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Article Cite This: ACS Omega 2018, 3, 6056−6065

Insights into Membrane Translocation of Protegrin Antimicrobial Peptides by Multistep Molecular Dynamics Simulations Pin-Kuang Lai* and Yiannis N. Kaznessis*,† Department of Chemical Engineering and Materials Science, University of Minnesota, 421 Washington Avenue SE, Minneapolis, Minnesota 55455, United States ABSTRACT: Protegrin-1 (PG-1) is a cationic arginine-rich antimicrobial peptide. It is widely accepted that PG-1 induces membrane disruption by forming pores that lead to cell death. However, the insertion mechanism for these highly cationic peptides into the hydrophobic membrane environment is still poorly understood at the molecular scale. It has previously been determined that the association of arginine guanidinium and lipid phosphate groups results in strong bidentate bonds that stabilize peptide−lipid complexes. It has also been suggested that arginine residues are able to drag phosphate groups as they insert inside the membrane to form a toroidal pore. However, whether bidentate bonds play a significant role in inducing a pore formation remains unclear. To investigate the role of bidentate complexes in PG-1 translocation, we conducted molecular dynamics simulations. Two computational electroporation methods were implemented to examine the translocation process. We found that PG-1 could insert into the membrane, provided the external electric potential is large enough to first induce a water column or a pore within the lipid bilayer membrane. We also found that the highly charged PG-1 is capable in itself of inducing molecular electroporation. Substitution of arginines with charge-equivalent lysines showed a markedly reduced tendency for insertion. This indicates that the guanidinium group likely facilitates PG-1 translocation. Potential of mean force calculations suggests that peptide insertion inside the hydrophobic environment of the membrane core is not favored. We found that formation of a water column or a pore might be a prerequisite for PG-1 translocation. We also found that PG-1 can stabilize the pore after insertion. We suggest that PG-1 could be a pore inducer and stabilizer. This work sheds some light on PG-1 translocation mechanisms at the molecular level. Methods presented in this study may be extended to other arginine-rich antimicrobial and cell-penetrating peptides.

1. INTRODUCTION Antimicrobial peptides (AMPs) are attracting widespread interest as an alternative to traditional antibiotic treatments and a potential solution for the emergence of resistance to antibiotics.1,2 There are over 2000 AMPs discovered in nature and documented in publicly available databases.3−5 Protegrin-1 (PG-1 RGGRLCYCRRRFCVCVGR-NH2) is a remarkably potent, naturally occurring cationic AMP, which has 18 amino acid residues and forms a β-hairpin structure (Figure 1). PG-1 was first isolated from porcine leukocytes.6 It has two intramolecular disulfide bonds and is rich in arginine (Arg). The atomic structure of PG-1 was determined from NMR experiments, in monomeric and dimeric forms and then in octameric pores inside lipid bilayers.7−9 The antimicrobial mechanism of PG-1 is proposed to be based on pore formation. It is believed that PG-1 binds onto membranes and inserts inside the hydrophobic core of lipid bilayer membranes, where it oligomerizes. Oligomers of PG-1 form pores, through which there is fast, uncontrollable ion transport. The transmembrane (TM) potential collapses rapidly because of this transport, and the ensuing osmotic lysis results in quick bacterial death.10−12 There is a plethora of molecular dynamics (MD) simulations conducted to investigate the mechanism of action of PG-1.10−16 © 2018 American Chemical Society

The majority of previous studies focused on certain stages of the pore formation process, such as early membrane surface binding and dimerization, or on the final pore structure. However, the process of PG-1 membrane translocation remains enigmatic from a molecular point of view because PG-1 has a +7 net charge at physiological pH and is thus not expected to interact favorably with the hydrophobic core of a lipid bilayer. In the experiment, the 13C−31P distances for PG-1 in the lipid membrane were found to be very short, which suggested the formation of bidentate complexes between guanidinium and lipid phosphate groups (see Figure 2).17−20 This complex is also commonly found in other Arg-rich AMPs and cellpenetrating peptides (CPPs).21 Therefore, it was suggested that the cationic Arg residues could drag the anionic phosphate groups as they insert into the hydrophobic part of the membrane, thereby facilitating a toroidal pore formation.17 However, it is still uncertain whether the association energy of a bidentate complex suffices to overcome the energy barrier for membrane insertion. In addition, toroidal pores have also been found in peptide-free systems.22,23 The presence of a bidentate Received: March 14, 2018 Accepted: May 8, 2018 Published: June 5, 2018 6056

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proposed that, provided the peptide is sufficiently charged, the bound peptides on the membrane suffice to trigger electroporation. Herein, we describe a series of MD simulations performed using different techniques. The adsorption of PG-1 on the membrane surface was simulated to confirm the presence of bidentate complexes. In addition, electroporation simulations were conducted to study the translocation process. This protocol has been applied before to study pore formation and transport of small molecules.27−29 Potential of mean force (PMF) calculations were finally conducted to examine the effect of bidentate complexes on the insertion energy barrier.

2. RESULTS AND DISCUSSION 2.1. Binding of PG-1 onto the POPE/POPG Bilayers. To investigate the interactions in the PG-1_POPE:POPG system, we performed three independent MD simulations (Sim 1, Sim 2, and Sim 3) by placing PG-1 2.0 nm above the bilayer surface initially. The simulations were carried out for 50 ns. Figure 3

Figure 1. Structure of PG-1. The arrows represent a β-hairpin structure pointing from the N-terminal to the C-terminal. Arg side chains are in magenta. Disulfide bonds are in yellow.

Figure 3. Distance along the membrane normal (z-axis) between the PG-1 center of mass and the average location of phosphorus atoms in the upper leaflet. Distance calculations are performed using all three sets of simulations for the PG-1 and POPE/POPG system.

illustrates the time trajectory of the center-of-mass distance between PG-1 and the average position of phosphorus atoms on the top leaflet. It can be observed that PG-1 rapidly binds onto the membrane surface in all simulations. Representative snapshots for Sim 2 are provided in Figure 4. During the simulations, PG-1 freely diffuses in the water subphase. We observed that the binding process has several stages. In the beginning (0−5 ns), PG-1 rotates its axis and moves toward the lipids rapidly. It interacts with the bilayer with either the two-terminal side or the β-turn side. Both sides include a cluster of Arg residues, which indicates that the

Figure 2. Schematic of the structure formed between the guanidinium and phosphate groups through combined electrostatic forces and bidentate hydrogen bonding.

complex might not then be necessary or sufficient for pore formation. Nevertheless, the experimental distance measurements showed only a result in the octameric pore state. To better understand the membrane insertion mechanism, intermediate structures and interactions must be investigated.21 There are a host of studies focusing on the translocation of peptides across the membrane. Fuertes et al.24 suggested that membrane-active peptides can serve as inducers and stabilizers of pores, the formation of which may also be induced by mechanical or electrical tensions. Sun et al.25 studied the Argrich CPPs of octa-arginine peptides. They applied external tension on the lipids to induce a pore for peptide translocation. They concluded that octa-arginine peptides can slow down the kinetic rate for pore closure of naturally occurring pores. Miteva et al.26 studied NK-lysin associated to a membrane and

Figure 4. Characteristic snapshots of one PG-1 with POPE/POPG simulation. The Arg residues are represented in sticks. The phosphorus atoms of POPE and POPG lipids are colored in gray and in purple, respectively. Phosphorus atoms within 0.3 nm are highlighted with orange color and their lipid tails are shown in sticks. 6057

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because of the hydrophobic environment of the lipids. This process is well-described by the solubility−diffusion mechanism.30 On the contrary, for hydrophilic molecules and ions, it appears that the formation of a water column or a pore (we use these two terms for contiguous water structures that span the lipid bilayer) can facilitate lipid flip-flop and subsequent transport of polar molecules.31 However, in conventional equilibrium MD simulations, it is challenging to observe pore formation because of the very long simulation time needed. There have been some studies where pore formation was observed, but these studies utilized relatively old force fields found to underestimate the free energy barrier for lipid flip-flop across the membrane.32−34 More recent atomistic simulations using CHARMM36 force fields estimate a much larger energy cost for lipid flip-flop. For example, in recent μs-long simulations, AMPs did not disrupt the lipid bilayer, and no pores were observed.35 It has been suggested that electroporation (i.e., the establishment of a potential across the membrane along with subsequent phenomena) is a reasonable method to induce pore formation. Moreover, there have been reports on the transport of molecules under TM potential using MD simulations. In this study, we apply electroporation to investigate the PG-1 translocation and attempt to link simulations to experimental results. We have carried out a set of simulations listed in Table 1 to investigate the effect of charge imbalanced methods, TM

positively charged residues are essential for the initial contact through electrostatic interactions. In the next stage (5−20 ns), the remaining part of the molecule fluctuates in water until additional Arg residues interact with the lipids. In the last stage (20−50 ns), PG-1 has barely any conformational change and inserts fully into the lipid head groups. In line with the aforementioned results, the lipid contact ratio presented in Figure 5 provides clear evidence that Arg residues

Figure 5. Lipid contact ratio of each PG-1 residue in the simulation. This ratio is defined as the number of nonhydrogen PG-1 atoms within 0.3 nm of POPE or POPG lipids divided by the total number of nonhydrogen atoms for each residue. Results are averaged over the last 20 ns of all simulations.

Table 1. List of Translocation Simulations Performed in This Worka

on both sides of the β-hairpin have the largest number of atoms in contact with the lipids. The average lipid contact is defined as the number of nonhydrogen PG-1 atoms within 0.3 nm of lipids divided by the total number of nonhydrogen atoms. It is noticed that the N-terminal Arg residue has the highest contact. This might be due to the longer length of the N-terminal side compared to the C-terminal side (see Figure 1), which allows the N-terminal Arg residue to insert more deeply into the bilayer. To further examine peptide−lipid interactions, Figure 6 presents the H-bonds formed between Arg residues and lipids.

case

charged residues

Arg6_10e_I Arg6_7e_I Arg6_7e_P Arg6_14e_I Arg6_14e_P Arg0_10e_I Arg1_10e_I Lys6_10e_I

Arg Arg Arg Arg Arg Arg Arg Lys

peptides (P/L ratio) 6 6 6 6 6 0 1 6

(6:160) (6:160) (6:160) (6:160) (6:160) (0:160) (1:160) (6:160)

ΔQ(e)

methods

10 7 7 14 14 10 10 10

ions ions peptides ions peptides ions ions ions

a

Arg represents the wild-type PG-1, while Lys indicates the chargeequivalent mutant. There are three PG-1 dimers in high P/L ratio simulation for a total of six peptides. The three dimers were placed parallel to the membrane surface initially. The charge imbalance can be generated by different numbers of ions (I) or peptides (P) in the two double bilayer systems. The charge imbalance for the peptide method (P) is generated by keeping three dimers in one system and subsequently removing dimers in another systems.

potential, P/L ratio, and bidentate bonds. Each system was simulated three separate times, starting from the same positions but using different initial velocities in the beginning of MD. 2.2.1. PG-1 Translocation under TM Potential. The Arg6_10e_I case displayed PG-1 translocation (Figure 7A) under ΔQ = 10e charge imbalance. At 1.0 ns, a water pore was formed with some lipids reorienting likely to stabilize the pore. The peptide started to translocate and at 2.7 ns, the Arg residue at position 4 (Arg4) reached the bilayer center, followed by the translocation of the N-terminal Arg residue (Arg1). This inserted state was also stable until the end of the 10 ns simulation. In this case, peptide translocation within the membrane was only partial. Furthermore, we observed that a strong bidentate hydrogen bond formed between the inserted Arg residue and lipid phosphate groups during the simulation.

Figure 6. Number of hydrogen bonds formed between the Arg residues and lipids. Results are averaged for all three simulations.

For the three simulations, the mean value of H-bonds in the last 20 ns is approximately 12. On average, each Arg residue forms two H-bonds through the bidentate hydrogen bonding (see Figure 2). 2.2. MD Simulations with TM Potential. Membrane transport of small nonpolar molecules is relatively favorable 6058

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Figure 7. Representative snapshots of the electroporation simulations for different cases. (A) Arg6_10e_I. (B) Arg6_14e_I. (C) Arg6_14e_P. (D) Arg0_10e_I. (E) Arg1_10e_I. (F) Lys6_10e_I. The description of each case is in Table 1.

cases, the system changes rapidly from no pore formation to one translocated dimer, as shown in Figure 7C. To have a finer control of the ΔQ, we focused on the charge imbalance method from ions only. 2.2.3. Effect of TM Potential. Figure 7A,B illustrates the results for Arg6_10e_I and Arg6_14e_I systems, respectively. For the Arg6_14e_I case, a large water pore was formed in the bilayer within several ps, and a few lipid flip-flops occurred. At 0.5 ns, one Arg residue side chain started to protrude into the water pore, and it reached the bilayer center by 1.0 ns. In addition, one PG-1 dimer initially parallel to the membrane near the pore opening rotated to align along the membrane normal. Shortly after the insertion of the Arg residue, the whole dimer translocated into the bilayer and spanned the entire pore. The inserted state remained stable until the end of the 10 ns simulation. In contrast, the Arg6_10e_I case only shows partial Arg insertions for PG-1, and the Arg6_7e_I case shows no pore formation and peptide translocation at all. Clearly, we can observe a TM voltage dependence on the pore formation and PG-1 translocation. 2.2.4. Effect of the P/L Ratio. Previous studies have shown that AMPs adopt two distinct orientations depending on the peptide/lipid ratio. In one, the orientation is parallel (at low ratio) and in another perpendicular (at high ratio) to the bilayer surface.36 The experimentally determined threshold P/L value for PG-1 is 1:30.37 The underlying molecular reasons for this are unclear. In this work, we performed simulations to examine the concentration dependence of PG-1 translocation at low P/

This indicates that the bidentate complex might be crucial for Arg insertion, which is consistent with experimental findings. The TM potential for the Arg6_10e_I allows only one or two Arg residues to translocate at a time. In the following analysis, the Arg6_10e_I case is regarded as a baseline for all other tests. 2.2.2. Comparison of Charge Imbalance Methods. For the Arg6_7e_I and Arg6_7e_P cases, there was no pore formation during 10 ns simulations, indicating that the TM potential generated by ΔQ = 7e was too small. Thereby, no other results are reported here. Generally, if the TM potential is above a threshold value, the pore formation can be observed in a few hundred picoseconds. Figure 7B,C illustrates PG-1 translocation with two different charge imbalance methods, Arg6_14e_I and Arg6_14e_P, respectively. Results were similar with both methods. In both cases, we observe a dimer translocating into the water pore from the membrane surface under ΔQ = 14e charge imbalance. For the Arg6_14e_I case, the TM potential is generated by ions in the water subphase. In contrast, for the Arg6_14e_P case, the TM potential is generated by the charges on the PG-1 itself, suggesting molecular electroporation.26 In other words, the highly charged PG-1 can generate sufficient TM potential to induce pore formation and facilitate subsequent translocation as if an external electric field is applied. This also provides evidence that PG-1 may serve as a pore inducer.24 Removing one peptide dimer from a bath results in a change of ΔQ = 7e per bilayer. From the Arg6_7e_P and Arg6_14e_P 6059

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Figure 8. Change of the charge difference ΔQ across each bilayer in the 10 ns simulations. Charge transfers due to PG-1 translocation are highlighted. The N-terminal Arg is denoted as Arg1, which has +2 charge. The Arg residue at position 4 is denoted as Arg4. All others are due to POPG flip-flop. Results are shown for the Arg6_10e_I, Arg1_10e_I and Lys6_10e_I systems. The points are added every 20 ps.

It has been shown in the experiment that Arg and Lys contribute distinctively to cell uptake for CPPs and antimicrobial potency for AMPs. For example, Mitchell et al. has shown that a synthetic CPP, polyarginine (Arg)n, enters the cell more efficiently than polylysine (Lys)n.39 In addition, Schmidt et al. reported that cryptdin-4 (Crp4), a potent bactericidal peptide, showed severely reduced antimicrobial activity for complete Lys-for-Arg substitution.40 Consistent with these results, our MD simulations provide molecular insight, demonstrating that the difference might be attributed to the impact of bidentate bonds on the propensity of peptide insertion in a lipid bilayer. 2.2.6. Discharging of TM Potential. As described in the Methods section, one drawback of electroporation by the charge imbalance method is that the imbalance is not reset during the simulation. The TM potential drops rapidly after the formation of a water pore and subsequent ion transfer. To focus on the interactions between PG-1 and lipids without a collapsing potential, we have added weak constraints to prevent ions from passing through the water pore. However, lipid flipflop of anionic palmitoyloleoylphosphatidylglycerol (POPG) or PG-1 translocation (i.e., Arg transfer) can also result in the TM potential being discharged. Because the focus of the study is the translocation of PG-1, along with lipid flip-flop, we did not restrict these motions with added constraints. It is instructive to show the change of ΔQ in different cases, as illustrated in Figure 8. One charge transfer will alter ΔQ by 2e. POPG flip-flop occurred within a few ns, and all observed flip-flop events were directed from Bath 1 (with excess negative charge) to Bath 2 (with excess positive charge), (see Figure 12.). Conversely, Arg residues always inserted in the reverse direction. The region of each bath was divided by the bilayer center. Phosphorus and Cζ atoms were used to determine the location of lipids and Arg residues in the baths. As can be observed in Figure 8, the discharging rate was much faster for the Arg6_10e_I system. It took less than 4 ns for the system to be fully discharged. For the Lys6_10e_I system, it took about 6 ns to discharge. The Arg1_10e_I system discharged the TM potential in more than 10 ns. Evidently, the discharging rate is faster for systems with the higher peptide concentration. It is also worth mentioning that the ion constraints implemented in this work could be relaxed and the ion-water swap techniques may be applied to maintain the constant voltage across the membrane. However, care must be taken because all transfers of ions, POPG lipids, or charged residues result in changes of ΔQ. The user could, in principle, reset the charge imbalance for all of these events. In practice, one must always be mindful of the artificial nature of the simulated systems and processes.

L (1:160) and at high P/L ratios (6:160). The high P/L ratio is above the threshold value indicated from the experiment. Figure 7D shows snapshots for the Arg0_10e_I case (pure membrane). With a TM potential, the pure membrane system can also form water pores or columns. We noticed that the pore size shrinks during the time interval between 3.0 and 10.0 ns. The stability of the pore will be discussed later. Figure 7E shows snapshots for the Arg1_10e_I case. At 1.0 ns, small water pores started to form because of the TM potential. At 3.0 ns, lipid head groups redistributed toward the membrane interior. By 10.0 ns, a toroidal-shaped pore developed. However, during these simulations, PG-1 remained bound in parallel to the membrane surface. This is in contrast to the case at the high P/L ratio where PG-1 molecules adopted a perpendicular orientation to the bilayer in an inserted state (Figure 7A,B). PG-1 stayed on the membrane surface for a low P/L ratio, likely because of the fact that the pore was formed relatively far from the peptide. This might be attributed to the stiffness of the bilayer in different domains. As previously shown, PG-1 forms extensive hydrogen bonds with lipids, which could increase the stability of this domain. It may be less likely for lipids in the vicinity of PG-1 to form a water defect, which may later grow into pores. Similar results have been reported by Reigada38 who simulated the electroporation process for heterogeneous bilayers composed of liquid-ordered and liquid-disordered regions. These authors found that pore formation occurs preferably in the disordered region. On the contrary, as the P/L ratio increases, the domain for the peptide-free lipid region shrinks. Under TM potential, pores may form around PG-1, and the ensuing lipid movement may trigger PG-1 translocation. 2.2.5. Effect of Substitution of Arg with Lys. Numerous investigations have been conducted to examine Arg-rich peptides. One particular analysis involved substituting Arg residues with charge-equivalent Lys residues. In our work, we substituted Arg residues in the Arg6_10e_I case with Lys residues in the Lys6_10e_I case. Figure 7F presents representative snapshots of the Lys6_10e_I system. Compared to wild-type PG-1, a striking difference was observed, in that the Lys mutant has a much reduced tendency for insertion. This finding provides compelling evidence that the Arg guanidinium group is key for PG-1 translocation. It lends support to the assumption that bidentate bonds in Arg−phosphate complexes play a significant role in aiding PG-1 insertion into the water pore. Indeed, NMR experiments have shown that Lys−phosphate interactions are less stable than Arg−phosphate interactions.21 This also indicates that PG-1 translocation is not simply driven by the force due to TM potential. 6060

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ACS Omega 2.3. PMF Calculation. It has been proposed that interactions between charged protein residues and lipid phosphate groups or water can provide the energy needed to overcome the insertion energy barrier for charged residues to insert inside the membrane.21 It is therefore interesting to study the thermodynamic driving forces of PG-1 insertion. A total of 3 μs umbrella sampling simulations were conducted to mimic the insertion processes of lipid, PG-1, and the lipid/PG-1 complex (Figure 9). The PMF profiles for these processes were obtained and compared (Figure 10).

It is proposed that formation of transient water pores is the rate-limiting step in the process of TM lipid flip-flop.41,42 Once a pore has been formed, the ensuing lipid translocation takes place on timescales of nanoseconds as observed from our electroporation simulations. We reason that water pore formation can not only increase lipid flip-flop rates but also facilitate the PG-1 translocation. It should be stressed that the insertion processes used for PMF calculations are not directly comparable to those of the electroporation processes. In the latter, a water pore forms first, and lipids reorient themselves before PG-1 insertion takes place. In the former, a water defect is formed as the forced insertion process takes place. It should also be stressed that the presented PMF calculations are of artificially built systems. The definition of the PMF zero level is entirely operational and only pertains to the presented cases. Consequently, these PMFs alone, without reactive trajectory sampling, can offer only very limited mechanistic interpretations. In other words, in the absence of simulations that accurately capture the vast phase space during the entire process of peptides moving from the bulk water subphase onto membranes and then inside the lipid core, any proposal of molecular mechanisms underlying biological phenomena must be considered tentative. Unfortunately, despite amazing improvements in both simulation software and hardware, such simulations are currently implausible. Nonetheless, MD simulations, for all their limitations, are often the only scientifically agreeable approach to capturing the vast complexity of biomolecular interactions in a way fit for forming sound hypotheses. 2.4. Stability of Water Pores with PG-1 Inserted. It is reported from the experiment that membrane pores induced by membrane-active peptides have a much longer lifetime than transient pores induced by thermal energy. Computer simulations also showed that many membrane-active peptides can stabilize membrane pores. This motivates us to study the role of PG-1 in pore stability. Figure 11A shows the dynamics of water pores for the pure membrane system (Arg0_10e_I) after we release the

Figure 9. Illustration of the pulling groups of (A) lipid, (B) PG-1, and (C) lipid/PG-1 complex at the bilayer center. The green spheres highlight the pulling atoms. For (A), the pulling atom is the phosphorus atom. For (B), the pulling atom is the Cζ atom of the Arg. For (C), the pulling atom is the phosphorus atom, while the bidentate hydrogen bonds are restrained by harmonic forces shown in yellow dashes.

Figure 10. PMFs for moving the pulling atom into the center of a POPE/POPG bilayer for the lipid, peptide, and bidentate complex. The PMFs were obtained from umbrella sampling calculations, while the pulling atom was restrained at a varying distance from the bilayer center of mass, in the direction normal to the plane of the bilayer. The error bars were determined by bootstrap analysis.

For the pure palmitoyloleoylphosphatidylethanolamine (POPE)/POPG bilayer, we obtained a relative free energy barrier for a single POPE flip-flop of 81.1 kJ/mol. The relative free energy barrier for a PG-1 Arg residue insertion was 94.9 kJ/mol. Figure 9A,B illustrates the lipid head and the Arg residue at bilayer center, respectively. We notice that there was no spontaneous pore formation. Only a water-filled membrane defect (water column from one side) occurred. The relative free energy barrier for the lipid/PG-1 complex was 124.8 kJ/mol. This free energy is still higher than that for a single lipid flip-flop. The actual timescale required for lipid flipflops ranges between minutes and hours.23 With a higher free energy barrier, it is expected that the timescale for the bidentate complex translocation may take even longer. Consequently, the argument that the bidentate complex facilitates pore formation by dragging phosphate groups as Arg residue inserts into the hydrophobic part of the membrane appears less convincing.17

Figure 11. Representative snapshots of the MD simulations for (A) pure membrane pores Arg0_10e_I and (B) membrane pores with PG1 inserted from the Arg6_10e_I system.

constraints on ions. It is observed that the transient water pore was resealed within 30 ns. In contrast, if there is a PG-1 inserted in a water pore (Arg6_10e_I), the pore remained open for the subsequent 200 ns, as shown in Figure 11B. This is similar to the kinetic stabilization of octa-arginine peptides reported by Sun et al.25 Therefore, we reason that PG-1 is able to stabilize and slow down the kinetic rate of water pore closure. This also provides evidence that PG-1 can serve as a pore stabilizer after insertion.24 6061

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ACS Omega 2.5. Proposed Mechanism for PG-1 Translocation. We have applied a computational electroporation method to study PG-1 translocation. The highly charged PG-1 generates molecular electroporation as if an external electric field is applied. Under conditions of a sufficient TM potential, a water pore or column will form across the membrane. There have been extensive studies on the collective motions of pore formation and lipid flip-flop.41,42 It has been proposed that formation of pores unavoidably results in lipid flip-flop. This is called the pore-mediated lipid flip-flop mechanism. In this study, we found that with the strong interactions of bidentate bonds in the guanidinium−phosphate complex, it is likely that the Arg residues might be dragged by the lipids during the diffusive TM transport through the water pores. This in turn may lead to PG-1 insertion and translocation. The charge-equivalent lysine mutant showed reduced permeability. This shows that PG-1 insertion is not simply driven by strong TM potential, and the bidentate complex may play an important role in translocation. Although we applied electroporation by imposing a charge imbalance to induce pore formation, it should be emphasized that transient pores may form under different circumstances. For example, the mechanical stress created by peptide aggregation is also considered to produce pores to relieve membrane line tension.25,43 In addition, we also showed that PG-1 insertion can stabilize water pores and slow down the kinetic rate of pore closure. We therefore propose that PG-1 may be capable of inducing pore formation by electrical or mechanical tension. The poremediated, lipid flip-flop driven PG-1 insertion process facilitates PG-1 translocation. The inserted PG-1 can further increase the stability of the water pores. The whole process from PG-1 inducing to stabilizing water pores may form a feedback loop, thereby highlighting a great example of collective motions in biological membranes.

4. METHODS AND THEORY 4.1. PG-1 Simulation System Preparation. The solvated peptide−membrane systems for various simulations were constructed using CHARMM-GUI.44−47 The initial structures of the PG-1 monomer and dimer were taken from the protein data bank (PDB IDs: 1PG1 and 1ZY6, respectively.7,8) The bilayer was modeled using the 3:1 mixture of POPE and POPG lipids, mimicking the components of the inner membrane of Gram-negative bacteria.48 There were 120 POPE molecules and 40 POPG molecules in the system. POPE is neutral, while the anionic POPG has a net (−1) charge. TIP3P waters49 were added on each side of the membrane with a thickness of approximately 3 nm. The systems were neutralized by adding potassium and chloride as counter ions. There were 40 potassium atoms, and the number of chloride atoms depended on the number of simulated peptides. To investigate the interactions of PG-1 with the POPE and POPG lipid bilayers, three sets of 50 ns simulations were performed. The PG-1 monomer was initially placed in the water subphase, approximately 2.0 nm away from the phosphorus atoms of the upper membrane leaflet. This system is denoted as PG-1_POPE:POPG. 4.2. Computational Electroporation Setup. The electroporation simulations were carried out to mimic a TM potential and to facilitate PG-1 translocation. The system was set up with a double bilayer configuration by duplicating a second bilayer on top of the original bilayer (Figure 12). PG-1 molecules were initially placed at the equilibrium position on the membrane (approximately 2.2 nm from the bilayer center) as determined by PG-1 membrane simulations described in section 2.1. A TM potential was generated by two different methods. The first one is by swapping ions and waters in Bath 1 and in Bath 2, creating a charge imbalance.50 The second one is to keep PG-1 in Bath 1 and partially remove PG-1 in Bath 2 to create a charge imbalance. The number of chloride is adjusted to maintain a neutral system. The number of ions remain the same for both baths. The former is considered as applying an external electric field, while the latter is regarded as an intrinsic electric field generated by the charges on the PG-1 itself. The charge difference is denoted as ΔQ. The whole system maintained zero net charge. Nine test cases were considered (listed in Table 1) to investigate the effect of four different factors on the PG-1 translocation: (1) the comparison of the two charge imbalance methods. (2) The influence of TM potential was studied by varying the charge difference ΔQ in the double bilayer system. (3) The concentration dependence of peptides was examined by changing the peptide to lipid (P/L) ratio. (4) The importance of the guanidinium group was investigated by substituting Arg with charge-equivalent Lys residues. One important drawback of the charge imbalance method is that the imbalance is not reset during the simulation. Once a charged particle transfers to other sides of the membrane, the system ΔQ changes. A protocol using a swapping method has been proposed to maintain a constant charge imbalance.51 In this procedure, the number of ions in the two solution baths is counted frequently and, if the number differs from the initial setup, a swapping action is carried out: an ion of one solution is exchanged by a water molecule of the other solution bath. However, this method only accounts for the change of charge imbalance due to ion flow. This approach has some limitations in our system. In addition to ions, there are anionic lipids,

3. CONCLUSIONS We have applied various MD protocols to study the interactions between PG-1 and lipids that underlie the peptide translocation process. Arg residues are crucial for PG-1 to bind on the membrane surface. An extensive network of bidentate bonds is formed between Arg guanidinium and lipid phosphate groups. Electroporation simulations illustrate that a strong TM potential results in water pore formation and the disruption of the membrane. The charge on the PG-1 is large enough to create TM potential to induce water pores. Lipid flip-flops and PG-1 translocation then take place in a few ns near the water pore. In addition, a threshold P/L appears to be necessary for the water pores to form in the vicinity of PG-1. Furthermore, the Arg to Lys substitution significantly reduces the insertion propensity, which strongly indicates that the bidentate hydrogen bonds facilitate PG-1 translocation. Moreover, the inserted PG-1 can stabilize water pores. PMF calculations show that the barrier for the bidentate complex is still high for insertion into the hydrophobic environment. We reason that PG-1 might be a pore inducer to facilitate water pore formation, which is a rate-limiting step for lipid flip-flop and for PG-1 translocation. After pore formation, lipids that flip-flop may drag PG-1 by virtue of the bidentate bond strength. The inserted PG-1 serves as a pore stabilizer after translocation. 6062

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window size was 0.1 nm. An umbrella potential of a force constant 3000 kJ mol−1 nm−2 was used for each window to hold the pulling groups together. A production run of 50 ns was performed for each window. The PMFs were evaluated using the g_wham tool in GROMACS.52 4.4. MD Simulation Details. All simulations in this work were conducted using the GROMACS 5.1 package.53 All-atom simulations were run at a temperature of 310.15 K using a Nosé−Hoover thermostat54,55 with a coupling time constant of 1.0 ps. The semi-isotropic pressure coupling method was used with both lateral and perpendicular pressures (both 1 atm) independently coupled to the Parrinello−Rahman barostat56 with a coupling time constant of 5 ps and a compressibility of 4.5 × 10−5. Periodic boundary conditions were employed. The simulation time step was 2 fs. The CHARMM36 force field57 was used for proteins and lipids, and the TIP3P model49 was used for waters. The cutoffs for the van der Waals interactions and electrostatic interactions calculated using the particle-mesh Ewald method58 were both 1.2 nm. The van der Waals interactions were smoothly turned off between 1.0 and 1.2 nm with the force-switching method. The LINCS algorithm59 was used to constrain all of the hydrogen bonds.



AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (P.-K.L.). *E-mail: [email protected] Phone: 651-503-2696. Fax: 612626-7246 (Y.N.K.). ORCID

Figure 12. Schematic representation of the double bilayer system with a charge imbalance ΔQ across each bilayer. The classical 3D periodic boundary condition is implemented. POPG lipids are shown in purple. POPE lipids are shown in gray. Potassium ions are shown in orange. Chloride ions are shown in green. Peptides on the membrane surface are shown in spherical beads. Waters are represented with a semitransparent surface.

Pin-Kuang Lai: 0000-0003-2894-3900 Yiannis N. Kaznessis: 0000-0002-5088-1104 Present Address †

General Probiotics Inc., 1000 Westgate Dr, Ste 122, St. Paul, MN 55114 (Y.N.K.). Notes

The authors declare no competing financial interest.



POPG and cationic PG-1 molecules, in the system. Any POPG lipid flip-flop or peptide transport through the membrane alters the imbalance as well. It is not clear how to best deal with some of these events. In this study, to avoid such situations, we added a weak one-dimensional constraint (force constant 100 kJ mol−1 nm−2) on the ion positions along the membrane normal (z-direction). This constraint still allows ions to diffuse in their original baths but prevents them from passing across the membrane. Although the system could still discharge because of translocation of lipids or peptides, time scales for such events are slow enough for the dynamic events of interest to take place well before the system is fully discharged, as shown in the Results and Discussion section. 4.3. PMF Calculation. To calculate the free energy of PG-1 translocation and to shed light on the importance of the bidentate complex, we performed umbrella sampling simulations for three systems. In system A, the lipid flip-flop free energy was obtained by pulling a phosphorus atom of a single lipid into the bilayer center. In system B, the energy cost of PG1 insertion was calculated by pulling a Cζ atom on an Arg residue into the bilayer center. In system C, the free energy of the combined lipid and PG-1 system was measured by pulling into the bilayer center a phosphorus atom on the lipid that has harmonic constraints on the bidentate hydrogen bonds. The reaction coordinate of the PMF was defined as the distance to the bilayer center along the bilayer normal. The

ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health (grant no. GM111358) and by a grant from the National Science Foundation (grant no. CBET-1412283). We acknowledge computational support from the Minnesota Supercomputing Institute (MSI) and from the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation grant no. ACI10535753. Support from the University of Minnesota Digital Technology Center and the University of Minnesota Institute for Engineering in Medicine is also acknowledged.



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