Protein Transduction Domains of HIV-1 and SIV TAT Interact with Charged Lipid Vesicles. Binding Mechanism and Thermodynamic Analysis

Abstract

Cell-penetrating peptides (CPPs) traverse cell membranes of cultured cells very efficiently by a mechanism not yet identified. Recent theories for the translocation suggest either the binding of the CPPs to extracellular glycosaminoglycans or the formation of inverted micelles with negatively charged lipids. In the present study, the binding of the protein transduction domains (PTD) of human (HIV-1) and simian immunodeficiency virus (SIV) TAT peptide (amino acid residues 47-57, electric charge z(p) = +8) to membranes containing various proportions of negatively charged lipid (POPG) is characterized. Monolayer expansion measurements demonstrate that TAT-PTD insertion between lipids requires loosely packed monolayer films. For densely packed monolayers (pi > 29 mN/m) and lipid bilayers, no insertion is possible, and binding occurs via electrostatic adsorption to the membrane surface. Light scattering experiments show an aggregation of anionic lipid vesicles when the electric surface charge is neutralized by TAT-PTD, the observed stoichiometry being close to the theoretical value of 1:8. Membrane binding was quantitated with isothermal titration calorimetry and three further methods. The reaction enthalpy is Delta H degrees approximately equal to -1.5 kcal/mol peptide and is almost temperature-independent with Delta C(p) degrees approximately 0 kcal/(mol K), indicating equal contributions of polar and hydrophobic interactions to the reaction heat capacity. The binding of TAT-PTD to the anionic membrane is described by an electrostatic attraction/chemical partition model. The electrostatic attraction energy, calculated with the Gouy-Chapman theory, accounts for approximately 80% of the binding energy. The overall binding constant, K(app), is approximately 10(3)-10(4) M(-1). The intrinsic binding constant (K(p)), corrected for electrostatic effects and describing the partitioning of the peptide between the lipid-water interface and the membrane, is small and is K(p) approximately 1-10 M(-1). Deuterium and phosphorus-31 nuclear magnetic resonance demonstrate that the lipid bilayer remains intact upon TAT-PTD binding. The NMR data provide no evidence for nonbilayer structures and also not for domain formation. This is further supported by the absence of dye efflux from single-walled lipid vesicles. The electrostatic interaction between TAT-PTD and anionic phosphatidylglycerol is strong enough to induce a change in the headgroup conformation of the anionic lipid, indicating a short-lived but distinct correlation between the TAT-PTD and the anionic lipids on the membrane outside. TAT-PTD has a much lower affinity for lipid membranes than for glycosaminoglycans, making the latter interaction a more probable pathway for CPP binding to biological membranes.

Cell-Penetrating HIV1 TAT Peptides Float on Model Lipid Bilayers

“…Previous detailed biophysical characterization of the structure and interactions of PTDs within membranes allowed important insights into the energetics of peptide translocation and the mechanisms of penetration processes (20,30,31). An especially well-studied PTD is the TAT peptide, and its cell penetrating capabilities have been characterized in detail (10 Numerous studies exist on the structures, which are adopted by PTDs in solution and in membranes, but not much is known about their mobility on or within membranes.…”

Evaluation of Cell-Penetrating Peptide/Adenovirus Particles for Transduction of CAR-Negative Cells

“…These studies demonstrated that transmembrane potential is required for the peptides to be absorbed by neutral and charged lipid bilayer systems (Terrone, Sang et al 2003;Ziegler, Blatter et al 2003;.…”

Structure and dynamics of cationic membrane peptides and proteins: Insights from solid‐state NMR

“…However, the actual energy barrier for TAT binding to anionic membranes is almost certainly much lower, because the Wimley-White interfacial scale was derived for neutral POPC bilayers and thus does not include electrostatic attraction between cationic peptides and anionic lipids. Indeed, calorimetric studies of the binding energy of HIV TAT to 25% anionic membranes 113,114 reported a favorable free energy of À5.2 kcal/mol and 80% of which was estimated to be due to electrostatic interaction. In addition to electrostatic attraction, H-bonding of the guanidinium ions to the lipid phosphates and water, as detected in the NMR spectra, 6 must further contribute to the stabilization of TAT at the membrane-water interface.…”

“…The high cationic density has several consequences. First, it drives CPP binding to the membrane surface, 113,114 after which Arg-phosphate interactions facilitate peptide insertion into the membrane by minimizing the exposure of the charged residues to the hydrophobic interior. Second, the Arg clusters can strongly interact with lipid headgroups on the distal surface of the bilayer to drive translocation.…”