Abstract
P-glycoprotein (ABCB1), a member of the ABC superfamily, functions as an ATP-driven multidrug efflux pump. The catalytic cycle of ABC proteins is believed to involve formation of a sandwich dimer in which two ATP molecules are bound at the interface of the nucleotide binding domains (NBDs). However, such dimers have only been observed in isolated NBD subunits and catalytically arrested mutants, and it is still not understood how ATP hydrolysis is coordinated between the two NBDs. We report for the first time the characterization of an asymmetric state of catalytically active native P-glycoprotein with two bound molecules of adenosine 5'-(gamma-thio)triphosphate (ATPgammaS), one of low affinity (K(d) 0.74 mm), and one "occluded" nucleotide of 120-fold higher affinity (K(d) 6 microm). ATPgammaS also interacts with P-glycoprotein with high affinity as assessed by inhibition of ATP hydrolysis and protection from covalent labeling of a Walker A Cys residue, whereas other non-hydrolyzable ATP analogues do not. Binding of ATPgammaS (but not ATP) causes Trp residue heterogeneity, as indicated by collisional quenching, suggesting that it may induce conformational asymmetry. Asymmetric ATPgammaS-bound P-glycoprotein does not display reduced binding affinity for drugs, implying that transport is not driven by ATP binding and likely takes place at a later stage of the catalytic cycle. We propose that this asymmetric state with two bound nucleotides represents the next intermediate on the path toward ATP hydrolysis after nucleotide binding, and an alternating sites mode of action is achieved by simultaneous switching of the two active sites between high and low affinity states.
Highlights
The ATP-binding cassette (ABC)2 protein superfamily is a very large and diverse group of active membrane transporters found in all organisms from bacteria to humans [1,2,3]
We report for the first time the characterization of an asymmetric state of catalytically active native P-glycoprotein with two bound molecules of adenosine 5-(␥-thio)triphosphate (ATP␥S), one of low affinity (Kd 0.74 mM), and one “occluded” nucleotide of 120-fold higher affinity (Kd 6 M)
We propose that this asymmetric state with two bound nucleotides represents the intermediate on the path toward ATP hydrolysis after nucleotide binding, and an alternating sites mode of action is achieved by simultaneous switching of the two active sites between high and low affinity states
Summary
ATP␥S was found to interact with Pgp with high affinity as assessed by inhibition of ATP hydrolysis and protection from covalent labeling of Cys residues in the NBDs. Binding of ATP␥S (but not ATP) was found to induce Trp residue heterogeneity, as indicated by collisional quenching, suggesting the existence of conformational asymmetry in the Pgp molecule. Binding of ATP␥S (but not ATP) was found to induce Trp residue heterogeneity, as indicated by collisional quenching, suggesting the existence of conformational asymmetry in the Pgp molecule This asymmetric occluded state containing two bound nucleotides still displays high affinity binding of several drug substrates and modulators, which has important implications for the catalytic and transport cycle of Pgp. We propose a site-switching mechanism that leads to alternation of catalysis between the two active sites
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