Molecular dynamics simulation in aqueous solution of the isolated peptides and BDNA
Before simulating the interaction between the different peptides and BDNA, the individual
model structures were relaxed along independent 200-ns simulations, performed in triplicate
(Table 1). To that end, the homology models obtained for the peptide structures were
first examined for overall quality. The Ramachandran plot for CLFampin, CLFcin and
CLFchimera revealed that 93.3%, 100.0% and 93.5% of the residues were situated within
the most favored region, respectively, whereas the remaining residues were found within
the additional allowed region.
Structural fluctuation analysis
Root-mean-square deviations from the initial structure of the peptide as a function
of simulation time and root-mean-square fluctuations of peptide residues are presented,
for one of the 200-ns replicates, in Figure 1. The behavior of these quantities in
the remaining replicates is consistent with the observations made here (see Additional
file 1: Figure S1 and Additional file 2: Figure S2). The RMSD values are stable after
the initial 100 ns, the larger peptide CLFchimera showing higher RMSD and RMSF values.
CLFchimera was obtained from the C-term-C-term fusion of CLFampin 265-284 and CLFcin17-30,
using a lysine (Lys21) as linker [
7]. Figure 1B shows that the global fluctuations of the corresponding sequences in
the shorter peptides are lower in general than in the fusion peptide, as expected
in light of the structures shown in Figure 1C. It is worth noting that the shorter
CLFcin adopts a more stable helical structure than CLFampin when isolated in solution,
to become more flexible in the fusion peptide. Structures from the stable part of
the 200 ns simulations with all residues in the most favored regions of the Ramachandran
plot were used as initial structures for the corresponding simulation of peptide-DNA
Figure 1. Structural fluctuation analysis. (A) RMSD as a function of time; (B) RMSF per residue; (C) Cartoon structure of CLFchimera
(C1), CLFampin (C2) and CLFcin (C3) at 0, 100 and 200 ns (red, blue and green, respectively). RMSD and RMSF quantities were computed for structures at 0.1-ns intervals from the
200-ns simulations after least square fitting to the initial structure using the backbone
Molecular dynamics simulation of the peptide-DNA systems
Simulations between CLFchimera, CLFcin and CLFampin and BDNA were performed for 200
ns in triplicate. To construct the system, the peptide was introduced in the BDNA
box at a random position and orientation. Center of mass distance (COM), hydrogen
bonds, salt bridges and contacting surface area between peptide and DNA were analyzed.
Center of mass distances
The center of mass distance between peptide and DNA was calculated as a function of
time (Figure 2A). Side view of snapshots of the first and last configurations are
shown in Figure 2B. In all three replicates, COM distances were initially around 3,
3 and 4 nm for CLFcin, CLFampin and CLFchimera, respectively. The peptides instantly
moved toward the DNA grooves and COM distances decreased rapidly. The three replicates
show some differential behavior in terms of final distance and convergence (Additional
file 3: Figure S3A and Additional file 4: Figure S4A), as well as in terms of position
and orientation (Additional file 3: Figure S3B and Additional file 4: Figure S4B),
suggesting that the binding is not specific, as demonstrated further below.
Figure 2. COM distance analysis. (A) COM distances between CLFcin, CLFampin and CLFchimera and DNA along 200 ns. (B)
Structures at times t=0 (cyan) and t=200ns (purple): (B1) CLFcin-DNA, (B2) CLampin-DNA,
and (B3) CLFchimera-DNA.
Number of hydrogen bonds and salt bridges
The number of hydrogen bonds between peptide and DNA showed significant variation
during simulation (Figure 3; Additional file 5: Figure S5 and Additional file 6: Figure
S6). The average number of hydrogen bonds in the second half of the three simulation
replicates (100-200 ns, 300 ns in total) was 5.66±0.23, 4.61±0.55 and 2.63±0.27 for
CLFchimera, CLFcin and CLFampin, respectively (see also Additional file 7: Table S1
for details), suggesting that CLFchimera establishes more stable interactions with
A representative snapshot of the CLFchimera-DNA interaction is illustrated in Figure
3D. In this frame, it can be seen that hydrogen-bonding interactions are mainly established
between positively charged residues of the peptide and the DNA-backbone phosphate
groups, which constitute also salt bridges.
Figure 3. Number of hydrogen bonds with DNA as a function of simulation time (200
ns). (A) CLFampin, (B) CLFcin, (C) CLFchimera. D: Snapshot at t = 135 ns of the CLFchimera-DNA system, indicating hydrogen bonds (red lines) and salt
bridges (yellow dashed).
Salt bridges also play a fundamental role in protein-ligand interactions [
</a>, <a href="#_ENREF_33">
</a>]. In several studies, a cutoff of 4 Å between N-O atom pairs has been used to define
salt bridge formation [<a href="#_ENREF_34">
34</a>, <a href="#_ENREF_35">
35</a>]. Here, we calculated salt bridges between P atoms from the nucleic-acid backbone
and N atoms from Lysine and Arginine residues, and thus used 5 Å as cutoff. The average
number of salt bridges in the second half of the three simulation replicates between
DNA and CLFchimera, CLFcin, CLFampin were 4.09±016, 3.17±0.28 and 1.71±0.44 (see Additional
file 7: Table S1 for details). Again, CLFchimera establishes more salt bridges with
DNA than the other two peptides.</p>
Contacting surface area
The solvent-accessible surface area was calculated with the Gromacs library [
36]. The contacting surface area can be then calculated using the following formula:
CSA = (SASA Peptide(s) + SASA DNA – SASA Peptide(s)-DNA)/2 [
37]. Initially, the CSA was close to zero due to the distance between peptides and DNA.
The evolution of the CSA is shown in Figure 4 for one of the simulation replicates
(see Additional file 8: Figure S7 for the other two). In all three replicates, the
CSA is stable after the initial 100 ns, indicating a stable interaction has been reached.
The average CSA in the period 100-200 ns is 5.92±0.41, 4.9±0.1, and 4.76±0.36 nm2 for the CLFchimera, CLFcin and CLFampin systems, respectively (see Additional file
7: Table S1 for details). The CSA is higher for CLFchimera than for the other two
peptides, in line with the observed interactions.
Figure 4. Contacting surface area between peptide and DNA along a 200 ns MD simulation.
MM/PBSA binding free energy estimate
The binding free energy was estimated using the MM/PBSA method. The results for the
period 100-200 ns in one of the replicates are presented in Table 2. As indicated
in the Methods section, particularly for this type of systems (high charge density),
the single-trajectory MM/PBSA approach represents a very crude estimate of the binding
free energy that, most certainly, severely overestimates the real value. Nevertheless, the calculations will be used here to qualitatively compare and rank
the different systems, which should be relatively safe given that the nature of the
interactions is the same in all cases. The results indicate that CLFchimera has the
lowest DNA-binding energy. The plot of the binding free energy along the period 100-200
ns in one of the replicates is shown in Figure 5 (see Additional file 9: Figure S8
for the other two replicates). No significant differences in the obtained binding
free energy values were observed among replicates (-786±2.545, -731±3.521 and -712±7.801
kJ/mol for CLFchimera; -340±4.437, -352±4.437 and -316±7.215 kJ/mol for CLFcin; -71±3.063,
-78±5.103 and -62±2.202 kJ/mol for CLFampin).
Table 2. Binding free energy for the three peptide-DNA systems calculated by the MM/PBSA
method (one simulation replicate).
Figure 5. Estimated binding free energy for the peptide-DNA systems. Calculated with the MM/PBSA method on the 100-200 ns period of one of the simulation
The free energy values for the CLFchimera-DNA system were decomposed into residue
contributions using the MmPbSaDecomp.py python script. The results, presented in Figure 6 for one of the simulation replicates,
indicate that residues LYS5, LYS9, LYS13, ARG16, LYS18, ARG27, LYS34 and LYS35 are more relevant
for binding. On the other hand, GLU12 and SER36 have a detrimental effect. The contributions
in the other two simulation replicates follow the same trends (Additional file 10: Figure S9).
Figure 6. Contribution to DNA binding free energies of amino-acid residues in CLFchimera.
Previous experimental studies revealed that substitution of positively charged residues
such as LYS269, LYS277 and LYS282 with alanine in bovine Lactoferrampin (LYS9, LYS13
and LYS18 in CLFchimera) resulted in a dramatic decrease in antimicrobial activity
39], a finding consistent with our in silico results (Figure 6). However, Karn et al. (2006) showed that substitution of GLU276
(GLU12 in CLFchimera) with glycine in bovine Lactoferrampin had no effect on increasing
antimicrobial activity [
38]. Several experimental studies regarding bovine Lactoferricin indicated that the
core hexapeptide “RRWQWR” in this peptide has a significant role in antimicrobial
40]. The first two amino acids from this central core in CLFchimera (ARG27 and ARG28)
made a considerable contribution to the interaction with DNA in our simulations (Figure
6); however, they were not as effective as other positively charged residues. Investigation
of minimum distances (averaged over the three replicates) showed that LYS5 and LYS35
were closest to DNA, 0.13±0.03 nm and 0.12±0.02nm, respectively (see Additional file 11: Figure S10).
As shown in Figure 6, GLU12 and SER36 play a major inhibiting role in the interaction
with DNA. Figure S9 shows that they displayed also the largest minimum distance to
DNA, with 0.64±0.13 nm and 0.57±0.99nm, respectively. This indicates that these residues
have a negative contribution to binding despite being the least close to the DNA.
Effect of CLFchimera concentration on DNA binding
To understand the effect of peptide concentration on DNA binding, we performed simulations
with 1, 2, 3 and 4 CLFchimera molecules and one DNA helix.
Number of hydrogen bonds and salt bridges
As expected, when peptide concentrations rose, the number of hydrogen bonds between
DNA and peptides increased but showed saturation (Figure 7 for one of the replicates
of each system; Additional file 12: Figure S11 and Additional file 13: Figure S12 for the other two replicates). The average number of hydrogen bonds in
the 100-200 ns period of the three replicates was 5.66±0.23, 9.66±0.56, 11.75±0.45, and 12.4±0.37 for 1, 2, 3 and 4 peptides, respectively (see Additional file 7: Table S1 for details). The corresponding average number of salt bridges was 4.09±0.16, 5.79±0.72, 6.50±0.24,
and 7.03±0.06, respectively (see Additional file 7: Table S1 for details). The largest change in the number of hydrogen bonds and salt bridges occurs in the
transition between one and two peptides, and shows saturation after three peptides.
Figure 7. Number of hydrogen bonds with DNA at different concentrations of CLFchimera.
(A) 1-CLFchimera, (B) 2-CLFchimera, (C) 3-CLFchimera, (D) 4-CLFchimera.
The average percentage of hydrogen bonds involving the DNA phosphate group revealed
that nearly all hydrogen bonding was formed between peptide side chains and DNA phosphate
groups (see Additional file 14: Table S2). As these groups are equal in all nucleic-acid
bases, we may conclude that the binding of CLFchimera to DNA is DNA-sequence unspecific.
For this reason, a potential DNA-related antimicrobial activity of this peptide could
consist in the disruption of replication. Uyterhoeven et al., 2008 reported that Buforin
II, another antimicrobial peptide, would probably target nucleic acids in a non-sequence-specific
14]. Sim et al., 2017 also examined the interaction of Buforin II and DesHDAP1 with DNA
and suggested that a large percentage of hydrogen bonds were formed between peptide
side chains and phosphate groups in the nucleic acid backbone (96.3% and 81.7%, respectively).
Then, they demonstrated experimentally that Buforin II and DesHDAP1 did not show signs
of sequence-specific DNA binding [
41]. In another study on a particular AMP derived from Chinese traditional edible housefly
larvae by Tang et al., 2009, it was revealed that the phosphate anion of the DNA double
helix is one of the binding sites in DNA-peptide interaction [
9]. The results of the present study are in accordance with all these previous findings.
On the other hand, an experimental study on the interaction between bovine Indolicidin
and DNA demonstrated that Indolicidin bound tightly to the ds[AT], ds[GC] and ds[AG]
sequences, but formed loose bonds with ds[GT] [
Contacting surface area
Results from the CSA analysis at different concentrations of CLFchimera revealed an
increase in CSA with peptide concentration (Figure 8 for one of the replicates of
each system and Additional file 15; Figure S13 for the other two replicates). The
average CSA (three replicates) for 1, 2, 3 and 4 peptides was 5.92±0.41, 10.30±0.83,
11.79±1.14 and 14.55±1.39 nm2, respectively (see Additional file 7: Table S1 for details). The highest increase
in CSA occurs for the transition between one and two peptides. However, the difference
between 3 and 4 peptides is slightly larger than could be expected from the previous
analysis of interactions, indicating that the increase in contact area is not proportionally
translated in additional specific interactions.
Figure 8. Contacting surface area at different concentrations of CLFchimera.
MM/PBSA binding free energy estimate
As shown in Table 3 (one of the replicates), the CLFchimera-DNA binding energy decreases
with increasing peptide concentration. The decrease in binding energy per peptide
is again largest in the transition from one to two peptides. The results for the different
replicates are in this regard consistent: -946+/-4.504, -927±9.31 and -894±5.056 kJ/mol
for 2-CLFchimera; -1004+/-4.007, -1020±9.23 and -975±8.09 kJ/mol for 3-CLFchimera;
-1027+/-15.718, -1071±12.21 and -1022±10.12 kJ/mol for 4-CLFchimera. The saturation
effect observed on going from three to four peptides could be partially due to repulsion
between the highly charged peptides, in addition to crowding at the sites favorable
for the interaction with DNA phosphate groups.
Table 3. Binding free energy estimated by the MM/PBSA method, for one replicate at
each CLFchimera concentration.
The binding energy contribution per amino-acid residue (Figure 9 for one replicate
and Additional file 16: Figure S14 for the other two) indicates that the residues
with important contributions are the same at the different concentrations. It also
shows that they are primarily responsible for the decrease in binding energy with
Figure 9. Contribution of amino-acid residues to DNA-binding energy at different concentrations of CLFchimera.
Table 2 and 3 demonstrate that complex formation and stability was highly correlated
with electrostatic interaction, as expected. Our MM/PBSA results conform to the results
obtained by Pandey et al., 2018 on similar systems [
A computational study by Khabiri et al., 2017 reported that changes in binding free
energy do not correlate strongly with salt bridges or hydrogen bonding in protein-DNA
43]. Contrary to their results, we show that for our peptide-DNA systems the electrostatic
energy and binding free energy are correlated with the number of hydrogen bonds and
salt bridges (Figure 10), with an R2 between binding free energy and the number of hydrogen bonds and salt bridges of
-0.92 and -0.95, respectively, and an R2 of -0.95 and -0.97 between electrostatic energy and hydrogen bonds and salt bridges,
Figure 10. Trends in the number of hydrogen bonds and salt bridges, and electrostatic,
van der Waals and binding energy in simulations of DNA with CLFampin, CLFcin and different
concentrations of CLFchimera.
Structural characterization of CLFchimera
To assess the conformational heterogeneity of the peptide, clustering analysis was
performed for all simulation trajectories. CLFchimera was thus distributed into 22
clusters. The conformation of the top seven clusters, covering 97% of all structures,
is shown in Figure 11. Clusters one and two comprised 65% of all structures. The analysis
shows that the helical segments of CLFchimera are stable along the simulations, with
major conformational changes affecting the turn between them.
Figure 11. Top seven, most-populated conformations of CLFchimera in the simulations.
Effect of CLFchimera concentration on DNA conformation
Analysis of RMSD and hydrogen bonds between the two strands of the DNA revealed that
the presence of 1, 2 and 3 molecules of CLFchimera had no significant effect on DNA
conformation during MD simulations. However, four copies of the peptide caused DNA
partial denaturation (Figure 12, A and B), significantly increasing the RMSD values
(Figure 12C) and decreasing the number of backbone hydrogen bonds (Figure 13) after
about 50 ns of simulation. This result was observed for two (out of three) replicates
of four copies of concentrations.
Figure 12. Monitoring of BDNA conformation during MD simulation. A: changes in BDNA conformation in the absence of the peptide. B: change of BDNA
conformation with presence of four copies of the peptide. C: RMSD analysis of the
DNA without and with four copies of the peptide.
Figure 13. Analysis of the number of DNA interstrand hydrogen bonds in the presence
of zero and four copies of CLFchimera.
To improve our understanding of the possible intracellular mechanisms of antimicrobial
peptides (AMPs) derived from camel lactoferrin, we planned an in silico study based on molecular dynamics simulation. In this study, DNA was selected as
a well-known intracellular target for AMPs. Overall, the simulation results indicated
that the chimeric peptide CLFchimera has a higher affinity for DNA than its component
peptides CLFcin and CLFampin. It is shown that the interaction between DNA and CLFchimera
follows a saturation curve with increasing peptide concentration, the largest gain
in binding free energy corresponding, for a 12-bp DNA, to the transition between one
and two peptide copies. Saturation is reached after three peptide copies, with four
copies inducing the denaturation of the DNA helix.
Binding free energy analysis revealed several lysine and arginine residues as critical
in the formation of CLFchimera-DNA complexes. It is in fact concluded that electrostatic interactions and particularly salt bridges
play a pivotal role in the interaction between CLFchimera and DNA. Moreover, nearly all hydrogen bonds between CLFchimera and DNA involved the backbone
phosphate groups of DNA, indicating that CLFchimera targets the nucleic acids in a
A conformational clustering analysis suggests that CLFchimera mostly performs its
activity through the interaction of its two α-helices, which maintained their secondary
structure throughout the simulations, with DNA. The outcomes of this study provide
insight into the structural and dynamic aspects of the interaction between Lactoferrin-derived
AMPs and DNA as well as new directions for the design of novel AMPs with intracellular