Decoding the membrane activity of the cyclotide kalata B1: the importance of phosphatidylethanolamine phospholipids and lipid organization on hemolytic and anti-HIV activities

Abstract

Cyclotides, a large family of cyclic peptides from plants, have a broad range of biological activities, including insecticidal, cytotoxic, and anti-HIV activities. In all of these activities, cell membranes seem likely to be the primary target for cyclotides. However, the mechanistic role of lipid membranes in the activity of cyclotides remains unclear. To determine the role of lipid organization in the activity of the prototypic cyclotide, kalata B1 (kB1), and synthetic analogs, their bioactivities and affinities for model membranes were evaluated. We found that the bioactivity of kB1 is dependent on the lipid composition of target cell membranes. In particular, the activity of kB1 requires specific interactions with phospholipids containing phosphatidylethanolamine (PE) headgroups but is further modulated by nonspecific peptide-lipid hydrophobic interactions, which are favored in raft-like membranes. Negatively charged phospholipids do not favor high kB1 affinity. This lipid selectivity explains trends in antimicrobial and hemolytic activities of kB1; it does not target bacterial cell walls, which are negatively charged and lacking PE-phospholipids but can insert in the membranes of red blood cells, which have a low PE content and raft domains in their outer layer. We further show that the anti-HIV activity of kB1 is the result of its ability to target and disrupt the membranes of HIV particles, which are raft-like membranes very rich in PE-phospholipids.

FIGURE 1.

Sequence and structure of kB1. A, the sequence of kB1 is shown. Backbone cyclization between Gly-1 and Asn-29 is illustrated with a black line, and the disulfide connectivity is shown in yellow. The segments between Cys residues are termed loops. B, a three-dimensional structure of kB1 (PDB ID 1nb1) shows the cyclic knotted nature of cyclotides. The six Cys are labeled, and backbone loops are identified. Blue and green circles show the position of Gly-1 and Trp-23 residues, respectively. The black arrow indicates the direction of the peptide chain. C, surface representation of the molecule is shown in two views, illustrating the hydrophobic (green), bioactive (red), and amendable faces (blue). The same color code is used in the primary sequence shown in panel A.

FIGURE 2.

Binding of kB1 to lipid membranes requires the presence of PE-phospholipids. A, the affinity of kB1 for the different lipid systems was studied using SPR and is compared by the amount of kB1 bound to the membrane at the end of association phase (after injection of 25 μm kB1 over the membrane surface during 170 s). The P/L ratio was calculated to normalize the response to the total amount of lipid deposited in the chip (1 response unit (RU) = 1 pg/mm² of peptide or lipid). B, sensorgrams were obtained upon injection of 25 μm kB1 over POPC or POPC/POPE (5%; 10 or 20%) during 180 s (association phase) and dissociation from the membranes followed for 600 s (dissociation phase). C, shown is the amount of the kB1 bound to POPC or POPC/POPE (5%; 10 or 20%) at the end of association phase versus the amount of POPE in the chip surface (half of the total amount of POPE was considered, as only half of the lipid is in the outer leaflet). D, sensorgrams were obtained upon injection of 25 μm kB1 onto membranes that mimic the outer membrane (LPS and POPC/LPS (20%)) or the inner (POPG/POPE (40/60%)) membrane of E. coli as well as native inner membrane.

FIGURE 3.

Activity of kB1 is correlated with affinity for the lipid bilayer. Native kB1, active mutants (R28K, T20K, N29K, and T20K/N29K), and inactive mutants (V4K, E7D, T8K, V10K, N15K, V16K, W23K, and V25K) are compared in their affinity for lipid membranes. A, sensorgrams obtained upon injection of 25 μm of kB1 or analogs onto POPC/POPE/Chol/SM (10/33/40%) membranes are shown. B, correlation between the amount of peptide bound to the membrane and the hemolytic activity of the different analogs are shown. The values were normalized for the response obtained with the native kB1 (see supplemental Fig. S2 and Table S1). RU, response unit.

FIGURE 4.

NMR monitored titration of kalata B1 with PE. A, two-dimensional total correlation spectra of kB1 (black) and kB1 after the addition of 67 mm PE (red) show examples of observed chemical shift changes. B, binding curves for selected resonances show an overall chemical shift change of >0.02 ppm within the studied range. Curves were fitted to the data using a one-to-one specific binding model. C, the PE binding surface of kalata B1 is shown. Residues with at least one resonance show a chemical shift change of >0.02 ppm upon binding to PE are shown in red. D, shown is the putative binding mode of PE to kB1.

FIGURE 5.

Membrane leakage and flip-flop induced by kB1. A, the fluorescence of CF was converted to the percentage of leakage and plotted as a function of P/L ratio. The efficiency of vesicle leakage induced by kB1 is compared for membrane with different lipid compositions. B, leakage from POPC/POPE/Chol/SM (10/33/40%) vesicles induced by kB1 is compared with the membrane-inactive V25K and the membrane-active N29K. C, outward movement of C6-NBD-PE and C6-NDB-PC phospholipids was compared for POPC and POPC/Chol/SM. NBD fluorescence quenching by Co²⁺ was followed upon titration with kB1. Quenching efficiency (represented by I₀/I, where I₀ is the initial fluorescence intensity, and I is the fluorescence obtained upon addition of peptide) is plotted as a function of P/L.

FIGURE 6.

kB1 binds to the RBCs membrane and induces changes in the RBCs shape. Images of RBCs were generated by atomic force microscopy for both control samples without the addition of peptide and after incubation with different kB1 concentrations.

FIGURE 7.

The effect of kB1 and its analogs on the shape of E. coli followed by atomic force microscopy. Three different concentrations of kB1, N29K, or V25K were incubated with E. coli during 2 h. The images show that at low micromolar concentrations, neither kB1 nor its analogs induce effect on E. coli. Effects on E. coli shape are only evident when a high concentration of peptide is incubated with the bacteria. No differences among the three peptides were detected.

FIGURE 8.

KB1 induces disruption of HIV membrane. A, activity of kB1 against HIV-1 NL4.3 or HIV-1 Clade is shown. A, immobilized virus particles were pretreated with kB1 for 1 h and washed off before being cultured with target TZM-bl cells. The error bars represent the S.D. B, KB1 is compared with the membrane-inactive V25K and the membrane-active N29K for their virucidal activity against NL4.3. The error bars represent S.D. For clarity, only the positive S.D. is represented in this panel. C, after treatment of NL4.3 virus particles with 50 μg/ml kB1, V25K, or N29K, viral proteins were detected by Western blot. A low level of viral capsid protein p24 for kB1- and N29K-treated virus compared with control treatment, but not for V25K, indicates removal of membrane envelope and disruption of the particle.