Page 134 - GPD-4-2

Gene & Protein in Disease Binding of 11q to DENV and WNV proteases

temperature at 300 K and the pressure at 1 atm. During residues and therefore have higher RMSF values. Internal
each step, hydrogen atoms were restrained by applying residues connected to disordered amino acids and residues
the SHAKE algorithm. An integration time step of 2 fs belonging to loop regions also possess higher RMSF values.
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was considered throughout the simulations. To account As these residues are far from the active site, they have
for the long-range electrostatic interactions, the particle– minimal effects on the complex stability. Nevertheless,
mesh Ewald approach was employed, and a threshold none of the terminal residues have RMSF values >20Å,
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of 10 Å was considered to account for the non-bonded and therefore, do not overreact to the solvent environment.
intermolecular interactions. Overall, the residues of WNV protease demonstrated

2.4. Binding free energy calculations higher fluctuations with peaks reaching up to ~18 Å, while
the residues of DENV protease exhibited lower residue
The Poisson–Boltzmann surface area continuum solvation fluctuations across the trajectory, generally remaining
(PBSA) method combined with the molecular mechanics below ~14 Å, with several regions showing noticeably
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energy (MM) of the AMBER 14 program was used reduced flexibility compared to WNV–NS2B–NS3–11q.
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to compute the Gibbs ∆G bind. The last 10 ns of the MD This, along with RMSD values, suggests that DENV
trajectory were used to extract 100 snapshots of each protease adopts a more rigid complex conformation, likely
complex at a pause of 100 ps to compute ∆G bind. For this contributing to enhanced binding affinity of 11q.
purpose, the water molecules and ions were stripped from
3.3. Radius of gyration
the MD trajectories. Equation I was used to compute ∆G bind
as follows: The radius of gyration (Rg) plots (Figure 4) provide insights
into the compactness and overall structural stability of
∆G bind = G cocmplex (minimized) - G protein (unbound,minimized) - G ligand the protein-ligand complexes during the 100 ns MD
(unbound,minimized) (I) simulation. As shown in Figure 4A, the DENV–NS2B–
In this case, ∆G bind represents the binding free energy, NS3–11q complex maintained Rg values ranging between
G complex(minimized) represents the MM/PBSA free energy of the ~16.5 Å and 17.5 Å throughout the simulation. Similarly,
minimized complex, G protein (unbound, minimized) represents the the WNV–NS2B–NS3–11q complex exhibited Rg values
MM/PBSA free energy of the minimized protein following lying between ~15.5 Å and 16.5 Å (Figure 4B). These results
indicate that during the simulations, both the proteases
its release from its bound ligand, and G ligand (unbound, minimized)
represents the MM/PBSA free energy of the minimized adopted a compact structure, and the compactness of the
ligand following its release from the complex. Since normal WNV protease is slightly higher than that of the DENV
mode analysis was not considered, entropy contributions protease. It also indicates that after ligand binding, the
were absent from the ∆G bind. folding structure of the proteases remained intact during
the simulations.
3. Results and discussion
3.4. Solvent accessible surface area (SASA)
3.1. Root mean square deviation (RMSD)
The SASA plots depicted in Figure 4 reveal that the
The RMSD of the protein C atoms of different residues proteases were well exposed to the solvent throughout
α
of the DENV and WNV proteases (Figure 3A), computed the simulations. The SASA values of the DENV protease
by considering the corresponding minimized complexes as were computed to be between ~8,500 Å and 10,000 Å ,
2
2
references, suggests that the protein does not move much suggesting a dynamic but moderate surface exposure
during the MD simulations from its initial conformation. during the simulation (Figure 4A). These variations may
RMSD did not cross 2.5Å, manifesting the protein stability correlate with subtle local conformational rearrangements,
upon ligand binding. If we compare structural variations of possibly to optimize ligand binding. In contrast, the WNV–
the protein C atoms in the WNV and DENV proteases, it NS2B–NS3 protease showed relatively constant SASA
α
is clear that the WNV protease has slightly higher RMSD values (~9,000 Å ) with minimal fluctuations (Figure 4B).
2
variations. This is because the WNV protease adjusts its
conformation to accommodate 11q in its active site and 3.5. The binding of 11q with DENV protease
hence is more flexible compared to the DENV protease. The average MD-simulated structure of the NS2B–NS3–
11q complex belonging to DENV (Figure 5A) reveals that
3.2. Root mean square fluctuation (RMSF) the ligand 11q remained intact in the active site of the
The RMSF of the protease residues (Figure 3B) suggest that protease throughout the simulations. Notably, the head
the terminal residues are more flexible than the internal benzyl group of 11q remained consistently anchored in

Volume 4 Issue 2 (2025) 5 doi: 10.36922/gpd.8293