title: 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.
authors.affiliation: Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India 110067.
authors.forename: Sudhakar
authors.initials: S
authors.lastname: Jha
authors.forename: Neerja
authors.initials: N
authors.lastname: Karnani
authors.forename: Suman K
authors.initials: SK
authors.lastname: Dhar
authors.forename: Kasturi
authors.initials: K
authors.lastname: Mukhopadhayay
authors.forename: Suneet
authors.initials: S
authors.lastname: Shukla
authors.forename: Preeti
authors.initials: P
authors.lastname: Saini
authors.forename: Gauranga
authors.initials: G
authors.lastname: Mukhopadhayay
authors.forename: Rajendra
authors.initials: R
authors.lastname: Prasad
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D002176:Candida albicans / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D005656:Fungal Proteins; D006868:Hydrolysis; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism*; D009841:Oligonucleotides / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
chemical_list: D018528:ATP-Binding Cassette Transporters; C094693:CDR1 protein, Candida albicans; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D009711:Nucleotides; D009841:Oligonucleotides; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D003545:Cysteine; D005033:Ethylmaleimide