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, C(m)EO(n), 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 demonstrated 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.