Kim Young-Chan, Snoberger Aaron, Schupp Jane, Smith David M
Department of Biochemistry, West Virginia University, 1 Medical Center Drive, Morgantown, West Virginia 26506, USA.
Nat Commun. 2015 Oct 14;6:8520. doi: 10.1038/ncomms9520.
The primary functions of the proteasome are driven by a highly allosteric ATPase complex. ATP binding to only two subunits in this hexameric complex triggers substrate binding, ATPase-20S association and 20S gate opening. However, it is unclear how ATP binding and hydrolysis spatially and temporally coordinates these allosteric effects to drive substrate translocation into the 20S. Here, we use FRET to show that the proteasomal ATPases from eukaryotes (RPTs) and archaea (PAN) bind ATP with high affinity at neighbouring subunits, which complements the well-established spiral-staircase topology of the 26S ATPases. We further show that two conserved arginine fingers in PAN located at the subunit interface work together as a single allosteric unit to mediate the allosteric effects of ATP binding, without altering the nucleotide-binding pattern. Rapid kinetics analysis also shows that ring resetting of a sequential hydrolysis mechanism can be explained by thermodynamic equilibrium binding of ATP. These data support a model whereby these two functionally distinct allosteric networks cooperate to translocate polypeptides into the 20S for degradation.
蛋白酶体的主要功能由高度变构的ATP酶复合体驱动。ATP仅与这个六聚体复合体中的两个亚基结合，就能触发底物结合、ATP酶-20S结合以及20S门控打开。然而，目前尚不清楚ATP结合和水解如何在空间和时间上协调这些变构效应，以驱动底物转运到20S中。在这里，我们利用荧光共振能量转移（FRET）表明，真核生物的蛋白酶体ATP酶（RPTs）和古细菌的蛋白酶体ATP酶（PAN）在相邻亚基处以高亲和力结合ATP，这补充了已确立的26S ATP酶的螺旋楼梯拓扑结构。我们进一步表明，PAN中位于亚基界面的两个保守精氨酸指作为一个单一的变构单元共同作用，以介导ATP结合的变构效应，而不改变核苷酸结合模式。快速动力学分析还表明，连续水解机制的环重置可以用ATP的热力学平衡结合来解释。这些数据支持了一个模型，即这两个功能不同的变构网络协同作用，将多肽转运到20S中进行降解。
