Molecular dynamics simulations suggest a mechanism for translocation of the HIV-1 TAT peptide across lipid membranes

Abstract

The recombinant HIV-1 Tat protein contains a small region corresponding to residues (47)YGRKKRRQRR(57)R, which is capable of translocating cargoes of different molecular sizes, such as proteins, DNA, RNA, or drugs, across the cell membrane in an apparently energy-independent manner. The pathway that these peptides follow for entry into the cell has been the subject of strong controversy for the last decade. This peptide is highly basic and hydrophilic. Therefore, a central question that any candidate mechanism has to answer is how this highly hydrophilic peptide is able to cross the hydrophobic barrier imposed by the cell membrane. We propose a mechanism for the spontaneous translocation of the Tat peptides across a lipid membrane. This mechanism involves strong interactions between the Tat peptides and the phosphate groups on both sides of the lipid bilayer, the insertion of charged side chains that nucleate the formation of a transient pore, followed by the translocation of the Tat peptides by diffusing on the pore surface. This mechanism explains how key ingredients, such as the cooperativity among the peptides, the large positive charge, and specifically the arginine amino acids, contribute to the uptake. The proposed mechanism also illustrates the importance of membrane fluctuations. Indeed, mechanisms that involve large fluctuations of the membrane structure, such as transient pores and the insertion of charged amino acid side chains, may be common and perhaps central to the functions of many membrane protein functions.

Fig. 1.

Binding of a Tat peptide to a zwitterionic lipid bilayer. (Upper) Snapshot obtained at the end of the 200-ns simulation (simulation A in Table 1). The peptide is partially exposed to water and surrounded by phosphates and carbonyl groups of the phospholipids. (Lower) Positively charged groups of the peptide bind to the phosphate and carbonyl groups of the phospholipid molecules. In this configuration, the Tat peptide binds to 14 phosphate groups.

Fig. 2.

Binding and translocation of Tat peptides (simulations A and D in Table 1). (a) Snapshot of the system comprising 72 DOPC lipids, 3,552 water molecules, and one Tat peptide after a 200-ns simulation. The phospholipid molecules are represented with transparent white surfaces, the phosphate atoms are in yellow spheres, the peptide molecules are in red, any water molecule at a distance of <3.5 Å from any phospholipid or amino acid atom is deep blue, and the rest of the water molecules appear as a pale blue transparent surface. (b–d) Snapshots of the system with four Tat peptides and the same number of DOPC lipids and water molecules as in a after 70, 140, and 200 ns, respectively.

Fig. 3.

Binding of Tat peptides to phosphate groups. The yellow spheres show phosphates of the proximal layer on which the Tat peptides (in red) are initially bound, and the phosphates of the distal layer are shown by green spheres. For clarity, other molecules are not shown. (Left) Simulation at 323 K (simulation A in Table 1). (Right) Simulation at 363 K (simulation F in Table 1). (Top) Side view of the membrane in which a translocating peptide is visible. (Middle) View of the plane of the membrane and four periodic replicas that only includes the peptides and phosphates of the proximal layer. (Bottom) View of the distal layer of the membrane. These pictures illustrate that the phosphates of both the proximal and distal layers are attracted to the peptides. Heterogeneity induced in the proximal layer with high and low densities of phosphates is visible. Several of the periodic replicas of the translocating peptide are indicated with a yellow ellipse. This peptide is bound to a smaller number of phosphate groups, compared with the rest of the peptides.

Fig. 4.

Area per lipid and thickness of the bilayer as a function of the number of peptides over the number of phospholipids (simulations A–D and H–L in Table 1). (a) Area per lipid. (b) Thickness of the lipid bilayer. Diamonds indicate results for the cases in which all of the Tat peptides are on the proximal layer. Squares indicate cases in which one Tat peptide is bound to the distal layer and all other peptides are on the proximal layer.

Fig. 5.

Snapshots at different times along the simulation of a system composed of 92 DOPC lipids, 8,795 water molecules, and four Tat peptides (simulation G in Table 1). Colors and representations are the same as in Fig. 2. (a) Position of the peptides before translocation. (b and c) Translocation of an arginine amino acid (b) toward the distal layer that nucleates the formation of a water-filled pore (c). (d) Snapshot at the same instant as c but from a direction perpendicular to the membrane, showing another perspective of the pore and translocating peptide.

Fig. 6.

Alternative view of the pore (Fig. 5d), including four periodic images of the system. Phosphate-depletion zones over the surface of the membrane and high concentration of phosphates around the Tat peptides can be clearly seen.