Stinson Benjamin M, Baytshtok Vladimir, Schmitz Karl R, Baker Tania A, Sauer Robert T
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
1] Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Nat Struct Mol Biol. 2015 May;22(5):411-6. doi: 10.1038/nsmb.3012. Epub 2015 Apr 13.
The hexameric AAA+ ring of Escherichia coli ClpX, an ATP-dependent machine for protein unfolding and translocation, functions with the ClpP peptidase to degrade target substrates. For efficient function, ClpX subunits must switch between nucleotide-loadable (L) and nucleotide-unloadable (U) conformations, but the roles of switching are uncertain. Moreover, it is controversial whether working AAA+-ring enzymes assume symmetric or asymmetric conformations. Here, we show that a covalent ClpX ring with one subunit locked in the U conformation catalyzes robust ATP hydrolysis, with each unlocked subunit able to bind and hydrolyze ATP, albeit with highly asymmetric position-specific affinities. Preventing U↔L interconversion in one subunit alters the cooperativity of ATP hydrolysis and reduces the efficiency of substrate binding, unfolding and degradation, showing that conformational switching enhances multiple aspects of wild-type ClpX function. These results support an asymmetric and probabilistic model of AAA+-ring activity.
大肠杆菌ClpX的六聚体AAA+环是一种依赖ATP的蛋白质解折叠和转运机器，它与ClpP肽酶共同作用以降解靶底物。为实现高效功能，ClpX亚基必须在可加载核苷酸（L）和不可卸载核苷酸（U）的构象之间切换，但其切换的作用尚不确定。此外，有争议的是，起作用的AAA+环酶是呈对称构象还是不对称构象。在此，我们表明，一个共价ClpX环，其中一个亚基锁定在U构象，能催化强劲的ATP水解，每个未锁定的亚基都能够结合并水解ATP，尽管具有高度不对称的位置特异性亲和力。阻止一个亚基中的U↔L相互转换会改变ATP水解的协同性，并降低底物结合、解折叠和降解的效率，这表明构象切换增强了野生型ClpX功能的多个方面。这些结果支持了AAA+环活性的不对称和概率模型。
