Dimerization of protegrin-1 in different environments

Abstract

The dimerization of the cationic β-hairpin antimicrobial peptide protegrin-1 (PG1) is investigated in three different environments: water, the surface of a lipid bilayer membrane, and the core of the membrane. PG1 is known to kill bacteria by forming oligomeric membrane pores, which permeabilize the cells. PG1 dimers are found in two distinct, parallel and antiparallel, conformations, known as important intermediate structural units of the active pore oligomers. What is not clear is the sequence of events from PG1 monomers in solution to pores inside membranes. The step we focus on in this work is the dimerization of PG1. In particular, we are interested in determining where PG1 dimerization is most favorable. We use extensive molecular dynamics simulations to determine the potential of mean force as a function of distance between two PG1 monomers in the aqueous subphase, the surface of model lipid bilayers and the interior of these bilayers. We investigate the two known distinct modes of dimerization that result in either a parallel or an antiparallel β-sheet orientation. The model bilayer membranes are composed of anionic palmitoyl-oleoyl-phosphatidylglycerol (POPG) and palmitoyl-oleoyl-phosphatidylethanolamine (POPE) in a 1:3 ratio (POPG:POPE). We find the parallel PG1 dimer association to be more favorable than the antiparallel one in water and inside the membrane. However, we observe that the antiparallel PG1 β-sheet dimer conformation is somewhat more stable than the parallel dimer association at the surface of the membrane. We explore the role of hydrogen bonds and ionic bridges in peptide dimerization in the three environments. Detailed knowledge of how networks of ionic bridges and hydrogen bonds contribute to peptide stability is essential for the purpose of understanding the mechanism of action for membrane-active peptides as well as for designing peptides which can modulate membrane properties. The findings are suggestive of the dominant pathways leading from individual PG1 molecules in solution to functional pores in bacterial membranes.

Keywords: dimerization; potential of mean force; protegrin.

Figure 1

Structure of the PG1 dimer in the parallel β-sheet arrangement, in an NCCN packing mode. The peptide backbones are shown as yellow ribbons. The solution Na⁺ and Cl⁻ counterions are shown as red and blue spheres, respectively. The peptide residues Arg and Cys are also shown as sticks. Water molecules have been removed for clarity.

Figure 2

A snapshot at the end of a 4 ns simulation of the PG1 dimer in the parallel β-sheet arrangement inside the membrane. Peptides are shown in blue NewCartoon representation. Water is shown as van der Waals spheres. The solution Na⁺ and Cl⁻ counterions are shown as small yellow and large green spheres, respectively. The peptide Cys and Arg residues are shown as sticks. The sidechain atoms of the other residues and the bilayer lipid atoms are omitted for clarity.

Figure 3

The potential of mean force, W(D), as a function of the distance between two peptides for the PG1 dimer in parallel (solid line) and antiparallel (dottet line) β-sheet arrangements in (a) water, (b) on the membrane surface. Each data point for W(D) represents the mean of eight 0.5 ns simulations. The PMF for the parallel and antiparallel arrangements inside the membrane (c) are also shown. For untiparallel structure (dotted line) each data point for W(D) represents the mean of eight 0.5 ns simulations. For parralel structure we have two plots. The dashed line represents the mean of eight 0.5 ns simulations, whereas solid line represents the mean of sixteen 0.5 ns simulations.

Figure 4

Average number, N_Cl of chloride counterions bound to PG1 peptides as a function of the distance D, between the two peptides, for the parallel and the antiparallel β-sheet arrangement (a) in water and (b) on the surface of the POPE/POPG membrane.

Figure 5

Average number N_O of anionic lipid headgroup oxygens binding to both PG1 peptides as a function of the distance D, between the two peptides in the parallel and the antiparallel β-sheet arrangements (a) on the surface of the POPE/POPG membrane and (b) inside the POPE/POPG membrane.

Figure 6

(a) Average number, N_Cl, of chloride counterions bound the both PG1 peptides. p: the parallel; a: the antiparallel β-sheet arrangement of the PG1 dimer. (b) Average number, N_O, of anionic lipid headgroup oxygens bound to the both PG1 peptides, on the surface of the POPE/POPG membrane and inserted in the POPE/POPG membrane. p: the parallel; a: the antiparallel β-sheet arrangement of the PG1 dimer.

Figure 7

Average number of endogenic (a) and exogenic (b) hydrogen bonds between two PG1 peptides in all three studied environments. p: the parallel; a: the antiparallel β-sheet arrangement of the PG1 dimer.