- Conformational study of the protegrin-1 (PG-1) dimer interaction with lipid bilayers and its effect

We simulated the Protegrin-1 (PG-1) dimers with different β-sheet arrangements in an aqueous solution and in a lipid bilayer composed of POPC. To create the PG-1 dimer, the two β-hairpins were initially assembled into β-sheets with both antiparallel (turn-next-to-tail) and parallel (turn-next-to-turn) β-sheet motifs in an NCCN packing mode [18]. The monomer β-hairpin conformations were obtained from NMR spectroscopy [3] and our previous PG-1 monomer simulations [22]. Although, the β-sheets of the PG-1 dimer have different monomer origins, the behavior of the β-sheets on the bilayer surface strongly depends on their topology, i.e parallel or antiparallel arrangement which is more important for the lipid interaction. Thus, the energetically stable PG-1 dimer conformation strongly depends on its surrounding environments. While the β-sheet conformations are less stable in the bulk water environment, both the antiparallel and parallel β-sheet conformations of the PG-1 dimer are well preserved at the amphipathic interface of the lipid bilayer, with the dimer interface of the antiparallel β-sheets being more stable. In all cases, the dimer interface is held tightly by at least two intermolecular backbone H-bonds between two cysteine residues at the C-terminal strands from each monomer. As suggested by the experimental observations [3,15], the dimeric β-sheet conformations on the lipid bilayer are stable when the β-sheets are associated with the lipids, since the strong electrostatic repulsion between the β-hairpin monomers can be shaded by the lipids. However, the β-sheet itself has a significant conformational change to avoid the repulsive force due to the neighboring arginine side chains. This leads to a twisted or cross β-sheet structure. Fig. 10 shows snapshots of the β-sheets of the PG-1 dimer at the end of the simulations. It can be seen from the figure that significant twist of the β-sheet conformation, especially at the loop and both termini regions, can be observed for all β-sheets on the bilayer surface.

In this work we wish to compare between different dimer conformations in different environments and to find the connection between the dimer conformation and the activity of the PG-1 dimer in the early stage of the membrane disruption. In this early stage, the PG-1 dimer is in contact with the bilayer surface, preparing the penetration into the hydrophobic core region of the lipid bilayer. Thus, our lipid simulations start with a PG-1 dimer that is initially located at the top of the lipid bilayer without giving any stress to the lipid bilayer. The dimer quickly moves to the amphipathic interface immediately following the start of the simulation. This indicates that our simulation results are independent of the initial dimer location with respect to the interface, since this kind of relaxation takes a very short time. If the PG-1 monomer or dimer is fully embedded in the lipid bilayer, the membrane disruption effects are enhanced. In this case, the initial position and orientation of the peptides in the lipid bilayer are crucial, since the peptides cause hydrophobic mismatch between the lipid bilayer and the PG-1, inducing a highly curved bilayer surface. In this paper, we do not target the dimer insertion into the lipid bilayer, since the timescale for such spontaneous dimer relocation may range from few hundreds of nanoseconds to a microsecond, which is far beyond the timescale of our simulations. In our simulation, the antiparallel PG-1 β-sheets bind within the top leaflet of a lipid bilayer with its apolar surface of the β-sheet containing the four disulfide bonds facing the bilayer surface, closely following the experimental suggestion [3]. Unlike the parallel PG-1 β-sheet with the symmetric β-sheet surfaces, the antiparallel PG-1 β-sheet has the asymmetry of the β-sheet surfaces. The antiparallel PG-1 β-sheet may bind the lipid bilayer with its four disulfide bonds away from the bilayer surface. However, the simulation results for the opposite-facing interaction of the antiparallel β-sheet are not expected to be different from the observations in our current setting, since most charged residues are located at both ends of the β-sheet of the PG-1 dimer and do not alter the charge distribution following the β-sheet flip. Electrostatic interactions between the positively charged residues of the PG-1 dimer and the lipid headgroups play an important role in the peptide/lipid interaction.

The simulations of the PG-1 β-sheets with different monomer β-hairpin conformations have shown that the behavior of the β-sheets on the bilayer surface strongly depends on the topology. For example, parallel β-sheets have smaller values of the peptide order parameter, stronger lipid interaction, and they induce a bilayer disruption effect. In the turn-next-to-turn association of the parallel β-sheet, charge distributions due to the positively charged arginine side chains are separated into two regions, β-turns and both termini. As compared to the flexible motions in the arginine residues in order to avoid electrostatic repulsion at both termini, the dynamic motions of the arginine side chains at the β-turns are stiffened due to the restricted backbone motions, clustering positively charges at the one side of the β-sheet. These confined repulsive forces by the positive charges at the β-turns induce distortion of the β-sheet plane, lying obliquely to the bilayer. As a result, one monomer of the parallel β-sheets interacts with the lipid more strongly than the other and has a larger lipid accessible surface area than the other as seen in Fig. 5 and 6. It seems that in the initial stage of the dimer invasion into the lipid bilayer, the oblique attack by the parallel β-sheets into the lipid bilayer increases the bilayer surface pressure and hence enhances the membrane disruption effects. The aggregation of PG-1 dimers into ordered aggregates accomplishes the membrane disrupting effects via forming a pore/channel in the membrane [20]. Based on our observations, we deduce that the parallel β-sheet is an active candidate to insert into the lipid bilayer and to be the sole repeat motif in the ordered aggregates as suggested by the solid-state NMR experiments [18,19].

In our previous PG-1 monomer simulations [22], PG-1 exhibited different features at the amphipathic interface of the lipid bilayers with different lipid compositions. In the presence of anionic lipids in the bilayer, PG-1 located more closely to the bilayer surface and interacted more strongly with the lipids. The local thinning of the lipid bilayer due to the PG-1 invasion was clearer in the lipid bilayer with anionic lipids. On the other hand, no thinning effect due to the PG-1 monomer was observed in the pure POPC lipid bilayer. In this paper, we do not observe any influence on the POPC lipid bilayer due to the antiparallel β-sheets. However, a slight bending or disruption of the lipid bilayer within the parallel β-sheets can be clearly seen. In fact antimicrobial activity of the peptide would be warranted in lipid bilayers containing anionic lipid. This is also our interest, to use the anionic lipid in the future. To date, many experimental studies have used the POPC bilayer systems [16,17,19]. However, recently, membrane containing anionic lipid has been used to demonstrate the formation of PG-1 pore in the membrane [20].

The model of PG-1 dimerization in the POPC lipid bilayer [17] has suggested that at low peptide concentration the PG-1 monomer likely remains on the bilayer surface and PG-1 dimers are inserted into the lipid bilayer. At high concentration, the dimer fraction increases, and the ordered aggregates form a toroidal pore in the lipid bilayer. However, there was no clear indication where the dimerization of PG-1 takes place to create the β-sheet structure. Our previous simulation showed that the monomer β-hairpin structure of PG-1 is well preserved at the amphipathic interface of the lipid bilayers [22]. Recently, solid-state NMR also showed that the PG-1 monomer is fully immersed in the lipid bilayer [27,28]. Based on these observations, the PG-1 dimerization can occur either on the surface of the lipid bilayer or in the interior of the bilayer, since the PG-1 monomers are populated in both environments. Furthermore, our current simulation showed that the PG-1 dimer in water exhibits a partially folded β-sheet conformation. The formation of the dimer in water implies that the dimerization can occur in many different environments and that as a seed in the formation of ordered aggregates, the PG-1 dimer can directly insert into the lipid bilayer. On the bilayer surface, the β-sheets of PG-1 dimer disturb the lipid order through strong electrostatic interaction between the polar headgroup and the charged side chain of the β-sheet, inducing local thinning, and facilitating dimeric insertion. In the interior of the lipid bilayer, the embedded β-sheet of the PG-1 dimer causes hydrophobic mismatch between the lipid bilayer and the PG-1 dimer, inducing highly curved bilayer surface. In both cases, the PG-1 dimer is responsible for the membrane disruption/thinning effects, irrespective of where the PG-1 β-sheet is located on/in the lipid bilayer. Continued investigations of PG-1 dimers embedded in the lipid bilayer and of ordered aggregates of the PG-1 complex in the setting of the lipid bilayers are being conducted to further our understanding of the complex behavior of membrane disruption.

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