3.3. Effect of mutations on thermodynamics parameters and
intrinsically disordered regions (IDRs)
We identified 795 substitutions, 46 deletions and 3 insertion in
SARS-CoV-2 Nsp1 genomic sequences. We found that 69 mutations occur in
17 major residues of the HTH motif identified by us. Among the 69
mutations, 11 mutations were found to occur in more than 10 sequenced
SARS-CoV-2 genomes. We found that 6 out of 11 substitutions in the HTH
motif had a ΔΔGDestablizing < 0 (Figure 5a).
Only one substitution (E159D) had a ΔΔGDestablizing< -1. On the contrary, substitutions such as M174K and T170I
had ΔΔGDestablizing > 1. Overall, ΔΔG
values below zero suggest that the mutation causes destabilization of
the protein; otherwise, it leads to stabilization in protein. We also
found that some of these mutations also lead to changes in
intra-molecular interactions: T170I and M174K, result in a new
hydrophobic interaction; T170I removes a hydrogen bond; M174K forms a
weak hydrogen bond, and M174K leads to form ionic interaction (Figure
5b). These results suggests that the changes in intra-molecular
interactions may be responsible for the increased or decreased stability
of protein.
The vibrational entropy energy (ΔΔSVib) analysis
revealed differences between the wild and the mutated variants. The
vibrational entropy provides an average of the configurational entropies
of the protein structure in single minima of the energy landscape[36,37]. The ΔΔSVib <
0 of mutant type indicates the rigidity of the Nsp1 structure while
ΔΔSVib > 0 shows the flexibility of the
structure. As shown in Figure 5c, we found positive
ΔΔSVib for R175H, L177F, L173P, E159K, and E159D
mutations, suggesting they stabilize the Nsp1 structure. On the other
hand, we found a most negative value of ΔΔSVib for
M174K (~ -2.48), suggesting that it may play a
significant role in destabilizing the structure of Nsp1. Such mutations
can increase or decrease the Nsp1 protein flexibility and affect the
speed and efficiency of its interaction with the ribosome when occuring
at the protein binding site.
In addition to 11 mutations, we identified W161 in the loop of HTH motif
as causing a turn in that motif, and it leads to the helix disruption.
This residue can also cause hydrophobic interactions (with F157) and
stabilize the protein structure. Several mutations are known for this
residue, including W161C, W161S, W161L, and W161R. Our study showed that
W161C has ΔΔGDestablizing < -1, which leads
to instability of the protein structure. Mutation F157S can also affect
the formation or loss of this hydrophobic interaction, which in turn
reduces the stability of the Nsp1 in that region.
As shown in Figure 5d, changes in binding affinity
(ΔΔGbinding) due to point mutations for the 11
residues of the Nsp1 HTH motif were predicted. Positive values
corresponded to N178S, R175H, L177F, M174K, L173P, N178K, F157L, and
D156N (destabilizers) and negative values corresponded to T170I, E159K,
and E159D (stabilizers), which led to decreased and increased binding
affinity of Nsp1, respectively.
Finally, positions 120-180 of Nsp1 were identified as intrinsically
disordered regions (IDRs), as determined by the VSL2 predictor (Figure
6). In line with this result, the PONDR VL3 predictor identified
positions 150-180 (Figure 6), approximately related to the C-terminal
protein. The SARS-CoV-2 Nsp1 positions 124-152 form a loop contains
G127, G130, G132, G133, G137, G146, and G150, which separates the HTH
motif from the rest of the protein.