Biophysical Insight on the Membrane Insertion of an Arginine-Rich Cell-Penetrating Peptide

Abstract

Cell-penetrating peptides (CPPs) are short peptides that can translocate and transport cargoes into the intracellular milieu by crossing biological membranes. The mode of interaction and internalization of cell-penetrating peptides has long been controversial. While their interaction with anionic membranes is quite well understood, the insertion and behavior of CPPs in zwitterionic membranes, a major lipid component of eukaryotic cell membranes, is poorly studied. Herein, we investigated the membrane insertion of RW16 into zwitterionic membranes, a versatile CPP that also presents antibacterial and antitumor activities. Using complementary approaches, including NMR spectroscopy, fluorescence spectroscopy, circular dichroism, and molecular dynamic simulations, we determined the high-resolution structure of RW16 and measured its membrane insertion and orientation properties into zwitterionic membranes. Altogether, these results contribute to explaining the versatile properties of this peptide toward zwitterionic lipids.

Keywords: NMR; cell-penetrating peptide; lipid model systems; membrane biophysics; molecular dynamics; peptide–lipid interaction.

Figure 1

RW16 and penetratin insertion in the membrane. (a) Sequence alignment of RW16 and penetratin. (b) Edmunson wheel of RW16 along the axis generated from Helixator, http://www.tcdb.org. (c) Inhibition rate (F₀/F) of RW16 and penetratin Trp fluorescence in buffer and in the presence of DOPC liposomes (P/L 1:50 mol:mol), with increasing concentrations of acrylamide. (d) Normalized accessibility factor (NAF) for penetratin and RW16 in presence of DOPC liposomes. Significance was tested with a Student’s t-test, where ** 0.001 < p < 0.01.

Figure 2

NMR spectroscopy of RW16 in DPC-d₃₈ micelles. (a–c) Chemical shift assignments of RW16 using a combination of 2D ¹H,¹³C-HSQC, 2D ¹H,¹H-TOCSY, and 2D ¹H,¹H-NOESY at 310 K. (a) Selected region of 2D ¹H,¹³C-HSQC, illustrating assigned δ1, ζ2, η2, ε3, and ζ3 ¹H-¹³C resonances for the 6 Trp residues. (b) Selected region of the 2D ¹H,¹H-TOCSY, highlighting assignment of the Trp ε1 ¹H resonances from the δ1 crosspeaks in the 2D ¹H,¹³C-HSQC. (c) Selected regions from the 2D ¹H,¹H-NOESY spectrum used to obtain distances for structure calculation, with representative NOE strips indicated for Trp ε1 ¹H nuclei.

Figure 3

Solution structure of micelle-bound RW16. (a) Selected regions from the 2D ¹H,¹H-NOESY spectrum used to obtain distances for structure calculation, with representative NOE strips indicated for all backbone amide ¹H^N nuclei. The upfield shifted side chain ¹Hγ resonance of Arg15 is also indicated, with NOE crosspeaks to Trp11 and Trp14. (b) Ensemble of 10 structures calculated for RW16 bound to DPC-d₃₈ micelles. The 6 Trp residues (orange) and 10 Arg residues (blue) are labeled. Note that the N-terminal biotin-aminopentanoic acid tag, although present in the sample, was not included in the structural models. The ensemble of structures has been deposited in the Protein Data Bank under accession number 6RQS.

Figure 4

RW16 peptide structure in contact with zwitterionic membranes. (a) ¹Hα chemical shifts compared to random coil ¹Hα predictions obtained from NMR data. (b) CD spectra of RW16 in phosphate buffer (black line) and in the presence of DPC micelles (gray line). (c) Fraction of helix calculated from the molecular dynamics (MD) simulations. The three states H (α-helix), G (3₁₀ helix), and I (π-helix) of the DSSP program were considered as part of the helix fraction (see Materials and Methods). After discarding the first 10 ns, each trajectory was cut into two blocks. Each value ± error was calculated as the mean and standard deviation over the six blocks respectively. (d) Snapshot of RW16 inserted in DOPC bilayer at t = 633 ns of MD trajectory 1. The C-terminus is on the left and N-terminus on the right. Trp are represented in orange, Arg in blue, the backbone is shown as an orange/blue ribbon and the lipids are drawn as spheres where carbon atoms are in cyan, oxygen in red, and nitrogen in blue.

Figure 5

Peptide insertion into the membrane. (a) Representative Trp fluorescence spectra of RW16 in the presence of DOPC or DOPC/BrPC liposomes. (b) Curve fitting calculated by distribution analysis (DA) or the parallax method (PM) for RW16 in zwitterionic vesicles. The data were averaged over four independent experiments and each value ± error represents the mean and standard deviation. (c) Density profiles along the perpendicular axis to the bilayer plane calculated by MD simulations corresponding to water molecules, DOPC molecules, peptides, and the overall system. (d) Close up from (c) on the Trp and Arg region of the peptide, and on the lipid subgroups.

Figure 6

Peptide tilting relative to the normal to the zwitterionic membrane plane calculated by NMR and MD. (a) Tilt of the peptide calculated from the difference in z position between the C-alpha of Arg2 and Arg13 calculated over the three trajectories. The histogram (gray) shows the average distribution of z over the three trajectories. The scheme on the right panel shows that a tilt <90° describes a deeper insertion of the N-terminus, while a tilt >90° describes a deeper insertion of the C-terminus. (b) Solvent accessibility for RW16 in DPC micelles, as measured by NMR spectroscopy using the paramagnetic probe Gd(DTPA-BMA). The resulting solvent paramagnetic relaxation enhancement values (sPRE; Figure S4) have been measured for several atoms in RW16 and shown as spheres. Atoms that are strongly affected (colored blue) by the added Gd(DTPA-BMA) are more solvent-exposed as compared to atoms that are less affected (colored white). The tilt of the peptide shown represents an estimate based on the observed data.

Figure 7

Side chain contacts between Arg15 and residues Trp10, Trp11, and Trp14. (a) COM-COM of side chain distances between Trp10 and Arg15 (left, W10–R15), Trp11 and Arg15 (middle, W11–R15), and Trp14 and Arg15 (right, W14–R15) for the three trajectories. (b) Surface density of RW16 amino acids showing a special arrangement of Arg15 and Arg–Trp π-cation interactions (left). Close-up on the pocket formed by Trp10, Trp11, and Trp14 around Arg15 (right). Structure generated with Pymol (PDB ID: 6RQS) [38].

Figure 8

Summary cartoon representing RW16 embedded in a zwitterionic membrane. Shown is the calculated secondary structure and orientation of the peptide with the Trp (orange) and Arg (blue) side chains in a zwitterionic membrane (gray) with the polar headgroups (dark gray) and the aliphatic chains (light gray).