Exploring the P-glycoprotein binding cavity with polyoxyethylene alkyl ethers - PubMed (original) (raw)

Exploring the P-glycoprotein binding cavity with polyoxyethylene alkyl ethers

Xiaochun Li-Blatter et al. Biophys J. 2010.

Abstract

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.

Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1

Free energies of binding of C12EOn (n = 3–10, 23) to membrane and transporter as a function of the number of ethoxyl groups (n) (T = 37°C). (A) Free energy of lipid-water partitioning, ΔGlw0 (▴); free energy of transporter-water binding (first binding region) ΔGtw(1)0 (□), free energy of transporter-water binding (second binding region) ΔGtw(2)0 (○), both derived from ATPase activity measurements. (B) Free energy of transporter-lipid binding (first binding region) ΔGtl(1)0 (■), (second binding region) ΔGtw(2)0 (●). Linear fits were performed in the range of n = 4–10. Values for ΔGtw(1)0 and ΔGtw(2)0 are the averages of at least five measurements.

Figure 2

Figure 2

Free energies of binding of CmEO8 (m = 10, 12, 14) to the membrane and to the transporter as a function of the number of methylene groups (m). Free energy of lipid-water partitioning, ΔGlw0 (▴) (measured at T = 25°C and calculated at 37°C), free energy of transporter-water binding (first binding region) ΔGtw(1)0 (▪), and free energy of transporter-water binding (second binding region) ΔGtw(2)0 (●) (T = 37°C).

Figure 3

Figure 3

P-gp ATPase activity measured as a function of concentration of C12EOn (n = 3–10, 23) in plasma membrane vesicles of NIH-MDR1-G185 cells (T = 37°C and pH 7.0). C12EO3 (▪), C12EO4 (★), C12EO5 (formula image), C12EO6 (▴), C12EO7 (formula image), C12EO8 (●), C12EO9 (♦), C12EO10 (◂), and C12EO23 (○). Data are expressed as the average of two measurements. (Solid lines) Fits to Eq. 4.

Figure 4

Figure 4

P-gp ATPase activity measured as a function of concentration of CmEO8 (m = 10, 12, 14) in plasma membrane vesicles of NIH-MDR1-G185 cells (T = 37°C and pH 7.0). C10EO8 (▪), C12EO8 (●), and C14EO8 (♦). Data are expressed as the average of two measurements. (Solid lines) Fits to Eq. 4.

Figure 5

Figure 5

Detergent mole fraction X(1) (▪) and X(2) (○) at the concentration of half-maximum activation, K(1), and inhibition, K(2), as a function of the number of ethoxyl groups, n, at 37°C.

Figure 6

Figure 6

Difference in the free energy of binding from water to the activating and inhibitory binding region of P-gp, ΔGtw(2)0 – ΔGtw(1)0, as a function of the cross-sectional area, _A_D, of allocrits, determined by surface activity measurements. Drugs measured previously (Fig. 6 in (16)) (○). E(n) corresponds to C12EOn (n = 3, 4, 5, 8, 10, 23) (■).

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