title: 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.
authors.affiliation: Department of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.
authors.forename: André
authors.initials: A
authors.lastname: Ziegler
authors.forename: Xiaochun Li
authors.initials: XL
authors.lastname: Blatter
authors.forename: Anna
authors.initials: A
authors.lastname: Seelig
authors.forename: Joachim
authors.initials: J
authors.lastname: Seelig
mesh_terms: D000818:Animals; D001665:Binding Sites; D002151:Calorimetry / Q000379:methods; D003903:Deuterium / Q000737:chemistry; D015696:Gene Products, tat / Q000737:chemistry* / Q000378:metabolism*; D015497:HIV-1 / Q000737:chemistry*; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism*; D008956:Models, Chemical; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010713:Phosphatidylcholines / Q000737:chemistry; D010715:Phosphatidylglycerols / Q000737:chemistry; D010759:Phosphorus Isotopes; D011312:Pressure; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012542:Scattering, Radiation; D015302:Simian Immunodeficiency Virus / Q000737:chemistry*; D055672:Static Electricity; D013499:Surface Properties; D013816:Thermodynamics; D054322:tat Gene Products, Human Immunodeficiency Virus
chemical_list: D015696:Gene Products, tat; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D010759:Phosphorus Isotopes; D054322:tat Gene Products, Human Immunodeficiency Virus; C060037:1-palmitoyl-2-oleoylglycero-3-phosphoglycerol; D003903:Deuterium; C028694:1-palmitoyl-2-oleoylphosphatidylcholine