Covalent modification of cysteine 193 impairs ATPase function of nucleotide-binding domain of a Candida drug efflux pump

Jha, Sudhakar ; Karnani, Neerja ; Lynn, Andrew M. ; Prasad, Rajendra (2003) Covalent modification of cysteine 193 impairs ATPase function of nucleotide-binding domain of a Candida drug efflux pump Biochemical and Biophysical Research Communications, 310 (3). pp. 869-875. ISSN 0006-291X

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Official URL: http://linkinghub.elsevier.com/retrieve/pii/S00062...

Related URL: http://dx.doi.org/10.1016/j.bbrc.2003.09.094

Abstract

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.

Item Type:Article
Source:Copyright of this article belongs to Elsevier Science.
Keywords:Candida albicans; ABC Transporter; Cdr1p; Nucleotide-binding Domain; Covalent Modification; Cysteine; ATP Hydrolysis; ATPase; N-ethylmaleimide; Structural Homology
ID Code:39358
Deposited On:12 May 2011 05:42
Last Modified:12 May 2011 05:42

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