Membrane-Embedded Anti-Cancer Peptide Causes a Minimal Structural Perturbation That Is Sufficient to Enhance Phospholipid Flip-Flop and Charge Permeation Rates

Abstract

A prime role of biological membranes is to form barriers for material transport into and out of cells. Membranes consist of phospholipids with polar heads, which are presented to the aqueous solutions, and hydrophobic tails that form the membrane core. This construct prevents the permeation of hydrophilic, well-solvated molecules across the lipid hydrophobic barrier. The barrier is not absolute, and several approaches are available for efficient translocation. Channels and pumps enable selective and efficient transport across membranes. Another transport mechanism is passive permeation, in which permeants, without assistance, directly transport across membranes. Passive transport is coupled to transient defects in the membrane structure that make crossing the hydrophobic bilayer easier-for example, displacements of head groups from aqueous solution-membrane interface into the membrane core. The defects, in turn, are rare unless assisted by passively permeating molecules such as cell-penetrating peptides that distort the membrane structure. One possible defect is a phospholipid molecule with a head pointing to the hydrophobic core. This membrane distortion allows head group flipping from one layer to the other. We show computationally, using atomically detailed simulations and the Milestoning theory, that the presence of a cell-penetrating peptide in a membrane greatly increases phospholipid flip-flop rate and hence defect formation and the permeability of membranes.

Keywords: cell penetrating peptide; membrane defect; membrane permeation.

Figure 1

A membrane with a hydrophobic core of DOPC lipids (cyan lines). Oxygen atoms of water molecules are shown as gray spheres, and the phosphate groups as yellow spheres (top) and orange spheres (bottom). Also shown is a single phospholipid molecule (green) with the phosphate head (red) migrating from the water–membrane interface to the membrane center from left to right.

Figure 2

Snapshots of a membrane with NAF-1^44–67 restrained to the center. The peptide is shown as a blue ribbon, which is partially helical. As in Figure 1, the lipid chains are in cyan. The lower phospholipid heads are in orange, while the top layer is in yellow. The mobile phospholipid is in green while the phosphorus atom is in red. Water molecules are in gray. The three snapshots from left to right illustrate the migration of the phospholipid from the water–membrane interface to the membrane center.

Figure 3

The free energy profile for the migration of a single phospholipid head from the water– membrane interface to the center of the membrane. The red curve is for the membrane containing at the center the NAF-1^44–67 peptide. The reaction coordinates are parameterized from 0 (phosphate head group at the membrane–water interface) to 1 (the phospholipid head group is at the center of the membrane). See the text for more details about the reaction coordinates.

Figure 4

The lower layer of the membrane is distorted, but the upper layer remains unperturbed when a single DOPC molecule is pulled to the center of the membrane from the lower layer. This observation suggests that the system is not in equilibrium, in which both sides of the membrane on either side of the permeating phospholipid should be similarly distorted. Hence, this simulation suffers from hysteresis. The color code is the same as in Figure 2.

Figure 5

The reaction coordinate used for the flipping of a DOPC phospholipid in a pure DOPC membrane. The figure shows the flipping phospholipid in green with its phosphorus atom in red. The phosphorus atoms are in yellow and orange for the upper and lower layers of the membrane, respectively. The distances between the phosphorus atoms closest to the flipping P atom are shown with blue lines. The difference between $d_{min}^{upper}$ and $d_{min}^{lower}$ is the reaction coordinate ($d_{diff}$) used in the Milestoning calculation. When the difference between the distances is zero, the phospholipid is at the center of the membrane. When the phospholipid is at a water–membrane interface, the absolute value of the difference is at its maximum. For the reaction coordinate, we define the maximal value of the difference as zero (no translocation of the phospholipid to the center) and the minimal distance (displaced phospholipid at the center) as one.

Figure 6

The number of additional phospholipid heads that are at the membrane center as a function of the position of the flipping phospholipid head, which we monitor along the reaction coordinate. Without the peptide at the membrane center, the number is less than 3 when the translocated phospholipid reaches the center. The peptide, with a charge of +5, supports a steady and larger number of phospholipids at the center (between five to seven).

Figure 7

The weighted number of phosphorus atoms at the membrane core along the normalized flipping coordinate. The weight is performed according to the respective milestone probability obtained in our calculations (Figure 3). It is the probability of the translocated phospholipid reaching a position along the reaction coordinate multiplied by the number of phospholipid heads that are at the membrane core (shown in Figure 6). We define the membrane core as the region of the membrane that is less than 1 nm from the membrane center along the z coordinate. This evaluation was performed using the 450 configurations extracted in every milestone (the error bars are obtained as the standard deviation of the average).

Figure 8

Similar to the previous figure (Figure 7), but counting the number of water molecules in the membrane core for the two membrane systems. A significant difference between the two figures is the permeation of water molecules into the pure membrane. The number of water molecules at the core is about 2 for the unperturbed pure membrane (the weighted number > 0 because the milestone probability is high when the normalized flipping coordinate is close to zero). When the flipping peptide is at the center, the number of water molecules at the core is about 35. However, the milestone probability is very small at that location and the plot looks flat close to one for the pure membrane.

Figure 9

The distance of the center of mass of the peptides NAF-1^44–67, NAF-1^47–67, and NAF-1^50–67 to the membrane center, obtained by 300 ns MD simulations after removing the harmonic restraint that forced the peptides to the membrane center. The simulations illustrate that the original peptide remains in the center while the shorter versions drift away from it.

Figure 10

Number of phosphate groups at the membrane core after removing the center of mass restraint and performing a 300 ns MD run for the three peptides NAF-1^44–67, NAF-1^47–67, and NAF-1^50–67. The membrane core is defined as the region within 1 nm of the bilayer center.