The research team led by Prof. ZHANG Kaiming from the University of Science and Technology of China (USTC) of the Chinese Academy of Sciences (CAS), and the collaborators, analyzed complete three-dimensional (3D) structure of the Lon protease, and revealed the molecular mechanisms by which Lon recognized and translocated protein substrates. These two studies were published in Journal of Biological Chemistry and Sciences Advances. 

Lon AAA+ protease (LonA) is a conserved adenosine 5’-triphosphate (ATP)-dependent protease in both prokaryotic and eukaryotic organelles. LonA assembles into a homohexamer, and each monomer contains an N-terminal domain, an intermediate ATPase domain, and a C-terminal protease domain. It plays an important role in cellular protein homeostasis by degrading damaged or misfolded proteins, thus preventing these unwanted proteins from forming toxic aggregates. 

Although multiple structural and functional features of LonA have been reported in previous studies, the mechanisms of its mode of actions are still unknown. Therefore, it is necessary to analyze the complete structure of the full-length Lon. 

By using cryo-electron microscopy (cryo-EM), the researchers determined the structure of LonA at 3.6-angstrom resolution in a substrate-engaged state. The obtained structure revealed the mechanism of a processive rotary translocation mediated by LonA-specific nucleotide-dependent allosteric coordination among the ATPase domains, which was induced by substrate binding. 

However, due to the lack of the understanding of the N-terminal region of Lon hexamer, the mechanism of how Lon works remains unclear. Therefore, the researchers analyzed complete 3D structures of the Lon protease in two substrate-bound states with a 2.4-angstrom resolution. They found that these structures served as a multipurpose platform that controlled the access of substrates to the AAA+ ring, provided a ruler-based mechanism for substrate selection, and acted as a pulley device to facilitate unfolding of the translocated substrate. 

The findings of these studies provide a complete framework for understanding the structural mechanisms of Lon and other AAA+ proteases with similar activity.