THE ADSORPTION OF PSEUDOMONAS-AERUGINOSA EXOTOXIN A TO PHOSPHOLIPID MONOLAYERS IS CONTROLLED BY PH AND SURFACE-POTENTIAL

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 similar to 4.6 nm(2) per molecule, up to a maximum density of one molecule per 2 8 nm(2) of lipid film, which corresponds roughly to the cross-sectiona l area of the toxin. This suggests that ETA molecules adsorb until the y contact each other, but insert only a small portion into the lipid f ilm. The kinetic process could be described by a Langmuir adsorption i sotherm. The apparent association and dissociation rate constants were determined, as were their dependence upon toxin concentration, membra ne composition, pH, and ionic strength. Two parameters were found to b e paramount for this interaction: pH and surface potential of the lipi d. It appears that ETA binding occurs only in a conformational state i nduced by low pH and is promoted by an electrostatic interaction betwe en a positively charged region of the protein and the negative charge of acidic phospholipids. On the basis of a simple model, the salient f eatures of ETA involved in its adsorption were derived: 1) the existen ce of a conformational state induced by the protonation of a group wit h pK 4.5 +/- 0.2; 2) a positive charge of 1.9 +/- 0.3 e.u. able to int eract with the surface potential of the membrane; 3) the fraction of p otential experienced by the protein in the activated state that preced es binding, similar to 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 con stant K-d similar to 90 nM.