Conserved Asp327 of walker B motif in the N-terminal nucleotide binding domain (NBD-1) of Cdr1p of Candida albicans has acquired a new role in ATP hydrolysis.
Conserved Asp327 of walker B motif in the N-terminal nucleotide binding domain (NBD-1) of Cdr1p of Candida albicans has acquired a new role in ATP hydrolysis.
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DOI：
10.1021/bi061535t
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发表时间：
2006-12
期刊：
影响因子：
2.9
通讯作者：
V. Rai;M. Gaur;S. Shukla;S. Shukla;S. Ambudkar;S. Komath;R. Prasad
中科院分区：
文献类型：
--
作者：
V. Rai;M. Gaur;S. Shukla;S. Shukla;S. Ambudkar;S. Komath;R. Prasad
Cdr1p核苷酸结合结构域（NBDs）的沃克A和B基序虽然几乎与所有ABC转运蛋白相同，但具有独特的取代。我们过去已经表明，Cdr1p N端NBD的沃克B基序中的色氨酸326和沃克A基序中的半胱氨酸193分别在ATP结合和水解中具有不同的作用。在本研究中，我们研究了N端NBD的沃克B基序中一个高度保守的天冬氨酸327的作用，它前面（色氨酸326）和后面（天冬酰胺328）有非典型的氨基酸取代，并将其与Cdr1p C端NBD中与其等效的高度保守的天冬氨酸1026进行了比较。我们观察到，通过D327N、D327A、D1026N、D1026A和D327N/D1026N取代去除负电荷，导致Cdr1p突变体变体在ATP酶活性和药物外排方面严重受损。重要的是，所有突变体变体在细胞表面表达以及光亲和药物类似物[125I] IAAP和[3H]叠氮平标记方面都表现出与野生型相似的特征。尽管Cdr1p D327N突变体变体与[α - 32P] 8 - 叠氮ATP具有相当的结合能力，但Cdr1p D1026N和Cdr1p D327N/D1026N突变体变体在核苷酸结合方面存在缺陷。从ATP酶活性的pH曲线进一步明显看出，两个保守的羧酸盐残基天冬氨酸327和天冬氨酸1026在功能上是不同的。Cdr1p D327N突变体变体在酸性pH下其残余ATP酶活性提高了约40%，而Cdr1p D1026N突变体变体没有这种pH效应。我们的实验数据表明，N端NBD的天冬氨酸327获得了一种新的作用，即在ATP水解中作为催化碱，这一作用通常是所有非真菌转运蛋白的沃克B基序中保守的天冬氨酸旁边的谷氨酸所具有的。
The Walker A and B motifs of nucleotide binding domains (NBDs) of Cdr1p though almost identical to all ABC transporters, has unique substitutions. We have shown in the past that Trp326 of Walker B and Cys193 of Walker A motifs of N-terminal NBD of Cdr1p have distinct roles in ATP binding and hydrolysis, respectively. In the present study, we have examined the role of a well conserved Asp327 in the Walker B motif of the N-terminal NBD, which is preceded (Trp326) and followed (Asn328) by atypical amino acid substitutions and compared it with its equivalent well conserved Asp1026 of the C-terminal NBD of Cdr1p. We observed that the removal of the negative charge by D327N, D327A, D1026N, D1026A, and D327N/D1026N substitutions, resulted in Cdr1p mutant variants that were severely impaired in ATPase activity and drug efflux. Importantly, all of the mutant variants showed characteristics similar to those of the wild type with respect to cell surface expression and photoaffinity drug analogue [125I] IAAP and [3H] azidopine labeling. Although the Cdr1p D327N mutant variant showed comparable binding with [alpha-32P] 8-azido ATP, Cdr1p D1026N and Cdr1p D327N/D1026N mutant variants were crippled in nucleotide binding. That the two conserved carboxylate residues Asp327 and Asp1026 are functionally different was further evident from the pH profile of ATPase activity. The Cdr1p D327N mutant variant showed approximately 40% enhancement of its residual ATPase activity at acidic pH, whereas no such pH effect was seen with the Cdr1p D1026N mutant variant. Our experimental data suggest that Asp327 of N-terminal NBD has acquired a new role to act as a catalytic base in ATP hydrolysis, a role normally conserved for Glu present adjacent to the conserved Asp in the Walker B motif of all the non-fungal transporters.