title: The adsorption of Pseudomonas aeruginosa exotoxin A to phospholipid monolayers is controlled by pH and surface potential.
abstract: The interaction of Pseudomonas aeruginosa exotoxin A (ETA) with lipid monolayers was studied by measuring the variation in surface pressure. ETA adsorbs to the monolayer, occupying an average area of approximately 4.6 nm2 per molecule, up to a maximum density of one molecule per 28 nm2 of lipid film, which corresponds roughly to the cross-sectional area of the toxin. This suggests that ETA molecules adsorb until they contact each other, but insert only a small portion into the lipid film. The kinetic process could be described by a Langmuir adsorption isotherm. The apparent association and dissociation rate constants were determined, as were their dependence upon toxin concentration, membrane composition, pH, and ionic strength. Two parameters were found to be paramount for this interaction: pH and surface potential of the lipid. It appears that ETA binding occurs only in a conformational state induced by low pH and is promoted by an electrostatic interaction between a positively charged region of the protein and the negative charge of acidic phospholipids. On the basis of a simple model, the salient features of ETA involved in its adsorption were derived: 1) the existence of a conformational state induced by the protonation of a group with pK 4.5 +/- 0.2; 2) a positive charge of 1.9 +/- 0.3 e.u. able to interact with the surface potential of the membrane; 3) the fraction of potential experienced by the protein in the activated state that precedes binding, approximately 80%; 4) the intrinsic adsorption and desorption rate constants, k(a)0 = (4.8 +/- 0.3) x 10(3) M(-1) s(-1) and k(d)0 = (4.4 +/- 0.4) x 10(-4) s(-1). These rate constants are independent of pH and lipid and buffer composition, and provide a dissociation constant Kd approximately 90 nM.
authors.affiliation: CNR-ITC Centro Fisica Stati Aggregati, Trento, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Nordera
authors.forename: M D
authors.initials: MD
authors.lastname: Serra
authors.forename: G
authors.initials: G
authors.lastname: Menestrina
mesh_terms: D036002:ADP Ribose Transferases; D000327:Adsorption; D001427:Bacterial Toxins / Q000737:chemistry; D001703:Biophysics / Q000379:methods; D005098:Exotoxins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry*; D008956:Models, Chemical; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D011550:Pseudomonas aeruginosa; D013499:Surface Properties; D013816:Thermodynamics; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D008081:Liposomes; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A