Molecular-motor coordination The proteasome is a cytosolic molecular machine that recognizes and degrades unneeded or damaged proteins that have been tagged with ubiquitin. A heterohexameric adenosine triphosphatase motor pulls the substrate into the proteolytic chamber, while at the same time, a protein located at the entrance of this motor removes the ubiquitin. De la Peña et al. trapped the substrate inside the motor by inhibiting removal of ubiquitin. This allowed them to determine cryo–electron microscopy structures in the presence of substrate and adenosine triphosphate (ATP). The findings distinguish three sequential conformational states that show how ATP binding, hydrolysis, and phosphate release are coordinated between the six subunits of the motor to cause the conformational changes that translocate the substrate through the proteasome. Science, this issue p. eaav0725 The structure of the yeast 26S proteasome reveals sequential states of ATP binding, hydrolysis, and substrate translocation. INTRODUCTION As the major protease in eukaryotic cells and the final component of the ubiquitin-proteasome system, the 26S proteasome is responsible for protein homeostasis and the regulation of numerous vital processes. Misfolded, damaged, or obsolete regulatory proteins are marked for degradation by the attachment of polyubiquitin chains, which bind to ubiquitin receptors of the proteasome. A heterohexameric ring of AAA+ (ATPases associated with diverse cellular activities) subunits then uses conserved pore loops to engage, mechanically unfold, and translocate protein substrates into a proteolytic core for cleavage while the deubiquitinase Rpn11 removes substrate-attached ubiquitin chains. RATIONALE Despite numerous structural and functional studies, the mechanisms by which adenosine triphosphate (ATP) hydrolysis drives the conformational changes responsible for protein degradation remained elusive. Structures of related homohexameric AAA+ motors, in which bound substrates were stabilized with ATP analogs or hydrolysis-eliminating mutations, revealed snapshots of ATPase subunits in different nucleotide states and spiral-staircase arrangements of pore loops around the substrate. These structures gave rise to “hand-over-hand” translocation models by inferring how individual subunits may progress through various substrate-binding conformations. However, the coordination of ATP-hydrolysis steps and their mechanochemical coupling to propelling substrate were unknown. RESULTS We present the cryo–electron microscopy (cryo-EM) structures of the actively ATP-hydrolyzing, substrate-engaged 26S proteasome with four distinct motor conformations. Stalling substrate translocation at a defined position by inhibiting deubiquitination led to trapped states in which the substrate-attached ubiquitin remains functionally bound to the Rpn11 deubiquitinase, and the scissile isopeptide bond of ubiquitin is aligned with the substrate-translocation trajectory through the AAA+ motor. Our structures suggest a ubiquitin capture mechanism, in which mechanical pulling on the substrate by the AAA+ motor delivers ubiquitin modifications directly into the Rpn11 catalytic groove and accelerates isopeptide cleavage for efficient, cotranslocational deubiquitination. These structures also show how the substrate polypeptide traverses from the Rpn11 deubiquitinase, through the AAA+ motor, and into the core peptidase. The proteasomal motor thereby adopts staircase arrangements with five substrate-engaged subunits and one disengaged subunit. Four of the substrate-engaged subunits are ATP bound, whereas the subunit at the bottom of the staircase and the disengaged subunit are bound to adenosine diphosphate (ADP). CONCLUSION Of the four distinct motor states we observed, three apparently represent sequential stages of ATP binding, hydrolysis, and substrate translocation and hence reveal the coordination of individual steps in the ATPase cycle and their mechanochemical coupling with translocation. ATP hydrolysis occurs in the fourth substrate-engaged subunit from the top, concomitantly with exchange of ADP for ATP in the disengaged subunit. The subsequent transition, which is likely triggered by phosphate release from the fourth, posthydrolysis subunit of the staircase, then involves major conformational changes of the entire ATPase hexamer. The bottom ADP-bound subunit is displaced and the previously disengaged subunit binds the substrate at the top of the staircase, while the four engaged subunits move downward as a rigid body and translocate substrate toward the peptidase. Our likely consecutive proteasome conformations, together with previously determined substrate-free structures, suggest a sequential progression of ATPase subunits through the ATP-hydrolysis cycle. We hypothesize that, in general, hexameric AAA+ translocases function by this sequential mechanism. Cryo-EM structures of the substrate-engaged 26S proteasome. (A) Substrate path through the proteasome, with ubiquitin bound to Rpn11 (left inset) and the substrate polypeptide traversing through the AAA+ motor into the core peptidase. (B) Schematic showing coordinated ATP hydrolysis and nucleotide exchange observed between consecutive motor states. (C) Substrate translocation is driven by changes in the spiral-staircase arrangement of pore loops, as indicated by arrows. The 26S proteasome is the primary eukaryotic degradation machine and thus is critically involved in numerous cellular processes. The heterohexameric adenosine triphosphatase (ATPase) motor of the proteasome unfolds and translocates targeted protein substrates into the open gate of a proteolytic core while a proteasomal deubiquitinase concomitantly removes substrate-attached ubiquitin chains. However, the mechanisms by which ATP hydrolysis drives the conformational changes responsible for these processes have remained elusive. Here we present the cryo–electron microscopy structures of four distinct conformational states of the actively ATP-hydrolyzing, substrate-engaged 26S proteasome. These structures reveal how mechanical substrate translocation accelerates deubiquitination and how ATP-binding, -hydrolysis, and phosphate-release events are coordinated within the AAA+ (ATPases associated with diverse cellular activities) motor to induce conformational changes and propel the substrate through the central pore.

中文翻译：

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底物参与的 26S 蛋白酶体结构揭示了 ATP 水解驱动的易位机制

分子-运动协调 蛋白酶体是一种细胞溶质分子机器，可识别和降解已用泛素标记的不需要或损坏的蛋白质。异六聚腺苷三磷酸酶马达将底物拉入蛋白水解室，同时，位于该马达入口处的蛋白质去除泛素。德拉佩尼亚等。通过抑制泛素的去除，将底物困在电机内。这使他们能够在存在底物和三磷酸腺苷 (ATP) 的情况下确定冷冻电子显微镜结构。研究结果区分了三个连续的构象状态，这些状态显示了 ATP 结合、水解和磷酸盐释放如何在马达的六个亚基之间协调，从而导致通过蛋白酶体转运底物的构象变化。科学，这个问题 p。eaav0725 酵母 26S 蛋白酶体的结构揭示了 ATP 结合、水解和底物易位的连续状态。引言 作为真核细胞中的主要蛋白酶和泛素-蛋白酶体系统的最终组成部分，26S 蛋白酶体负责蛋白质稳态和许多重要过程的调节。错误折叠、损坏或过时的调节蛋白通过与蛋白酶体的泛素受体结合的多泛素链的附着而被标记为降解。AAA+（与多种细胞活动相关的 ATP 酶）亚基的异六聚体环然后使用保守的孔环来接合、机械展开并将蛋白质底物转移到蛋白水解核心中进行切割，而去泛素化酶 Rpn11 去除底物附着的泛素链。基本原理尽管进行了大量结构和功能研究，但三磷酸腺苷 (ATP) 水解驱动导致蛋白质降解的构象变化的机制仍然难以捉摸。相关的 homohexameric AAA+ 马达的结构，其中结合的底物用 ATP 类似物或水解消除突变稳定，揭示了不同核苷酸状态的 ATPase 亚基的快照和底物周围孔环的螺旋楼梯排列。通过推断单个亚基如何通过各种底物结合构象，这些结构产生了“手交手”易位模型。然而，ATP 水解步骤的协调及其与推进底物的机械化学耦合是未知的。结果我们展示了具有四种不同运动构象的主动 ATP 水解、底物接合的 26S 蛋白酶体的冷冻电子显微镜 (cryo-EM) 结构。通过抑制去泛素化使底物易位停滞在定义的位置导致被困状态，其中底物附着的泛素在功能上仍与 Rpn11 去泛素酶结合，并且泛素的易裂异肽键通过 AAA+ 马达与底物易位轨迹对齐。我们的结构表明了一种泛素捕获机制，其中通过 AAA+ 马达对底物的机械拉动将泛素修饰直接传递到 Rpn11 催化凹槽中，并加速异肽裂解以实现高效的共易位去泛素化。这些结构还显示了底物多肽如何从 Rpn11 去泛素化酶穿过 AAA+ 马达并进入核心肽酶。蛋白酶体马达因此采用阶梯式排列，具有五个与底物接合的亚基和一个脱离的亚基。四个与底物结合的亚基与 ATP 结合，而楼梯底部的亚基和脱离的亚基与二磷酸腺苷 (ADP) 结合。结论 在我们观察到的四种不同运动状态中，三种显然代表了 ATP 结合、水解和底物易位的连续阶段，因此揭示了 ATPase 循环中各个步骤的协调及其与易位的机械化学耦合。ATP 水解发生在从顶部数第四个与底物结合的亚基中，伴随着分离的亚基中 ADP 与 ATP 的交换。随后的转变很可能由楼梯的第四个水解后亚基释放磷酸盐触发，然后涉及整个 ATPase 六聚体的主要构象变化。底部的 ADP 结合亚基被置换，先前脱离的亚基与楼梯顶部的底物结合，而四个接合的亚基作为刚体向下移动，并将底物移向肽酶。我们可能的连续蛋白酶体构象，连同先前确定的无底物结构，表明 ATP 酶亚基通过 ATP 水解循环的顺序进展。我们假设，一般来说，六聚 AAA+ 转位酶通过这种顺序机制起作用。与底物结合的 26S 蛋白酶体的冷冻电镜结构。(A) 通过蛋白酶体的底物路径，泛素与 Rpn11（左插图）结合，底物多肽穿过 AAA+ 马达进入核心肽酶。(B) 示意图显示了在连续运动状态之间观察到的协调 ATP 水解和核苷酸交换。(C) 底物易位是由孔环的螺旋楼梯排列的变化驱动的，如箭头所示。26S 蛋白酶体是主要的真核生物降解机器，因此与许多细胞过程密切相关。蛋白酶体的异六聚腺苷三磷酸酶 (ATPase) 马达展开并将目标蛋白质底物转移到蛋白水解核心的开放门中，而蛋白酶体去泛素化酶同时去除底物附着的泛素链。然而，ATP水解驱动负责这些过程的构象变化的机制仍然难以捉摸。在这里，我们展示了主动 ATP 水解、与底物结合的 26S 蛋白酶体的四种不同构象状态的冷冻电子显微镜结构。这些结构揭示了机械底物易位如何加速去泛素化以及 ATP 结合、-水解、