The ATPase Associated with diverse cellular Activities (AAA+) family of proteases play crucial roles in cellular proteolysis and stress responses. Like other AAA+ proteases, the Lon protease is known to be allosterically regulated by nucleotide and substrate binding. Although it was originally classified as a DNA binding protein, the impact of DNA binding on Lon activity is unclear. In this study, we characterize the regulation of Lon by single-stranded DNA (ssDNA) binding and serendipitously identify general activation strategies for Lon. Upon binding to ssDNA, Lon's ATP hydrolysis rate increases due to improved nucleotide binding, leading to enhanced degradation of protein substrates, including physiologically important targets. We demonstrate that mutations in basic residues that are crucial for Lon's DNA binding not only reduce ssDNA binding but result in charge-specific consequences on Lon activity. Introducing negative charge at these sites induces activation akin to that induced by ssDNA binding, whereas neutralizing the charge reduces Lon's activity. Based on single molecule measurements, we find this change in activity correlated with changes in Lon oligomerization. Our study provides insights into the complex regulation of the Lon protease driven by electrostatic contributions from either DNA binding or mutations.

中文翻译：

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通过 ssDNA 结合和局部电荷变化对 Lon 蛋白酶进行变构调节。


与多种细胞活动相关的 ATP 酶 （AAA+） 蛋白酶家族在细胞蛋白水解和应激反应中起着至关重要的作用。与其他 AAA+ 蛋白酶一样，已知 Lon 蛋白酶受核苷酸和底物结合的变构调节。虽然它最初被归类为 DNA 结合蛋白，但 DNA 结合对 Lon 活性的影响尚不清楚。在这项研究中，我们表征了单链 DNA （ssDNA） 结合对 Lon 的调节，并偶然确定了 Lon 的一般激活策略。与 ssDNA 结合后，由于核苷酸结合的改善，Lon 的 ATP 水解速率增加，导致蛋白质底物（包括生理上重要的靶标）的降解增强。我们证明，对 Lon 的 DNA 结合至关重要的碱性残基突变不仅会减少 ssDNA 结合，还会对 Lon 活性产生电荷特异性影响。在这些位点引入负电荷会诱导类似于 ssDNA 结合诱导的激活，而中和电荷会降低 Lon 的活性。基于单分子测量，我们发现这种活性变化与 Lon 寡聚化的变化相关。我们的研究提供了对 DNA 结合或突变的静电作用驱动的 Lon 蛋白酶复杂调控的见解。