Covalent modification of cysteine 193 impairs ATPase function of nucleotide-binding domain of a Candida drug efflux pump.
N-ethylmaleimide (NEM) impairs the ATPase function of N-terminal NBD of Candida drug resistance gene product Cdr1p. To identify the reactive cysteine(s) for such a contribution, we adopted a three-arm approach that included covalent modification, cysteine mutagenesis, and structure homology modeling. The covalent modification results clearly indicate the ability of NEM and iodoacetic acid (IAA) to potently inhibit the ATPase activity of N-terminal NBD. Since this domain contains five cysteine residues in its sequence, we mutated each and found four of these (C325A, C363A, C402A, and C462A) to stay sensitive to NEM/IAA modification and influence ATPase activity, while C193A mutation completely abrogated the catalytic function. The structural homology modeling data further validate these biochemical findings by ruling out any plausible interactions within the cysteine residues, and deriving the importance of Cys-193 in lying at a bond length clearly feasible to interact with ATP and divalent cation to critically influence ATP hydrolysis.
Jha
Sudhakar
S
Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
Karnani
Neerja
N
Lynn
Andrew M
AM
Prasad
Rajendra
R
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002175:Candida / Q000378:metabolism*; D002384:Catalysis; D002412:Cations; D003545:Cysteine / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D006868:Hydrolysis; D019807:Iodoacetic Acid / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D002412:Cations; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D003545:Cysteine; D005033:Ethylmaleimide; D019807:Iodoacetic Acid