The ATP-dependent Lon protease belongs to a unique group of proteases that bind DNA. Eukaryotic Lon is a homo-oligomeric ring-shaped complex localized to the mitochondrial matrix. In vitro, human Lon binds specifically to a single-stranded GT-rich DNA sequence overlapping the light strand promoter of human mitochondrial DNA (mtDNA). We demonstrate that Lon binds GTrich DNA sequences found throughout the heavy strand of mtDNA and that it also interacts specifically with GU-rich RNA. ATP inhibits the binding of Lon to DNA or RNA, whereas the presence of protein substrate increases the DNA binding affinity of Lon 3.5-fold. We show that nucleotide inhibition and protein substrate stimulation coordinately regulate DNA binding. In contrast to the wild type enzyme, a Lon mutant lacking both ATPase and protease activity binds nucleic acid; however, protein substrate fails to stimulate binding. These results suggest that conformational changes in the Lon holoenzyme induced by nucleotide and protein substrate modulate the binding affinity for single-stranded mtDNA and RNA in vivo. Co-immunoprecipitation experiments show that Lon interacts with mtDNA polymerase ␥ and the Twinkle helicase, which are components of mitochondrial nucleoids. Taken together, these results suggest that Lon participates directly in the metabolism of mtDNA.The ATP-dependent Lon (La) protease is a multi-functional enzyme conserved from archaea to mammalian mitochondria (1-6). Mitochondrial Lon is a homo-oligomeric complex in which each monomer carries separate sites for the binding and hydrolysis of both ATP and protein substrate (7,8). Lon selectively degrades abnormal polypeptides, thus serving a quality control function in protein biogenesis (9 -12). The energy requirement of Lon is mechanistically similar to that described for other ATP-dependent proteases such as ClpAP and the proteasome (13)(14)(15)(16)(17)(18)(19)(20). The binding of substrate polypeptide to Lon stimulates its ATPase activity (21). ATP hydrolysis is required for the processive unfolding of a substrate that permits peptide bond cleavage. Conformational changes within the Lon holoenzyme likely coincide with the cycle of ATP binding and hydrolysis, ADP dissociation, and ATP rebinding, as well as with the binding and hydrolysis of protein substrate.Lon belongs to a unique group of proteases that also bind DNA. Other proteases or protease components that bind DNA include the human adenovirus proteinase (AVP), 1 the adipocyte-enhancer binding protein 1 (AEBP1), Gal6p/bleomycin hydrolase, and the 19 S particle of the 26 S proteasome. AVP requires two co-factors for maximal protease activity: viral DNA and a viral peptide produced by AVP proteolysis. It is proposed that AVP utilizes its nonspecific DNA binding activity to locate its viral protein substrates (22). By contrast, the AEBP1 repressor binds specifically to the adipocyte enhancer 1 element. The carboxypeptidase activity of AEBP1 is stimulated by adipocyte enhancer 1 element DNA and is required for transcriptional repression...

Mechanistic studies of ATP-dependent proteolysis demonstrate that substrate unfolding is a prerequisite for processive peptide bond hydrolysis. We show that mitochondrial Lon also degrades folded proteins and initiates substrate cleavage non-processively. Two mitochondrial substrates with known or homology-derived three-dimensional structures were used: the mitochondrial processing peptidase ␣-subunit (MPP␣) and the steroidogenic acute regulatory protein (StAR). Peptides generated during a time course of Lon-mediated proteolysis were identified and mapped within the primary, secondary, and tertiary structure of the substrate. Initiating cleavages occurred preferentially between hydrophobic amino acids located within highly charged environments at the surface of the folded protein. Subsequent cleavages proceeded sequentially along the primary polypeptide sequence. We propose that Lon recognizes specific surface determinants or folds, initiates proteolysis at solvent-accessible sites, and generates unfolded polypeptides that are then processively degraded.

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 (hLon) are linked to serious diseases including myopathies, paraplegia, and cancer. Here, we present the first 3D structure of full-length hLon using cryo-electron microscopy. hLon 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 closedring 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 hLon mutant lacking the first 156 amino acids are severely disturbed, showing that hLon's N-terminal domains are crucial for the overall structure of the hLon, maintaining a conformation allowing its proper functioning.Human Lon (hLon, P36776) is a mitochondrial AAA + protein (ATPases Associated with diverse cellular Activities) belonging to the LonA protease subfamily 1 , which plays a crucial role in the maintenance of mitochondrial homeostasis. Its primary function is the degradation of misfolded, oxidatively modified and regulatory proteins 2 , but it also participates in the maintenance of mitochondrial DNA 3 and possesses a chaperone activity important for the proper assembly of protein complexes 4 . Changes in hLon expression have been linked to severe diseases, including epilepsy, myopathy, paraplegia, and cancer 5 . In several cancerous tissues, overexpression of hLon promoted proliferation of cancer cells 6 by remodeling their mitochondrial functions 7 while its down-regulation led to apoptosis and cell death 8 . Silencing of hLon or pharmacologically inhibiting its activity has therefore been considered as a new target for the development of anticancer drugs 9 .Like other ATPases, Lon's activities are accompanied by conformational changes induced by ATP binding and hydrolysis 10,11 . Early biochemical studies revealed that the binding of protein substrates by Lon stimulates its ATPase and peptidase activities and that this activation is likely to be allosteric 12,13 . Menon and Goldberg 12 first suggested a substrate-induced proteolytic mechanism, in which the default state of Lon is its inactive, ADP-bound form preventing accidental degradation of cellular proteins. Upon substrate binding, this form releases its ADP molecules and binds ATP, which is followed by its rapid hydrolysis and the cleavage of peptide bonds. In this mechanism, Lon can bind and hydrolyze ATP as long as the substrate binding sites are occupied. More recently, the idea that Lon's ATPase and protease activities are under allosteric...

ATP-dependent proteases are crucial for cellular homeostasis. By degrading short-lived regulatory proteins, they play an important role in the control of many cellular pathways and, through the degradation of abnormally misfolded proteins, protect the cell from a buildup of aggregates. Disruption or disregulation of mammalian mitochondrial Lon protease leads to severe changes in the cell, linked with carcinogenesis, apoptosis, and necrosis. Here we present the structure of the proteolytic domain of human mitochondrial Lon at 2 Å resolution. The fold resembles those of the three previously determined Lon proteolytic domains from Escherichia coli, Methanococcus jannaschii, and Archaeoglobus fulgidus. There are six protomers in the asymmetric unit, four arranged as two dimers. The intersubunit interactions within the two dimers are similar to those between adjacent subunits of the hexameric ring of E. coli Lon, suggesting that the human Lon proteolytic domain also forms hexamers. The active site contains a 3 10 helix attached to the N-terminal end of a-helix 2, which leads to the insertion of Asp852 into the active site, as seen in M. jannaschii. Structural considerations make it likely that this conformation is proteolytically inactive. When comparing the intersubunit interactions of human with those of E. coli Lon taken with biochemical data leads us to propose a mechanism relating the formation of Lon oligomers with a conformational shift in the active site region coupled to a movement of a loop in the oligomer interface, converting the proteolytically inactive form seen here to the active one in the E. coli hexamer.