Zhang Xujia, Stoffels Katinka, Wurzbacher Stephanie, Schoofs Geert, Pfeifer Günter, Banerjee Tisha, Parret Annabel H A, Baumeister Wolfgang, De Mot René, Zwickl Peter
Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, D-82152 Martinsried, Germany.
J Struct Biol. 2004 Apr-May;146(1-2):155-65. doi: 10.1016/j.jsb.2003.10.020.
Deletion mutants of the Rhodococcus erythropolis ARC AAA ATPase were generated and characterized by biochemical analysis and electron microscopy. Based on sequence comparisons the ARC protein was divided into three consecutive regions, the N-terminal coiled coil, the central ARC-specific inter domain and the C-terminal AAA domain. When the ARC AAA domain was expressed separately it formed aggregates of undefined structure. However, when the AAA domain was expressed in conjunction with the preceeding inter domain, but without the N-terminal coiled coil, high-molecular weight-complexes were formed (ARC-DeltaCC) which showed an N-ethylmaleimide-sensitive ATPase activity. In 2D crystallization experiments the ARC-DeltaCC particles yielded crystals nearly identical to those formed by the wild-type ARC complexes. Thus, the N-terminal coiled coil, which was proposed to have a role in the assembly of and/or interaction between the eukaryotic AAA ATPases in the 26S proteasome, is neither essential for assembly nor for ATP hydrolysis of the ARC ATPase. The N-terminal domain of related AAA ATPases mediates the interaction with substrates or co-factors, suggesting a regulatory function for the N-terminal coiled coil of the ARC ATPase. Surprisingly, the mutant ARC protein ARC-DeltaAAA consisting of the N-terminal coiled coil and the central inter domain, but deleted for the C-terminal AAA domain, was shown to form a dodecameric complex with sixfold symmetry. This suggests an important role of the inter domain for the ordered assembly of the ARC ATPase.
通过生化分析和电子显微镜对红平红球菌ARC AAA ATP酶的缺失突变体进行了构建和表征。基于序列比较，ARC蛋白被分为三个连续区域，即N端卷曲螺旋、中央ARC特异性结构域间区域和C端AAA结构域。当单独表达ARC AAA结构域时，它形成了结构不明确的聚集体。然而，当AAA结构域与前面的结构域间区域一起表达，但没有N端卷曲螺旋时，形成了高分子量复合物（ARC-ΔCC），其显示出对N-乙基马来酰亚胺敏感的ATP酶活性。在二维结晶实验中，ARC-ΔCC颗粒产生的晶体与野生型ARC复合物形成的晶体几乎相同。因此，在26S蛋白酶体中被认为在真核AAA ATP酶的组装和/或相互作用中起作用的N端卷曲螺旋，对于ARC ATP酶的组装和ATP水解都不是必需的。相关AAA ATP酶的N端结构域介导与底物或辅助因子的相互作用，这表明ARC ATP酶的N端卷曲螺旋具有调节功能。令人惊讶的是，由N端卷曲螺旋和中央结构域间区域组成但缺失C端AAA结构域的突变ARC蛋白ARC-ΔAAA，被证明形成了具有六重对称性的十二聚体复合物。这表明结构域间区域在ARC ATP酶的有序组装中起重要作用。
