Purification and Characterization of the N-Terminal Nucleotide Binding Domain of an ABC Drug Transporter of Candida albicans:  Uncommon Cysteine 193 of Walker A Is Critical for ATP Hydrolysis

Abstract

The Candida drug resistance protein Cdr1p (approximately 170 kDa) is a member of ATP binding cassette (ABC) superfamily of drug transporters, characterized by the presence of 2 nucleotide binding domains (NBD) and 12 transmembrane segments (TMS). NBDs of these transporters are the hub of ATP hydrolysis activity, and their sequence contains a conserved Walker A motif (GxxGxGKS/T). Mutations of the lysine residue within this motif abrogate the ability of NBDs to hydrolyze ATP. Interestingly, the sequence alignments of Cdr1p NBDs with other bacterial and eukaryotic transporters reveal that its N-terminal NBD contains an unusual Walker A sequence (GRPGAGCST), as the invariant lysine is replaced by a cysteine. In an attempt to understand the significance of this uncommon positioning of cysteine within the Walker A motif, we for the first time have purified and characterized the N-terminal NBD (encompassing first N-terminal 512 amino acids) of Cdr1p as well as its C193A mutant protein. The purified NBD-512 protein could exist as an independent functional general ribonucleoside triphosphatase with strong divalent cation dependence. It exhibited ATPase activity with an apparent K(m) in the 0.8-1.0 mM range and V(max) in the range of 147-160 nmol min(-)(1) (mg of protein)(-)(1). NBD-512-associated ATPase activity was also sensitive to inhibitors such as vanadate, azide, and NEM. The Mut-NBD-512 protein (C193A) showed a severe impairment in its ability to hydrolyze ATP (95%); however, no significant effect on ATP (TNP-ATP) binding was observed. Our results show that C193 is critical for N-terminal NBD-mediated ATP hydrolysis and represents a unique feature distinguishing the ATP-dependent functionality of the ABC transporters of fungi from those found in bacteria and other eukaryotes.

Chimeras of the ABC drug transporter Cdr1p reveal functional indispensability of transmembrane domains and nucleotide-binding domains, but transmembrane segment 12 is replaceable with the corresponding homologous region of the non-drug transporter Cdr3p

“…Our previous study has shown (Jha et al, 2003a(Jha et al, , b, 2004) that the unique positioning of the catalytically important residues C193 in Walker A of NBD1 and K901 in Walker A of NBD2 could not be exchanged. Hence a construct like CDR1-1C/1NGFP was not used; instead, to address this question, two constructs were instead made, one having Cdr1p with cytoplasmic hydrophilic domains comprising two N-terminal NBDs and another with two Cterminal NBDs (Fig.…”

How azide inhibits ATP hydrolysis by the F-ATPases

“…In addition to the F-ATPases, a number of other ATPases are inhibited by azide. They include the ABC transporters (34,35), the preprotein translocase SecA (36,37), DNA topoisomerase II␣ (38), and ecto-ATPases (39,40). In each case, azide probably enhances the inhibitory effect of ADP by stabilizing the ADP-bound state, and this enhancement has been demonstrated for the SecA translocase (41).…”

Cytosolic Action of Phytochelatin Synthase

“…MCB is typically conjugated to GSH in the cytosol and subsequently sequestered in the vacuole. In order to evaluate the consequence of inhibiting the vacuolar sequestration of MCB, we treated cells with azide, a potent inhibitor of F-ATPases (Bowler et al, 2006) and of ABC transporters (Dallas et al, 2003;Jha et al, 2003). Confocal images of MCB-challenged cell suspension cultures of Arabidopsis show that the rapid accumulation of the fluorescent signal in the vacuole was prevented in the presence of azide ( Fig.…”