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

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• 作者：Sudhakar Jha ; Neerja Karnani ; Suman K. Dhar ; Kasturi Mukhopadhayay ; Suneet Shukla ; Preeti Saini ; Gauranga Mukhopadhayay ; and Rajendra Prasad
• 刊名：Biochemistry
• 出版年：2003
• 出版时间：September 16, 2003
• 年：2003
• 卷：42
• 期：36
• 页码：10822 - 10832
• 全文大小：362K
• 年卷期：v.42,no.36(September 16, 2003)
• ISSN：1520-4995

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 K_m in the 0.8-1.0mM range and V_max 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.