- Conformational study of the protegrin-1 (PG-1) dimer interaction with lipid bilayers and its effect
Protegrin-1 (PG-1) is composed of 18 amino-acids (RGGRL-CYCRR-RFCVC-VGR) with a high content of cysteine (Cys) and positively charged arginine (Arg) residues. The peptide displays an antimicrobial activity , and thus is a very potent antibiotic peptide . A nuclear magnetic resonance (NMR) study has determined the monomeric structure of PG-1 in solution. The peptide forms a β-hairpin conformation and is stabilized by two disulfide bonds between the cysteine residues . Formation of the two disulfide bonds is crucial for the biological activity of PG-1, since the activity can be restored by stabilizing the peptide structure , and the ability to create pores in the membrane depends on its secondary structure . As a cytolytic peptide, PG-1 shares common features with other antimicrobial peptides. These include (i) membrane disruption by forming a pore/channel that leads to cell death [6,7], and (ii) the cationic nature of the peptide which adapts to the amphipathic characteristics [8-10]. However, PG-1 is distinguished from other antimicrobial peptides in that it adopts a β-sheet motif , while most antimicrobial peptides have an α-helical structure [11-14].
The major role of PG-1 in the microorganism is the formation of an oligomeric structure in the membrane that creates a pore/channel. It has been suggested that the self-association of PG-1 into a dimeric β-sheet can take place in two ways: an antiparallel β-sheet with a turn-next-to-tail association or a parallel β-sheet with a turn-next-to-turn association . These models initially failed to account for the stable β-sheet conformation of the PG-1 dimer, since instability due to the large electrostatic repulsion between the positively charged arginine residues that cluster at the edge of the dimer interface was encountered. However, the β-sheet dimers can be stabilized when they are associated with lipids, since the interaction of the arginine side chain with the polar lipid head can screen the electrostatic repulsion between neighboring arginine residues. A high-resolution proton NMR study has demonstrated that PG-1 forms a dimeric β-sheet structure in the presence of dodecylphosphocholine (DPC) micelles . The turn-next-to-tail association of PG-1 gives rise to an antiparallel β-sheet, in which six intermolecular backbone hydrogen bonds (H-bonds) are formed between the C-terminal β-strands from each monomer. The β-sheet structure is stable, since the interaction of the arginine side chain with DPC micelle dismisses the electrostatic repulsion.
Oligomerization or aggregation of PG-1 dimers into ordered aggregates is responsible for the membrane disrupting effects. It has been reported that PG-1 is immobilized in palmitoyl-oleyl-phosphatidylcholine (POPC) lipid bilayers, suggesting aggregation in the lipid bilayer . The results from NMR experiments using 19F spin diffusion showed that PG-1 dimers are populated in the POPC bilayer, while the dimer fraction is reduced as the peptide concentration decreases . The model of Tang et al. for the PG-1 aggregation from 2D solid-state NMR experiments on PG-1 aggregates in phosphate buffer saline (PBS) solution suggested that a parallel β-sheet in an NCCN (where N and C represent N- and C-termini, respectively) packing mode is the sole repeat motif in the ordered aggregates . The recent rotational-echo double-resonance solid-state NMR experiments determined that the PG-1 dimer structure is a β-sheet in the NCCN organization bound to the POPC bilayer . In the presence of anionic lipid in the bilayers, PG-1 is well inserted and begins to aggregate to form an annular pore, while PG-1 aggregates into β-sheets on the surface of POPC/cholesterol biayers .
The formation of the PG-1 dimer is important for pore formation in the lipid bilayer, since the dimer can be regarded as the primary unit for assembly into the ordered aggregates. Experimental observations for other cytolytic peptides have verified that dimer seeding accelerates the formation of ordered aggregates, such as fibrils made by β-amyloids and prions . However, in order to understand the molecular mechanism for pore formation and the biological activity of PG-1, one still needs to elucidate a number of crucial points: (i) how do the PG-1 monomers interact to form a β-sheet dimer; (ii) where is the β-sheet of the PG-1 dimer created; and (iii) how does the β-sheet of the PG-1 dimer interact with the lipid bilayer on the molecular level. Computer simulations of detailed atomic models can be a powerful approach to the understanding of the complex protein/lipid system and provide information complementary to experimental approaches. In this paper, we performed extensive molecular dynamics (MD) simulations of the β-sheet of PG-1 dimer in explicit water, salt, and lipid bilayers composed of POPC lipids. Our long term goal is to provide a detailed mechanism of the membrane disrupting effects by cationic peptides with β-sheet structure. The PG-1 dimer can have different β-sheet arrangements in the NCCN packing mode. The conformations are stable at the amphipathic interface of the lipid bilayer, which is consistent with the experimental suggestions [3,15]. We observe membrane disruption effects by the β-sheet, depending on the β-sheet arrangement.
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