Catalytic cycling of human mitochondrial Lon protease

The mitochondrial Lon protease (LonP1) regulates mitochondrial health by removing redundant proteins from the mitochondrial matrix. We determined LonP1 in eight nucleotide-dependent conformational states by cry-oelectron microscopy (cryo-EM). The flexible assembly of N-terminal domains had 3-fold symmetry, and its orientation depended on the conformational state. We show that a conserved structural motif around T803 with a high similarity to the trypsin catalytic triad is essential for proteolysis. We show that LonP1 is not regu-lated by redox potential, despite the presence of two conserved cysteines at disulfide-bonding distance in its unfoldase core. Our data indicate how sequential ATP hydrolysis controls substrate protein translocation in a 6-fold binding change mechanism. Substrate protein translocation, rather than ATP hydrolysis, is a rate -limiting step, suggesting that LonP1 is a Brownian ratchet with ATP hydrolysis preventing translocation reversal. 3-fold rocking motions of the flexible N-domain assembly may assist thermal unfolding of the sub-strate protein.

Automated summary

The mitochondrial Lon protease, LonP1, plays a crucial role in maintaining mitochondrial health by removing redundant proteins from the mitochondrial matrix. Cryo-EM analysis revealed eight nucleotide-dependent conformational states of LonP1. The study also showed how sequential ATP hydrolysis controls the translocation of substrate proteins in a 6-fold binding change mechanism.