Molecular basis of the adaptor domains from AAA-ATPases for locating organelles.

AAA-ATP酶是一种从细菌到人类高度保守的分子机器，它以ATP依赖的方式改变底物蛋白的构象和/或多聚化状态。AAA-ATP酶的特征在于在其分子的中心区域具有一个或几个“AAA”结构域，而各种N-末端结构域可能负责区分其特定功能。我推测，其中一些不仅负责其底物特异性，而且还负责其亚细胞定位。为了解决这一问题，我们采用了所谓的“蛋白质结构域解剖”的方法，首先从PEX 1的N端区域鉴定了一个新的结构域。令人惊讶的是，尽管发现与VCP和NSF的序列同一性低于15%，但PEX 1 NTD的结构与那些相应的结构域非常相似。第二，我们已经确定了从Vps 4 I型AAA-ATP酶的N-末端MIT结构域及其远缘同源物katanin p60的溶液结构。katanin p60的N端结构域与卷曲螺旋区相连，在结构分析前应设计卷曲螺旋区。其他一些AAA-ATP酶需要穿梭因子分子而不是其自身的接头结构域。从其中一个，我们确定了乌巴结构域从Dsk 2蛋白，19 S蛋白体的衔接子，在复合物与泛素。此外，还利用溶液核磁共振技术比较了K48和K63连接的四泛蛋白链的分子刚性和柔性，最终发现所研究的五个系统中有两个结构域与磷酸肌醇具有亲和力。这表明这些结构域具有与细胞器膜结合的功能。

AAA-ATPases are well conserved molecular machines from bacteria to human, which alters the conformations and/or multimerization states of substrate proteins with an ATP-depending manner. AAA-ATPases are characterized as harboring one or several "AAA" domains at their central region of the molecules, whereas various N-terminus domains may be responsible for differentiating their specific functions. I have hypothesized that some of them can be responsible not only for their substrate specificity, but also for their subcellular location. In order to solve this problem, so-called "protein domain anatomy" approach was taken.As a result, first we have identified a novel domain from the PEX1 N-terminal region. Surprisingly, although below 15% of sequence identity found to VCP and NSF, the structure of PEX1NTD is very similar to those corresponding domains. Second, we have identified the solution structures from N-terminal MIT domain of Vps4 type I AAA-ATPase and its distant homolog katanin p60. These domains adopted into an up-and-down three helix bundle fold with a characteristic cleft on the molecular surface, which may act as a ligand binding site, katanin p60 N-terminal domain was associated with a coiled-coil region, which should be designed prior to structural analysis. Some other AAA-ATPases require a shuttle factor molecule instead of its own adaptor domain. From one of them, we determined the solution structure of UBA domain from Dsk2 protein, an adaptor of 19S proteosome, in complex with ubiquitin. In addition, molecular rigidity and flexibility of K48- and K63-linked tetra ubiquitin chain were compared by using solution NMR.Finally, two domains of the five examined systems were found to have affinities to phosphoinositides. This suggested the domains share a function for associating organellar membranes.