Potential energy stabilization and RMS deviations indicated the attainment of system equilibrium during the simulation. System equilibrium occurred at 250 ps and coordinates were collected every 0.2 ps between 500 ps and 10 ns during the simulation. Panel A of shows the RMS deviations of DNA in the simulation of the complex (pink) and the simulation of the corresponding uncomplexed or free DNA (blue). Complexed DNA shows a consistently lower average RMS deviation relative to the free DNA (2.05 Å vs. 5.35 Å), suggesting increased flexibility of free DNA. Panel B shows the RMS deviations of bound and free netropsin. In this case, the behavior of complexed netropsin is much closer to that of free netropsin, suggesting that when netropsin resides in the DNA minor groove it experiences relatively unhampered motional freedom.
A. RMS deviations of complexed (pink) and free DNA (blue). B. RMS deviations of complexed (pink) and free (blue) netropsin.
Weighted Average Structures and Netropsin Binding Orientation
shows the weighted average structures for the netropsin/DNA complex and free DNA. The weighted average structures were determined from the coordinate sets whose structures were closest to the calculated average structure. As can be seen, two netropsin molecules are bound in the DNA minor groove. Netropsin1 (Nt1) is shown in yellow and the netropsin2 (Nt2) is shown in green.
Average structures of the netropsin/DNA complex (left) and the free DNA (right) from simulations of complex and free DNA. Hydrogen atoms were removed for clarity.
The schematic to the right in represents the binding orientation of the two netropsins to the DNA. The guanidinium terminus of each netropsin was directed towards the dyad of the oligonucleotide structure in a head-to-head orientation. The schematic on the left in represents the hydrogen-bonding patterns observed during the simulation. During the simulation, any hydrogen bond that existed for more than 5% of the time is presented as a dashed line. As shown, the patterns of H-bond interactions between DNA and the two netropsins were found to be identical.
Hydrogen-bonding patterns (left) and binding orientations (right) of netropsins bound to d(CTTAATTCGAATTAAG)2.
The binding energies of the two netropsins bound to d(CTTAATTCGAATTAAG)2
were calculated using the average potential energies for the complex (EComplex
), free DNA (EDNA-Free
) and free netropsin (ENetropsin-Free
). The resulting netropsin binding energy was calculated according to the following equation:
and found to be −2821.18 kcal (mol)−1
. This result indicated the increased stability of the complex structure relative to the structures of the free components.
It was of interest to evaluate the binding energy of a second netropsin to the complex when one netropsin has already been bound. These calculations were carried out as indicated by the equations below:
Results from these evaluations are shown in . The calculated binding energies of Nt1 and Nt2 are −1365.17 kcal (mol)−1 and −1382.90 kcal (mol)−1, respectively.
Netropsin Binding Energies Determined in the Simulation of Two Netropsins Bound to d(CTTAATTCGAATTAAG)2a
Dynamic Behavior of Netropsin
Netropsin may be considered to consist of four rigid planes connected by three flexible linkers. The planes are indicated in as the colored areas. During the simulation, these planes may rotate relative to each other, as indicated by the angles 1, 2, and 3 defined in . shows the rotation of angles 1, 2, and 3 observed during the simulation.
The left (free netropsin), middle (DNA-bound Nt1), and right (DNA-bound Nt2) columns show the rotations of angle 1 (bottom row), 2 (middle row), and 3 (top row), respectively.
These results indicate that angle 3 shows significant flexibility in free netropsin, while both bound netropsins are much less flexible. This suggests that the DNA site provides some constraint to this portion of the bound netropsin molecule. Angle 2 shows rigidity in both the free and the bound netropsins, suggesting that this region of the molecule is naturally rigid independent of its bound state; this further suggests an element of ligand structural pre-organization that may assist in DNA binding. Free netropsin shows significant movement in angle 1 while the bound netropsins are again much less mobile as observed for angle 3. However, different from angle 3, the motion that is observed in angle 1 suggests a patterned sampling of space by angle 1. Additionally, the pattern of motion in Nt1 is different from that observed in Nt2. Analysis of the simulation trajectories shown in indicated that the guanidinium terminus of Nt2 is often in a downward folded conformation and the guanidinium terminus of Nt1 is in a more extended conformation.
Dynamic behavior of the guanidinium termini of Nt1 and Nt2 (left) and a typical structure with bent conformation of Nt1 and extended conformation of Nt2 at their guanidinium termini, respectively.
Minor Groove Width Fluctuations
As shown in , the width of the minor groove is defined as the closest distance between phosphates along the duplex strand. The schematic on the right in illustrates minor groove widths where the guanidinium (pink) and amidinium (blue) termini of the two netropsins are located. The fluctuation of the minor groove width in these areas is shown in the plots on the left of . As can be seen, the amidinium and guanidinium termini of the two bound netropsins behave differently during the simulation. Particularly, in the case of Nt1 there seems to be some patterned behavior in minor groove width changes, indicated by a decrease in angle 1 around 4000 ps. In the case of Nt2 the changes are more subtle, but the differences in the minor groove width at the two termini of the netropsin appear to diverge as the simulation proceeds.
The width of DNA minor groove is defined (arrows) as the closest distance between two phosphates.
Fig. (8) The arrow lines on the right show the definition of the width of the minor groove at two places where the guanidinium and amidinium termini of the two bound netropsins are located. The plots to the left show the minor groove width fluctuation at the guanidinium (more ...)
The X-ray study [9
] found that the minor groove width contributes to the orientation of a bound netropsin with the guanidium terminus bound to the narrower part in the center of the sequence and the amidinium terminus bound to the wider part at the end of the sequence. Our molecular dynamics simulation results are thus in good agreement with these experimental results. Moreover, it should be noted that while the two netropsins are bound to identical sites in the palindromic DNA, their dynamic behaviors differ. Therefore, it appears that small molecule recognition and binding by DNA may be governed by mechanism(s) that are much more complex than initially anticipated and may represent unexpected challenges in genome-targeted drug design that go well beyond simple sequence readout.