Delta SARS-CoV-2 s2m Structure, Dynamics, and Entropy: Consequences of the G15U Mutation

Abstract

Bioinformatic analysis of the Delta SARS-CoV-2 genome reveals a single nucleotide mutation (G15U) in the stem-loop II motif (s2m) relative to ancestral SARS-CoV-2. Despite sequence similarity, unexpected differences between SARS-CoV-2 and Delta SARS-CoV-2 s2m homodimerization experiments require the discovery of unknown structural and thermodynamic changes necessary to rationalize the data. Using our reported SARS-CoV-2 s2m model, we induced the G15U substitution and performed 3.5 microseconds of unbiased molecular dynamics simulation at 283 and 310 K. The resultant Delta s2m adopted a secondary structure consistent with our reported NMR data, resulting in significant deviations in the tertiary structure and dynamics from our SARS-CoV-2 s2m model. First, we find differences in the overall three-dimensional structure, where the characteristic 90° L-shaped kink of the SARS-CoV-2 s2m did not form in the Delta s2m resulting in a “linear” hairpin with limited bending dynamics. Delta s2m helical parameters are calculated to align closely with A-form RNA, effectively eliminating a hinge point to form the L-shape kink by correcting an upper stem defect in SARS-CoV-2 induced by a noncanonical and dynamic G:A base pair. Ultimately, the shape difference rationalizes the migration differences in reported electrophoresis experiments. Second, increased fluctuation of the Delta s2m palindromic sequence, within the terminal loop, compared to SARS-CoV-2 s2m results in an estimated increase of entropy of 6.8 kcal/mol at 310 K relative to the SARS-CoV-2 s2m. The entropic difference offers a unique perspective on why the Delta s2m homodimerizes less spontaneously, forming fewer kissing dimers and extended duplexes compared to SARS-CoV-2. In this work, both the L-shape reduction and palindromic entropic penalty provides an explanation of our reported in vitro electrophoresis homodimerization results. Ultimately, the structural, dynamical, and entropic differences between the SARS-CoV-2 s2m and Delta s2m serve to establish a foundation for future studies of the s2m function in the viral lifecycle.

Effect of the SARS-CoV-2 Delta-associated G15U mutation on the s2m element dimerization and its interactions with miR-1307-3p

“…A difference in kissing dimer conformation is more probable as the monomers of these two s2m elements have a different shape based on their different migration in the TBM gel (Figure 6 right, arrows 1 and 1'). Moreover, our MD simulation findings show that the monomer s2m G15U is linear, while the original s2m monomer is kinked; this is consistent with this hypothesis (Makowski et al 2023). Despite these observed differences in the s2m G15U kissing dimer versus the s2m reference kissing dimer, we further demonstrate that this mutation does not affect the ability of the viral N protein to convert the s2m G15U kissing dimer to a stable duplex structure, which is the in vivo process of duplex formation.…”

“…Similarly, it is interesting to note that, despite having the same size, the monomer bands for the s2m reference and s2m G15U migrate differently in the TBM gel (Figure 6, right, arrow 1 and 1'), suggesting that they have different monomer conformations in the presence of MgCl 2 . Our MD simulations for the s2m revealed that it has a kinked 3D structure; however, the additional U15-A29 base pair in the upper stem of s2m G15U results in a linear structure that lacks the L-shaped kink, rationalizing the increased migration of s2m G15U through the TBM gel as compared to the original s2m form (Kensinger et al 2022;Makowski et al 2023). Thus, we cannot determine if the single dimer band observed for the s2m G15U in the TBM gel (Figure 6, right, arrow 3) originates from a mixture of linear kissing dimer and duplex conformations which will migrate at the same position, or from the duplex conformation formed spontaneously from an unstable kissing dimer during incubation and migration through the gel (Shetty et al 2010).…”

NMR characterization and ligand binding site of the stem loop 2 motif (s2m) from the Delta variant of SARS-CoV-2

“…These ambiguities, however, do not lead to a wrong secondary structure. In fact, followup MD simulations are in agreement with our dynamic NMR data (Makowski et al 2023).…”