Van't Hoff Analysis of ATP Binding to Human P-glycoprotein (ABCB1)

Abstract

ATP binding to ABC transport proteins is an established step in the mechanism by which substrates are transported. Basal ATPase activity (activity seen in the absence of transported drugs) also facilitates multidrug transport by ABC proteins (Al-Shawi et al. 2003, J. Biol. Chem. 278, 52629). Therefore, we aimed to develop a non-perturbing method for detecting structural changes of P-glycoprotein associated with ATP binding during turnover. To accomplish this we analyzed heat capacity changes during ATP binding to P-glycoprotein by generating van't Hoff relationships for KMATP values for ATP binding and KiAMPPNP values for AMPPNP binding (non-hydrolysable analog of ATP).The rate of ATP hydrolysis by purified P-glycoprotein was measured as a function of ATP and AMPPNP concentrations at different temperatures from 23 to 35°C, in the presence or absence of saturating concentration of drug. Plots of Ln(KaATP) as a function of reciprocal absolute temperature in the presence of verapamil, valinomycin, colchicine and in absence of drug were fit by a nonlinear van't Hoff equation. Changes in heat capacity were derived from the fits. For basal activity, the Ln(KaATP) increased with reciprocal temperature. The heat capacity change was positive. In the presence of verapamil the relationship declined and the heat capacity change was negative. Results obtained with the other drugs were similar. This suggests that P-glycoprotein became more hydrated on ATP binding during basal activity and adopted a more open form. In contrast, in the presence of verapamil P-glycoprotein became less hydrated and adopted a more compact form. Overall, the data support our contention that there are two structurally distinct reaction pathways for ATP hydrolysis by ABC transporters and further help clarify the coupling mechanism of ATP hydrolysis to substrate transport. Supported by NIH grant GM52502.