Jha Sudhakar, Karnani Neerja, Lynn Andrew M, Prasad Rajendra
Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
Biochem Biophys Res Commun. 2003 Oct 24;310(3):869-75. doi: 10.1016/j.bbrc.2003.09.094.
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.
N-乙基马来酰亚胺（NEM）会损害白色念珠菌耐药基因产物Cdr1p的N端核苷结合域（NBD）的ATP酶功能。为了确定对此有作用的反应性半胱氨酸，我们采用了一种三管齐下的方法，包括共价修饰、半胱氨酸诱变和结构同源性建模。共价修饰结果清楚地表明NEM和碘乙酸（IAA）能够有效抑制N端NBD的ATP酶活性。由于该结构域在其序列中包含五个半胱氨酸残基，我们对每个残基进行了突变，发现其中四个（C325A、C363A、C402A和C462A）对NEM/IAA修饰仍敏感并影响ATP酶活性，而C193A突变则完全消除了催化功能。结构同源性建模数据通过排除半胱氨酸残基内任何可能的相互作用，并得出Cys-193在与ATP和二价阳离子相互作用的键长位置上的重要性，从而能够与ATP和二价阳离子相互作用以严重影响ATP水解，进一步验证了这些生化研究结果。
