Exploring the P-Glycoprotein Binding Cavity with Polyoxyethylene Alkyl Ethers

P glycoprotein (ABCB1) moves allocrits from the cytosolic to the extracellular membrane leaflet preventing their intrusion into the cytosol It is generally accepted that allocrit binding from water to the cavity lined by the transmembrane domains occurs in two steps a lipid water partitioning step and a cavity binding step in the lipid membrane whereby hydrogen bond (i e weak electrostatic) interactions play a crucial role The remaining key question was whether hydrophobic interactions also play a role for allocrit binding to the cavity To answer this question we chose polyoxyethylene alkyl ethers CmEOn varying in the number of methylene and ethoxyl residues as model allocrits Using isothermal titration calorimetry we showed that the lipid water partitioning step was purely hydrophobic increasing linearly with the number of methylene and decreasing with the number of ethoxyl residues respectively Using in addition ATPase activity measurements we demon strated that allocrit binding to the cavity required minimally two ethoxyl residues and increased linearly with the number of ethoxyl residues The analysis provides the first direct evidence to our knowledge that allocrit binding to the cavity is purely electrostatic apparently without any hydrophobic contribution While the polar part of allocrits forms weak electrostatic interactions with the cavity the hydrophobic part seems to remain associated with the lipid membrane The interplay between the two types of interactions is most likely essential for allocrit flipping