Transient Kinetics of Nucleotide Binding to the P-glycoprotein Multidrug Transporter

Abstract

The P-glycoprotein multidrug transporter (Pgp) is a member of the ABC (ATP-binding cassette) superfamily of proteins, and contains two highly conserved cytosolic nucleotide-binding domains (NBDs). These domains couple ATP hydrolysis to the export of a variety of hydrophobic natural products, chemotherapeutic drugs and peptides. Pgp ATPase activity has been extensively characterized, but many details of the catalytic cycle of ATP hydrolysis remain unexplored. The fluorescent nucleotide analogue, TNP-ADP, interacts with the NBDs of Pgp, and the stoichiometry and affinity of binding were previously determined in our laboratory. Transient kinetic methods are commonly used to investigate the mechanism of molecular processes over a time-scale from milliseconds to hundreds of seconds. In this work, we studied the binding of TNP-ADP to Pgp using rapid stopped-flow kinetics. In these experiments, the binding reaction was monitored by following different fluorescent signals, under pseudo-first-order conditions (excess ligand). The time course of TNP-ADP binding displayed five relaxation times (τi's) over a time-span of 300 sec, which correspond to at least five different transitions. All the relaxation times presented a strong temperature-dependence. The time course of the reaction was analyzed by the computational tool Global Kinetic Explorer (KinTek, USA), using several models of sequential isomerizations after/before the fast bimolecular binding reaction. Also, the parameters were analyzed using the matrix projection operator technique for kinetic data (Bujalowski et al., 2000, J. Mol. Biol. 295:831). Both approaches provide, for the first time, information about the rate constants and fluorescent properties of the diverse intermediates formed during binding of TNP-ADP to Pgp. Elucidation of the details of the interaction of nucleotides with Pgp is of prime importance for formulation of a detailed mechanism of action of the transporter. This research is supported by a grant from the Canadian Cancer Society.