Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN 37235, USA.
Cell Rep. 2023 Sep 26;42(9):113061. doi: 10.1016/j.celrep.2023.113061. Epub 2023 Sep 1.
Lon is a widely distributed AAA+ (ATPases associated with diverse cellular activities) protease known for degrading poorly folded and damaged proteins and is often classified as a weak protein unfoldase. Here, using a Lon-degron pair from Mesoplasma florum (MfLon and MfssrA, respectively), we perform ensemble and single-molecule experiments to elucidate the molecular mechanisms underpinning MfLon function. Notably, we find that MfLon unfolds and degrades stably folded substrates and that translocation of these unfolded polypeptides occurs with a ∼6-amino-acid step size. Moreover, the time required to hydrolyze one ATP corresponds to the dwell time between steps, indicating that one step occurs per ATP-hydrolysis-fueled "power stroke." Comparison of MfLon to related AAA+ enzymes now provides strong evidence that HCLR-clade enzymes function using a shared power-stroke mechanism and, surprisingly, that MfLon is more processive than ClpXP and ClpAP. We propose that ample unfoldase strength and substantial processivity are features that contribute to the Lon family's evolutionary success.
Lon 是一种广泛分布的 AAA+（与多种细胞活动相关的 ATPases）蛋白酶，以降解错误折叠和受损的蛋白质而闻名，通常被归类为弱的蛋白质 unfoldase。在这里，我们使用来自 Mesoplasma florum 的 Lon-degron 对（分别为 MfLon 和 MfssrA），进行了组合和单分子实验，以阐明支持 MfLon 功能的分子机制。值得注意的是，我们发现 MfLon 可以展开和降解稳定折叠的底物，并且这些展开的多肽的易位以大约 6 个氨基酸的步幅进行。此外，水解一个 ATP 所需的时间与步与步之间的停留时间相对应，表明每一次 ATP 水解驱动的“动力冲程”都发生一步。将 MfLon 与相关的 AAA+酶进行比较，现在提供了强有力的证据，证明 HCLR 簇酶使用共享的动力冲程机制发挥作用，并且令人惊讶的是，MfLon 比 ClpXP 和 ClpAP 更具连续性。我们提出，充足的 unfoldase 强度和实质性的连续性是 Lon 家族进化成功的特征。
