D614G is located at the hinge region of the spike protein
The conformational transition between closed and open states of spike protein is mediated by large scale motions of RBD. Comparison of closed (PDB code: 6VXX) and 1-RBD up conformation (PDB code: 6VYB)  of spike trimer structures shows that apart from RBD that undergoes large movements, NTD and CTD2 domains have considerable Cα deviations with RMSD values of 1.6Å and 3.4Å, respectively (Fig. 1A). However, CTD3 that clasps the position 614 in the S1 region superposes well (RMSD 0.6Å), hinting that it likely facilitates domain-domain motions. To understand the association of 614th position to the domain motions, we performed GNM-NMA for spike protein trimer. In the Gaussian modes, hinges have the characteristics of crossover of displacement profiles from negative to positive or vice versa. The slowest Gaussian modes of protomers in spike protein trimer representing closed and 1-RBD up conformations show that displacement profiles of CTD3 are around zero, indicating the domain is steady while the tethered NTD and RBD exhibit notable displacements (Fig. 1B). Consistently in both conformations, displacement profiles crossover around Lys310-Phe318 and Gly593-Val618 in the S1, indicating they serve as hinges (vertical cyan bars in Fig. 1B). The hinge-1 (Lys-310-Phe318) mediates NTD-RBD motions while the hinge-2 (Gly593-Val618) mediates domain motions between RBD and S2 region (Fig. 1B). Notably, hinge-2 harbours the position of dominant D614G substitution. Given that glycine is a highly flexible residue, the replacement of aspartate to glycine potentially influences the flexibility of hinge-2, which aids the RBD-S2 motion essential for the conformational transition.
D614G substitution strengthens intra- and inter-protomer interactions
Further, we probed the effect of glycine substitution on the energetics of local inter-residue contacts. This can be quantified as frustration of a residue or inter-residue contacts. We calculated frustration index of residues and inter-residue contacts for two variants of spike protein viz. aspartate or glycine at the 614th position. The result shows that frustration index of aspartate in the spike protein (SD614) is -1.25, -1.25 and -1.30 for three protomers in the closed conformation (red lines in Fig. 2A). The frustration index of aspartate in the 1-RBD up conformation is -1.24, -1.31 and -1.28 for three protomers (red lines in Fig. 2B). Hence, in both the conformations aspartate is highly frustrated. Conversely, in glycine variant (SG614), the residue is neutrally frustrated with frustration index of -0.48, -0.42 and -0.46 for protomers in the closed conformation and -0.50, -0.35 and -0.37 for protomers in the 1-RBD up conformation (blue lines in Fig. 2). Recently, structures of spike protein with D614G substitution were released in PDB (November 4, 2020) . When we analyze them, we found that glycine has frustration index of -0.55, -0.19 and -0.88 for protomers in closed conformation and -0.75, -0.39 and -0.31 for protomers in 1-RBD up conformation (PDB codes: 7KDK and 7KDL). These result imply that residue frustration at the 614th position has become neutral upon glycine substitution.
In the spike protein of both conformations (SD614), aspartate is involved in intra-protomer contacts (with residues Ser591, Gly593 and Gly594) as well as in inter-protomer contacts (with Asn616, Arg646, Ser735, Thr859 and Pro862) through direct, long-range electrostatic or water-mediated interactions. The mutational frustration index indicates that all the 8 contacts are highly frustrated (Fig. 3, top panel). However, in the closed conformation of SG614 variant, glycine contacts with Leu611 and Cys649 of the same protomer and Pro862 of the adjacent protomer. Except inter-protomer contact through Pro862, the other intra-protomer contacts are minimally frustrated (Fig. 3, top left panel). Likewise, in the 1-RBD up conformation of SG614 variant, glycine has the same contact pattern, as observed in the closed conformation in addition to contact with Asn616. Of these four contacts, two contacts involving Leu611 and Cys649 are minimally frustrated (Fig. 3, top right panel). Similar observation is seen for mutational indices of contacts involving glycine at 614th position in the cryo-EM structures of D614G variant of spike protein (PDB codes: 7KDK and 7KDL, Supplementary Table S1A). Overall, the number of highly frustrated contacts are reduced upon aspartate to glycine substitution.
Next, we calculated configurational frustration index that indicates how favourable the native contact between two residues relative to other possible contacts those two residues can have. Results show that in the closed conformation, aspartate (SD614) has one minimally frustrated contact with Arg646 (Fig. 3, left bottom panel). Whereas, glycine (SG614) has six minimally frustrated contacts with residues Ser591, Gly593, Asn616, Thr645 and Arg646 of the same protomer and Thr859 of a preceding protomer in the clock-wise direction (Fig. 3, left bottom panel). Similar trend is seen for 1-RBD up conformation in which aspartate (SD614) has a highly frustrated contact with Gly593 while glycine (SG614) has the same contact but minimally frustrated besides three minimally frustrated contacts with other residues (Thr645, Arg646 and Thr859) (Fig. 3, right bottom panel). These observations are common among three protomers present in the spike protein trimer and corroborate with configurational indices calculated from cryo-EM structures of spike protein having D614G substitution (Supplementary Table S1B). Hence, glycine has more favourable contacts than aspartate. Overall, calculations of single residue, mutational and configurational frustrations reveal that glycine substitution modifies local interaction energy in the favourable direction.
D614G substitution increases the thermodynamic stability of spike protein trimer
If the reduction of frustration in the local interaction energies is significant upon aspartate to glycine substitution, it can have an influence on the thermodynamic stability of the spike protein trimer. To examine this, we calculated difference in the total free energy of trimer between SD614 and SG614 variants using FoldX package . Results show that the free energy difference (ΔΔG) is -2.6 kcal/mol for the closed conformation and -2.0 kcal/mol for the 1-RBD up conformation. To affirm this, we performed reverse mutation in the cryo-EM structures of spike protein with D614G substitution (PDB codes: 7KDK and 7KDL). Result shows that the energy differences are positive values i.e., 6.6 kcal/mol and 6.3 kcal/mol for closed and 1-RBD up conformations respectively, indicating destabilizing effect of reverse mutation. These energy differences are higher than the reasonable threshold value of ±0.5 kcal/mol . Hence, the stabilizing effect of glycine substitution in local environment significantly increases the overall stability of spike protein trimer. Together, these results imply that enhanced stability of SG614 may increase the availability of functional form of spike protein trimer and consequent in higher infectivity compared to the SD614 as observed in the recent experimental studies [15,16,19,24].