P-glycoprotein Is Stably Inhibited by Vanadate-induced Trapping of Nucleotide at a Single Catalytic Site

Abstract

P-glycoprotein (Pgp or multidrug-resistance protein) shows drug-stimulated ATPase activity. The catalytic sites are known to be of low affinity and specificity for nucleotides. From the sequence, two nucleotide sites are predicted per Pgp molecule. Using plasma membranes from a multidrug-resistant Chinese hamster ovary cell line, which are highly enriched in Pgp, we show that vanadate-induced trapping of nucleotide at a single catalytic site produces stably inhibited Pgp, with t 1/2 for reactivation of ATPase activity of 84 min at 37 degrees C and >30 h at 4 degrees C. Reactivation of ATPase correlated with release of trapped nucleotide. Concentrations of MgATP and MgADP required to produce 50% inhibition were 9 and 15 microM, respectively, thus the apparent affinity for nucleotide is greatly increased by vanadate-trapping. The trapped nucleotide species was ADP. Divalent Cation was required, with magnesium, manganese, and cobalt all effective: cobalt yielded a very stable inhibited species, t1/2 at 37 degrees C = 18 h. No photocleavage of Pgp was observed after vanadate trapping with MgATP, nor was UV-induced photolabeling of Pgp by trapped adenine nucleotide observed. Vanadate-trapping with 8-azido-ATP followed by UV irradiation caused permanent inactivation and specific labeling of Pgp. Vanadate-induced inhibition was also shown with pure, reconstituted Pgp, with similar characteristics to those in plasma membranes. Vanadate trapping overcomes technical difficulties posed by lack of high affinity nucleotide-binding site(s) or a covalent enzyme-phosphate catalytic intermediate in Pgp. The finding that vanadate trapping of nucleotide at just one site/Pgp is sufficient to give full inhibition at ATPase activity shows that the two predicted nucleotide sites can not function independently as catalytic sites.

Structure and function of ABC transporters: the ATP switch provides flexible control

“…It therefore seems that the conformational changes in the TMDs, at different steps in the catalytic cycle, may have different consequences for the different drug-binding sites for P-gp [68,69], TAP [70] and the histidine permease [71]. For some ABC transporters, the two pockets hydrolyse ATP non-simultaneously, for example, P-gp [22,52,63,72], MutS [36], and the bacterial histidine [50,61] and maltose [73] transporters. Consistent with this, the two nucleotidebinding pockets of a transporter frequently exhibit structural asymmetry even for HisP [29] and HlyB-NBD [40] where the two NBDs are identical in sequence.…”

Exploiting Reaction Intermediates of the ATPase Reaction to Elucidate the Mechanism of Transport by P-glycoprotein (ABCB1)

“…B, the apparent K m (8-azido-ATP/ADP) for occlusion of nucleotide was determined as described in A above with the following differences. The Pgps were incubated with increasing concentrations of 8-azido-[␣- 32 sis allow the nucleotide diphosphate to be occluded in the presence of Vi (3,18). Vi mimics the pentacovalent phosphorus, and the long lived, noncovalent ternary complex Pgp⅐MgADP⅐Vi is analogous to the catalytic transition state, Pgp⅐MgADP⅐P i (18,34,35 Fig.…”

“…The Pgps were incubated with increasing concentrations of 8-azido-[␣- 32 sis allow the nucleotide diphosphate to be occluded in the presence of Vi (3,18). Vi mimics the pentacovalent phosphorus, and the long lived, noncovalent ternary complex Pgp⅐MgADP⅐Vi is analogous to the catalytic transition state, Pgp⅐MgADP⅐P i (18,34,35 Fig. 2A shows that both wild-type and the E556Q/E1201Q mutant Pgps have comparable affinities (11.07 Ϯ 0.4 M and 10.7 Ϯ 0.28 M, respectively).…”

“…17). The Vi-induced, ADP-trapped state of Pgp has been well characterized (18,19), and is believed to be comparable with the posthydrolysis, Pgp⅐ADP⅐P i reaction intermediate. Moreover, recent studies have found that mutating the highly conserved glutamates (Glu 556 and Glu 1201 in human Pgp) in the Walker B region results in a phenotype wherein the protein shows no transport function and minimal steady-state ATP hydrolysis but can occlude the nucleotide (20).…”

Membrane topology and mutational analysis of Escherichia coli CydDC, an ABC-type cysteine exporter required for cytochrome assembly

“…This change in CydD was sufficient to disrupt function, as assessed by failure to restore growth of a cydD mutant under selective conditions and total lack of cytochrome d spectral signals (not shown). P-glycoprotein (the multidrug-resistance protein) indicate that the two ATP-binding sites cannot function independently and a strong cooperative interaction occurs between the two (Urbatsch et al, 1995). The requirement for two ABC domains in CydDC is demonstrated by the fact that the directed G388K/K389Q mutation of CydD in the nucleotide-binding domain disrupts function.…”