The N-terminal domain plays a crucial role in the structure of a full-length human mitochondrial Lon protease

Lon is an essential, multitasking AAA + protease regulating many cellular processes in species across all kingdoms of life. Altered expression levels of the human mitochondrial Lon protease ( h Lon) are linked to serious diseases including myopathies, paraplegia, and cancer. Here, we present the first 3D structure of full-length h Lon using cryo-electron microscopy. h Lon has a unique three-dimensional structure, in which the proteolytic and ATP-binding domains (AP-domain) form a hexameric chamber, while the N-terminal domain is arranged as a trimer of dimers. These two domains are linked by a narrow trimeric channel composed likely of coiled-coil helices. In the presence of AMP-PNP, the AP-domain has a closed-ring conformation and its N-terminal entry gate appears closed, but in ADP binding, it switches to a lock-washer conformation and its N-terminal gate opens, which is accompanied by a rearrangement of the N-terminal domain. We have also found that both the enzymatic activities and the 3D structure of a h Lon mutant lacking the first 156 amino acids are severely disturbed, showing that h Lon’s N-terminal domains are crucial for the overall structure of the h Lon, maintaining a conformation allowing its proper functioning.