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 cryoelectron 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 regulated 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 substrate protein. [Display omitted] •The full structure of LonP1 has been determined in eight conformations•A molecular movie of LonP1 in action is presented•LonP1 is a molecular ratchet•LonP1 may have a second proteolytic site Mohammed et al. report the structure of LonP1 in eight different conformations. These reveal essential details of how this vital mitochondrial protease recognizes and digests redundant and damaged proteins.