Summer Heike, Bruderer Roland, Weber-Ban Eilika
Institute of Molecular Biology and Biophysics, ETH Zürich, 8093 Zürich, Switzerland.
J Struct Biol. 2006 Oct;156(1):120-9. doi: 10.1016/j.jsb.2006.01.010. Epub 2006 Mar 9.
We investigated a new archaeal member of the AAA+ protein family (ATPases associated with various cellular activities) which is found in all methanogenic archaea and the sulphate-reducer Archaeoglobus fulgidus. These proteins cluster to COG1223 predicted to form a subgroup of the AAA+ ATPases. The gene from A. fulgidus codes for a protein of 40 kDa monomeric molecular weight, which we overexpressed in Escherichia coli and purified to homogeneity. The protein forms ring-shaped complexes with a diameter of 125A as determined by electron microscopy. Using sedimentation equilibrium analysis we demonstrate that it assembles into hexamers over a wide concentration range both in presence and absence of ATP. As suggested by homology to other members of the AAA+ family, the complex binds and hydrolyzes ATP. Michaelis-Menten analysis revealed a k(cat) of 118 min(-1) and a K(M) of 1.4 mM at 78 degrees C. This hyperthermophilic archaeal ATPase is stable to 86 degrees C and the ATPase activity is maximal at this temperature. The protein is most homologous to the AAA-domain of FtsH from bacteria, while the N-terminal domain shows predicted structural homology to members of the CDC48 family of AAA proteins. Possible roles of this new AAA+ protein are discussed.
我们研究了AAA+蛋白家族（与多种细胞活动相关的ATP酶）中的一个新古菌成员，该成员存在于所有产甲烷古菌和硫酸盐还原菌嗜热栖热菌中。这些蛋白聚集到预测形成AAA+ ATP酶一个亚组的COG1223中。嗜热栖热菌的基因编码一种单体分子量为40 kDa的蛋白，我们在大肠杆菌中对其进行了过表达并纯化至同质。通过电子显微镜确定，该蛋白形成直径为125Å的环形复合物。使用沉降平衡分析，我们证明它在存在和不存在ATP的情况下，在很宽的浓度范围内都组装成六聚体。与AAA+家族的其他成员同源性表明，该复合物结合并水解ATP。米氏分析显示，在78℃时，k(cat)为118 min(-1)，K(M)为1.4 mM。这种嗜热古菌ATP酶在86℃时稳定，且ATP酶活性在此温度下最大。该蛋白与细菌FtsH的AAA结构域最同源，而N端结构域与AAA蛋白CDC48家族成员在预测结构上具有同源性。本文讨论了这种新的AAA+蛋白的可能作用。
