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

AAA-ATPase是一种从细菌到人类都非常保守的分子机器，它以依赖于ATP的方式改变底物蛋白质的构象和/或多聚化状态。AAA-ATPase的特征是在其分子的中心区域含有一个或多个“AAA”结构域，而不同的N-末端结构域可能负责区分它们的特定功能。我假设它们中的一些不仅可以对它们的底物专一性负责，还可以对它们的亚细胞位置负责。为了解决这个问题，我们采用了蛋白质结构域解剖的方法。结果，我们首先从PEX1的N-末端区域中鉴定了一个新的结构域。令人惊讶的是，尽管PEX1NTD与VCP和NSF的序列同源性低于15%，但其结构与相应的结构域非常相似。其次，我们鉴定了Vps4I型AAA-ATPase的N-末端MIT结构域及其远端同源蛋白katanin p60的溶液结构。这些结构域形成一个上下三股螺旋的束状折叠结构，分子表面有一个特征性的裂隙，可能作为配基结合部位，katanin p60的N-末端结构域与一个螺旋卷曲区域相关联，这一区域应该在结构分析之前设计。其他一些AAA-ATPase需要一个穿梭因子分子，而不是它自己的适配域。其中，我们确定了19S蛋白体适配子Dsk2蛋白与泛素的络合物中的UBA结构域的溶液结构。此外，用溶液核磁共振比较了K48和K63连接的四泛素链的分子刚性和柔性，最后发现五个系统中的两个结构域与磷脂酰肌醇有亲和力。这表明这些结构域具有结合细胞膜的功能。

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.