Stanton Destini A, Ellis Emily A, Cruse Mariah R, Jedlinski Rafael, Kraut Daniel A
Department of Chemistry, Villanova University, Villanova, PA, 19085, USA.
Department of Chemistry, Villanova University, Villanova, PA, 19085, USA.
Biochem Biophys Res Commun. 2023 Oct 15;677:162-167. doi: 10.1016/j.bbrc.2023.08.025. Epub 2023 Aug 12.
The 26S proteasome is responsible for the unfolding and degradation of intracellular proteins in eukaryotes. A hexameric ring of ATPases (Rpt1-Rpt6) grabs onto substrates and unfolds them by pulling them through a central pore and translocating them into the 20S degradation chamber. A set of pore loops containing a so-called aromatic paddle motif in each Rpt subunit is believed to be important for the proteasome's ability to unfold and translocate substrates. Based on structural and mechanistic experiments, paddles from adjacent Rpt subunits, which are arrayed in a spiral staircase conformation, grip and pull on the substrate in a hand-over-hand type mechanism, disengaging at the bottom of the staircase and re-engaging at the top. We tested the contribution of the aromatic paddles to unfolding substrates of differing stabilities by mutating the paddles singly or in combination. For an easy-to-unfold substrate (a circular permutant of green fluorescent protein; GFP), mutations had little effect on degradation rates. For a substrate with moderate stability (enhanced GFP), there were modest effects of individual mutations on GFP unfolding rates, and alternating aromatic paddle mutants had a larger detrimental effect on unfolding than sequential mutants. For a more stable substrate (superfolder GFP), unfolding is overall slower, and multiple simultaneous mutations essentially prevent unfolding. Our results highlight the context-dependent need for grip during unfolding, support the hand-over-hand model for substrate unfolding and translocation, and suggest that for hard-to-unfold substrates, it is important to have simultaneous strong contacts to the substrate for unfolding to occur. The results also suggest a kinetic proofreading model, where substrates that cannot be easily unfolded are instead clipped, removing the initiation region and preventing futile unfolding attempts.
26S蛋白酶体负责真核生物细胞内蛋白质的解折叠和降解。由ATP酶组成的六聚体环（Rpt1 - Rpt6）抓住底物，通过将它们拉过中心孔并将其转运到20S降解腔室来使其解折叠。每个Rpt亚基中一组含有所谓芳香桨状基序的孔环被认为对蛋白酶体解折叠和转运底物的能力很重要。基于结构和机制实验，以螺旋楼梯构象排列的相邻Rpt亚基的桨状结构以接力式机制抓住并拉动底物，在楼梯底部脱离并在顶部重新结合。我们通过单独或组合突变桨状结构来测试芳香桨状结构对不同稳定性底物解折叠的贡献。对于易于解折叠的底物（绿色荧光蛋白的环状置换变体；GFP），突变对降解速率影响很小。对于具有中等稳定性的底物（增强型GFP），单个突变对GFP解折叠速率有适度影响，交替的芳香桨状突变体对解折叠的有害影响比连续突变体更大。对于更稳定的底物（超级折叠GFP），解折叠总体较慢，多个同时发生的突变基本上阻止了解折叠。我们的结果突出了解折叠过程中对抓握的上下文依赖性需求，支持底物解折叠和转运的接力模型，并表明对于难以解折叠的底物，重要的是与底物同时有强接触才能发生解折叠。结果还提出了一种动力学校对模型，即不易解折叠的底物反而被剪切，去除起始区域并防止徒劳的解折叠尝试。
