SARS-CoV-2 spike protein with D614G substitution has become the dominant variant in the ongoing COVID-19 pandemic. Several studies to characterize the new virus expressing G614 variant show that it exhibits increased infectivity compared to the ancestral virus having D614 spike protein. Here, using in-silico mutagenesis and energy calculations, we analyzed inter-residue interaction energies and thermodynamic stability of the dominant (G614) and the ancestral (D614) variants of spike protein trimer in closed and partially open conformations. We find that the local interactions mediated by aspartate at the 614th position are energetically frustrated and create unfavourable environment. Whereas, glycine at the same position confers energetically favourable environment and strengthens intra- as well as inter-protomer association. Such changes in the local interaction energies enhance the thermodynamic stability of the spike protein trimer as free energy difference (ΔΔG) upon glycine substitution is -2.6 kcal/mol for closed conformation and -2.0 kcal/mol for open conformation. Our results on the structural and energetic basis of enhanced stability hint that G614 may confer increased availability of functional form of spike protein trimer and consequent in higher infectivity than the D614 variant.

The evolution of homologous and functionally equivalent multiprotein assemblies is intriguing considering sequence divergence of constituent proteins. Here, we studied the implications of protein sequence divergence on the structure, dynamics and function of homologous yeast and human SF3b spliceosomal subcomplexes. Human and yeast SF3b comprise of 7 and 6 proteins respectively, with all yeast proteins homologous to their human counterparts at moderate sequence identity. SF3b6, an additional component in the human SF3b, interacts with the N-terminal extension of SF3b1 while the yeast homologue Hsh155 lacks the equivalent region. Through detailed homology studies, we show that SF3b6 is absent not only in yeast but in multiple lineages of eukaryotes implying that it is critical in specific organisms. We probed for the potential role of SF3b6 in the spliceosome assembled form through structural and flexibility analyses. By analysing normal modes derived from anisotropic network models of SF3b1, we demonstrate that when SF3b1 is bound to SF3b6, similarities in the magnitude of residue motions (0.86) and inter-residue correlated motions (0.94) with Hsh155 are significantly higher than when SF3b1 is considered in isolation (0.21 and 0.89 respectively). We observed that SF3b6 promotes functionally relevant ‘open-to-close’ transition in SF3b1 by enhancing concerted residue motions. Such motions are found to occur in the Hsh155 without SF3b6. The presence of SF3b6 influences motions of 16 residues that interact with U2 snRNA/branchpoint duplex and supports the participation of its interface residues in long-range communication in the SF3b1. These results advocate that SF3b6 potentially acts as an allosteric regulator of SF3b1 for BPS selection and might play a role in alternative splicing. Furthermore, we observe variability in the relative orientation of SF3b4 and in the local structure of three β-propeller domains of SF3b3 with reference to their yeast counterparts. Such differences influence the inter-protein interactions of SF3b between these two organisms. Together, our findings highlight features of SF3b evolution and suggests that the human SF3b may have evolved sophisticated mechanisms to fine tune its molecular function.

Mutations in the spike protein of SARS-CoV-2 are the major causes for the modulation of ongoing COVID-19 infection. Currently, the D614G substitution in the spike protein has become dominant worldwide. It is associated with higher infectivity than the ancestral (D614) variant. We demonstrate using Gaussian network model-based normal mode analysis that the D614G substitution occurs at the hinge region that facilitates domain-domain motions between receptor binding domain and S2 region of the spike protein. Computer-aided mutagenesis and inter-residue energy calculations reveal that contacts involving D614 are energetically frustrated. However, contacts involving G614 are energetically favourable, implying the substitution strengthens residue contacts that are formed within as well as between protomers. We also find that the free energy difference (ΔΔG) between two variants is -2.6 kcal/mol for closed and -2.0 kcal/mol for 1-RBD up conformation. Thus, the hermodynamic stability has increased upon D614G substitution. Whereas the reverse mutation in spike protein structures having G614 substitution has resulted in the free energy differences of 6.6 kcal/mol and 6.3 kcal/mol for closed and 1-RBD up conformations, respectively, indicating that the overall thermodynamic stability has decreased. These results suggest that the D614G substitution modulates the flexibility of spike protein and confers enhanced thermodynamic stability irrespective of conformational states. This data concurs with the known information demonstrating increased availability of the functional form of spike protein trimer upon D614G substitution.