactions among the residues of the loop domain (intra-loop interactions: residues N439-S477 mapped to residues G485-H505) and loop-RBD interactions (residues C336-C432 mapped to residues N439-L518) (Figs. 5 and 6, respectively). The intra-loop interactions include three new hydrogen bonds between Y475 and E485, L455 and P491, and Y453 and R493 (Supplemental 3). The loop-RBD interactions include new hydrogen bonds between residues N422-R454, D420-L461, L424-F464, E406-Y495, and a significantly enhanced loop domain residue R466 interaction with residues A352-N354. This latter interaction is highly conspicuous in the RMSF data, as specifically marked in Fig. 2 and plays a key role in maintaining the flap open state of Omicron’s RBD.
It is interesting that none of the ten RBD primary binding site mutations of Omicron are directly associated with internal energy interaction increases shown in Figs 5 and 6. To pinpoint conformational changes, the average structures of Omicron and WT after 0.3 microseconds of total simulation were superimposed as shown in Fig. 7.
The key conformational differences are the enhanced beta strand in the ½ beta barrel structure due to S373P mutation and a slightly enhanced helix associated with N440K that creates a new helical “staple” N437-K440. Note that the mutation K417N was not demonstrated to increase or decrease the helicity of its associated WT structure.
Recently, using Atomic Force Microscopy (AFM) and mutational screening, the mutation S373P was experimentally shown to be primarily responsible for an observed overall, significant increase in the biomechanical stability of the isolated Omicron RBD19. The enhanced β strand increases the stability of the ½ β-barrel structural motif and the overall mechanical stability of the Omicron RBD consistent with our observations here.
In addition to the importance of S373P, mutation E484A may help enhance the open flap conformation along with its enhanced ACE2 binding (Table 1.) Specifically, the mutation E484A involves a significant Ramachandran angle transitions from a β sheet domain to an atypical left-handed alpha helix as shown in Fig. 8. Left-handed helices, although unusual, have been linked to stability and ligand binding20. It is observed that F486 and N487, which immediately neighbor the disulfide bond C488-C480, transition out of their αL state from WT to Omicron, but are not involved in ACE2 binding (Table 1). From a protein engineering perspective, it is interesting to see enhanced stability and binding created in the center of the short disulfide region via the E484A αL transition mutation.
We also examined ACE2 bound state to both WT (PDB ID: 6M1721) and Omicron (PDB ID: 7T9L22) to investigate conformational differences. As shown in Fig 9A., the bound state conformations of the RBD are nearly identical between WT and Omicron. Additionally, as shown in Fig. 9B, the free Omicron structure after 0.3 μsec. simulation is nearly identical to these bound state conformations.
The alignment observed for the bound state structures was further verified with independent experimental structure files WT PDB ID: 6M0J23 and Omicron PDB ID: 7U0N24, as shown in Fig. 10.
It appears possible, therefore, that Omicron RBD may have improved “fitness”, in addition to increased binding energies, due to a more biomechanically stable conformation with a likeness to the bound state structures of the RBD to ACE2. Of course, the need to create stable antigens was a critical part of the development of a spike protein antigen (so-called S2P in its prefusion state) as vaccines to the coronavirus family of viruses16,25. Here we have shown one possible “roadmap” to developing stable antigens by examining protein conformations in the bound state form of the protein and the subsequent study of site directed mutations necessary to stabilize this structure in its free, unbound state. The results shown here for the more stable and bound-state like Omicron RBD are also consistent with the cross-reactivity demonstrated for Omicron based vaccines to the previous WT strain26,27.
Note Added in Proof: During the period of this study, a plethora of Omicron sub-lineages have appeared in global circulation as noted in the Introduction section. Currently, at the submission of this manuscript, the XBB.1.5 and JN.1 variant are the dominant circulating Omicron sub variants. The RBD mutations associated with these: S486P and the FLIP mutations L455F and F456L are directly associated with the dominant stabilizing interactions given in Figs. 5 and 6. More study is needed to determine if these “new” mutations represent additional conformational stability enhancing mutations for the Omicron RBD as hinted by this study.