文摘
The Candida drug resistance protein Cdr1p (~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 hydrolysisactivity, and their sequence contains a conserved Walker A motif (GxxGxGKS/T). Mutations of the lysineresidue within this motif abrogate the ability of NBDs to hydrolyze ATP. Interestingly, the sequencealignments of Cdr1p NBDs with other bacterial and eukaryotic transporters reveal that its N-terminalNBD contains an unusual Walker A sequence (GRPGAGCST), as the invariant lysine is replaced by acysteine. In an attempt to understand the significance of this uncommon positioning of cysteine withinthe 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. Thepurified NBD-512 protein could exist as an independent functional general ribonucleoside triphosphatasewith strong divalent cation dependence. It exhibited ATPase activity with an apparent Km in the 0.8-1.0mM range and Vmax in the range of 147-160 nmol min-1 (mg of protein)-1. NBD-512-associated ATPaseactivity 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 significanteffect on ATP (TNP-ATP) binding was observed. Our results show that C193 is critical for N-terminalNBD-mediated ATP hydrolysis and represents a unique feature distinguishing the ATP-dependentfunctionality of the ABC transporters of fungi from those found in bacteria and other eukaryotes.