Classification of ATP-dependent proteases Lon and comparison of the active sites of their proteolytic domains.

ATP-dependent Lon proteases belong to the superfamily of AAA+ proteins. Until recently, the identity of the residues involved in their proteolytic active sites was not elucidated. However, the putative catalytic Ser-Lys dyad was recently suggested through sequence comparison of more than 100 Lon proteases from various sources. The presence of the catalytic dyad was experimentally confirmed by site-directed mutagenesis of the Escherichia coli Lon protease and by determination of the crystal structure of its proteolytic domain. Furthermore, this extensive sequence analysis allowed the definition of two subfamilies of Lon proteases, LonA and LonB, based on the consensus sequences in the active sites of their proteolytic domains. These differences strictly associate with the specific characteristics of their AAA+ modules, as well as with the presence or absence of an N-terminal domain.     

A Lon-like protease with no ATP-powered unfolding activity

Lon proteases are a family of ATP-dependent proteases involved in protein quality control, with a unique proteolytic domain and an AAA(+) (ATPases associated with various cellular activities) module accommodated within a single polypeptide chain. They were classified into two types as either the ubiquitous soluble LonA or membrane-inserted archaeal LonB. In addition to the energy-dependent forms, a number of medically and ecologically important groups of bacteria encode a third type of Lon-like proteins in which the conserved proteolytic domain is fused to a large N-terminal fragment lacking canonical AAA(+) motifs. Here we showed that these Lon-like proteases formed a clade distinct from LonA and LonB. Characterization of one such Lon-like protease from Meiothermus taiwanensis indicated that it formed a hexameric assembly with a hollow chamber similar to LonA/B. The enzyme was devoid of ATPase activity but retained an ability to bind symmetrically six nucleotides per hexamer; accordingly, structure-based alignment suggested possible existence of a non-functional AAA-like domain. The enzyme degraded unstructured or unfolded protein and peptide substrates, but not well-folded proteins, in ATP-independent manner. These results highlight a new type of Lon proteases that may be involved in breakdown of excessive damage or unfolded proteins during stress conditions without consumption of energy.     

Atomic-resolution crystal structure of the proteolytic domain of Archaeoglobus fulgidus lon reveals the conformational variability in the active sites of lon proteases

The atomic-resolution crystal structure of the proteolytic domain (P-domain, residues 415-621) of Archaeoglobus fulgidus B-type Lon protease (wtAfLonB) and the structures of several mutants have revealed significant differences in the conformation of the active-site residues when compared to other known Lon P-domains, despite the conservation of the overall fold. The catalytic Ser509 is facing the solvent and is distant from Lys552, the other member of the catalytic dyad. Instead, the adjacent Asp508 forms an ion pair with the catalytic lysine residue. Glu506, an analog of the putative third catalytic residue from a related Methanococcus jannaschii LonB, also faces the solvent and does not interact with the catalytic dyad. We have established that full-length wtAfLonB is proteolytically active in an ATP-dependent manner. The loss of enzymatic activity of the S509A mutant confirms the functional significance of this residue, while retention of considerable level of activity by the D508A and E506A mutants rules out their critical involvement in catalysis. In contrast to the full-length enzymes, all individually purified P-domains (wild-type and mutants) were inactive, and the mutations had no influence on the active-site structure. These findings raise the possibility that, although isolated proteolytic domains of both AfLonB and E.coli LonA are able to assemble into expected functional hexamers, the presence of the other domains, as well as substrate binding, may be needed to stabilize the productive conformation of their active sites. Thus, the observed conformational variability may reflect the differences in the stability of active-site structures for the proteolytic counterparts of single-chain Lon versus independently folded proteolytic subunits of two-chain AAA+ proteases.     

The Thermoplasma acidophilum Lon protease has a Ser-Lys dyad active site.

A gene with significant similarity to bacterial Lon proteases was identified during the sequencing of the genome of the thermoacidophilic archaeon Thermoplasma acidophilum. Protein sequence comparison revealed that Thermoplasma Lon protease (TaLon) is more similar to the LonB proteases restricted to Gram-positive bacteria than to the widely distributed bacterial LonA. However, the active site residues of the protease and ATPase domain are highly conserved in all Lon proteases. Using site-directed mutagenesis we show here that TaLon and EcLon, and probably all other Lon proteases, contain a Ser-Lys dyad active site. The TaLon active site mutants were fully assembled and, similar to TaLon wild-type, displayed an apparent molar mass of 430 kDa upon gelfiltration. This would be consistent with a hexameric complex and indeed electron micrographs of TaLon revealed ring-shaped particles, although of unknown symmetry. Comparison of the ATPase activity of Lon wild-type from Thermoplasma or Escherichia coli with respective protease active site mutants revealed differences in Km and V values. This suggests that in the course of protein degradation by wild-type Lon the protease domain might influence the activity of the ATPase domain.     

The active site of a lon protease from Methanococcus jannaschii distinctly differs from the canonical catalytic Dyad of Lon proteases

ATP-dependent Lon proteases catalyze the degradation of various regulatory proteins and abnormal proteins within cells. Methanococcus jannaschii Lon (Mj-Lon) is a homologue of Escherichia coli Lon (Ec-Lon) but has two transmembrane helices within its N-terminal ATPase domain. We solved the crystal structure of the proteolytic domain of Mj-Lon using multiwavelength anomalous dispersion, refining it to 1.9-angstroms resolution. The structure displays an overall fold conserved in the proteolytic domain of Ec-Lon; however, the active site shows uniquely configured catalytic Ser-Lys-Asp residues that are not seen in Ec-Lon, which contains a catalytic dyad. In Mj-Lon, the C-terminal half of the beta4-alpha2 segment is an alpha-helix, whereas it is a beta-strand in Ec-Lon. Consequently, the configurations of the active sites differ due to the formation of a salt bridge between Asp-547 and Lys-593 in Mj-Lon. Moreover, unlike Ec-Lon, Mj-Lon has a buried cavity in the region of the active site containing three water molecules, one of which is hydrogen-bonded to catalytic Ser-550. The geometry and environment of the active site residues in Mj-Lon suggest that the charged Lys-593 assists in lowering the pK(a) of the Ser-550 hydroxyl group via its electrostatic potential, and the water in the cavity acts as a proton acceptor during catalysis. Extensive sequence alignment and comparison of the structures of the proteolytic domains clearly indicate that Lon proteases can be classified into two groups depending on active site configuration and the presence of DGPSA or (D/E)GDSA consensus sequences, as represented by Ec-Lon and Mj-Lon.     

Forms of LonB protease from Archaeoglobus fulgidus devoid of the transmembrane domain: The contribution of the quaternary structure to the regulation of enzyme proteolytic activity

Deletion of the transmembrane domain (TM-domain) of Archaeoglobus fulgidus LonB protease (Archaeoglobus fulgidus (AfLon)) was shown to result in uncontrollable activation of the enzyme proteolytic site and in vivo autolysis yielding a stable and functionally inactive fragment consisting of both α-helical and proteolytic domains (αP). The ΔTM-AfLon-S509A enzyme form, obtained by site-directed mutagenesis of the catalytic Ser residue, is capable of recombination with the αP fragment. The mixed oligomers were shown to be proteolytically active, which indicates a crucial role of subunit interactions in the activation of the AfLon proteolytic site. The thermophilic nature of AfLon protease was found to be due to the special features of the enzyme activity regulation, the structure of ATPase domain, and the quaternary structure.     

Crystal structure of Lon protease: molecular architecture of gated entry to a sequestered degradation chamber

Lon proteases are distributed in all kingdoms of life and are required for survival of cells under stress. Lon is a tandem fusion of an AAA+ molecular chaperone and a protease with a serine‐lysine catalytic dyad. We report the 2.0‐Å resolution crystal structure of Thermococcus onnurineus NA1 Lon (TonLon). The structure is a three‐tiered hexagonal cylinder with a large sequestered chamber accessible through an axial channel. Conserved loops extending from the AAA+ domain combine with an insertion domain containing the membrane anchor to form an apical domain that serves as a gate governing substrate access to an internal unfolding and degradation chamber. Alternating AAA+ domains are in tight‐ and weak‐binding nucleotide states with different domain orientations and intersubunit contacts, reflecting intramolecular dynamics during ATP‐driven protein unfolding and translocation. The bowl‐shaped proteolytic chamber is contiguous with the chaperone chamber allowing internalized proteins direct access to the proteolytic sites without further gating restrictions.     

A novel view on the architecture of the non-catalytic N-terminal region of ATP-dependent LonA proteases

ATP-Dependent Lon-proteases are components of the protein quality control system, which maintains cellular proteome. The Lon family consists of two subfamilies A and B, differing in subunit architecture and intracellular location. We propose here a reinterpretation of the domain organization of the non-catalytic N-terminal region of LonA proteases. Using Escherichia coli LonA protease (EcLon) as an example, it has been shown that a fragment (αN domain) located between the N-terminal domain and the AAA⁺ module is similar to the α1 domain of the first AAA⁺ module of chaperone-disaggregase ClpB. A coiled-coil (CC) region included in the αN domain of LonA is similar to the M domain of ClpB chaperones, which is inserted into the α1 domain. This region is suggested to adopt the structure similar to the propeller-like (PL) domain. The typical architecture of the N-terminal region of LonA proteases is postulated to be characterized by the obligatory presence of a PL domain, included in the αN domain, but may vary in the length and topology of the preceding N-terminal domain, which can have in some cases a more complex structure than in EcLon.     

Crystal Structures of Bacillus subtilis Lon Protease

Lon ATP-dependent proteases are key components of the protein quality control systems of bacterial cells and eukaryotic organelles. Eubacterial Lon proteases contain an N-terminal domain, an ATPase domain, and a protease domain, all in one polypeptide chain. The N-terminal domain is thought to be involved in substrate recognition, the ATPase domain in substrate unfolding and translocation into the protease chamber, and the protease domain in the hydrolysis of polypeptides into small peptide fragments. Like other AAA+ ATPases and self-compartmentalising proteases, Lon functions as an oligomeric complex, although the subunit stoichiometry is currently unclear. Here, we present crystal structures of truncated versions of Lon protease from Bacillus subtilis (BsLon), which reveal previously unknown architectural features of Lon complexes. Our analytical ultracentrifugation and electron microscopy show different oligomerisation of Lon proteases from two different bacterial species, Aquifex aeolicus and B. subtilis. The structure of BsLon-AP shows a hexameric complex consisting of a small part of the N-terminal domain, the ATPase, and protease domains. The structure shows the approximate arrangement of the three functional domains of Lon. It also reveals a resemblance between the architecture of Lon proteases and the bacterial proteasome-like protease HslUV. Our second structure, BsLon-N, represents the first 209 amino acids of the N-terminal domain of BsLon and consists of a globular domain, similar in structure to the E. coli Lon N-terminal domain, and an additional four-helix bundle, which is part of a predicted coiled-coil region. An unexpected dimeric interaction between BsLon-N monomers reveals the possibility that Lon complexes may be stabilised by coiled-coil interactions between neighbouring N-terminal domains. Together, BsLon-N and BsLon-AP are 36 amino acids short of offering a complete picture of a full-length Lon protease.     

Cryo-EM structure of hexameric yeast Lon protease (PIM1) highlights the importance of conserved structural elements

Lon protease is a conserved ATP-dependent serine protease composed of an AAA+ domain that mechanically unfolds substrates and a serine protease domain that degrades these unfolded substrates. In yeast, dysregulation of Lon protease (PIM1) attenuates lifespan and leads to gross mitochondrial morphological perturbations. Although structures of the bacterial and human Lon protease reveal a hexameric assembly, yeast PIM1 was speculated to form a heptameric assembly and is uniquely characterized by a ∼50-residue insertion between the ATPase and protease domains. To further understand the yeast-specific properties of PIM1, we determined a high-resolution cryo-electron microscopy structure of PIM1 in a substrate-translocating state. Here, we reveal that PIM1 forms a hexamer, conserved with that of bacterial and human Lon proteases, wherein the ATPase domains form a canonical closed spiral that enables pore loop residues to translocate substrates to the protease chamber. In the substrate-translocating state, PIM1 protease domains form a planar protease chamber in an active conformation and are uniquely characterized by a ∼15-residue C-terminal extension. These additional C-terminal residues form an α-helix located along the base of the protease domain. Finally, we did not observe density for the yeast-specific insertion between the ATPase and protease domains, likely due to high conformational flexibility. Biochemical studies to investigate the insertion using constructs that truncated or replaced the insertion with a glycine-serine linker suggest that the yeast-specific insertion is dispensable for PIM1’s enzymatic function. Altogether, our structural and biochemical studies highlight unique components of PIM1 machinery and demonstrate evolutionary conservation of Lon protease function.     

A Catalytic Ser–Lys Dyad in the Active Site of the ATP-Dependent Lon Protease from Escherichia coli

Two subfamilies of Lon proteases that differ in the structure of fragments containing the catalytically active Ser residue were revealed by the comparison of more than sixty sequences of Lon proteases from various sources. The absence of the classic catalytic triad in the active site of Lon proteases was confirmed. The catalytic site of Lon proteases was shown to be represented by the Ser–Lys dyad.     

Molecular basis for ATPase-powered substrate translocation by the Lon AAA+ protease

The Lon AAA+ (adenosine triphosphatases associated with diverse cellular activities) protease (LonA) converts ATP-fuelled conformational changes into sufficient mechanical force to drive translocation of a substrate into a hexameric proteolytic chamber. To understand the structural basis for the substrate translocation process, we determined the cryo-electron microscopy (cryo-EM) structure of Meiothermus taiwanensis LonA (MtaLonA) in a substrate-engaged state at 3.6 Å resolution. Our data indicate that substrate interactions are mediated by the dual pore loops of the ATPase domains, organized in spiral staircase arrangement from four consecutive protomers in different ATP-binding and hydrolysis states. However, a closed AAA+ ring is maintained by two disengaged ADP-bound protomers transiting between the lowest and highest position. This structure reveals a processive rotary translocation mechanism mediated by LonA-specific nucleotide-dependent allosteric coordination among the ATPase domains, which is induced by substrate binding.     

Structure of the catalytic domain of the human mitochondrial Lon protease: Proposed relation of oligomer formation and activity

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 3₁₀ helix attached to the N‐terminal end of α‐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.     

Protein unfolding and degradation by the AAA+ Lon protease

AAA+ proteases employ a hexameric ring that harnesses the energy of ATP binding and hydrolysis to unfold native substrates and translocate the unfolded polypeptide into an interior compartment for degradation. What determines the ability of different AAA+ enzymes to unfold and thus degrade different native protein substrates is currently uncertain. Here, we explore the ability of the E. coli Lon protease to unfold and degrade model protein substrates beginning at N-terminal, C-terminal, or internal degrons. Lon has historically been viewed as a weak unfoldase, but we demonstrate robust and processive unfolding/degradation of some substrates with very stable protein domains, including mDHFR and titin(I27) . For some native substrates, Lon is a more active unfoldase than related AAA+ proteases, including ClpXP and ClpAP. For other substrates, this relationship is reversed. Thus, unfolding activity does not appear to be an intrinsic enzymatic property. Instead, it depends on the specific protease and substrate, suggesting that evolution has diversified rather than optimized the protein unfolding activities of different AAA+ proteases.     

Catalytic dyad Ser-Lys at the active site of Escherichia coli ATP-dependent Lon-proteinase

Two subfamilies of Lon proteases that differ in the structure of the fragments containing the catalytically active Ser residue were revealed by the comparison of more than sixty sequences of Lon proteases from various sources. The absence of the classic catalytic triad in the active site of Lon proteases was confirmed. The catalytic site of Lon proteases was shown to be represented by the Ser-Lys dyad.     

Polypeptide stimulators of the Ms-Lon protease

Both the peptidase activity against small fluorescent peptide substrates and the ATPase activity of Lon (La) proteases are stimulated by unstructured proteins such as alpha-casein. This stimulation reveals the simultaneous interaction of Lon with two proteolytic substrates--alpha-casein and the peptide substrate. To understand the cellular function of this stimulation, it is important to determine the physical properties of Lon stimulators. The abilities of compositionally simple random copolymers of amino acids (rcAAs) to stimulate the peptidase and ATPase activities of the Lon protease from Mycobacterium smegmatis (Ms-Lon) and its N-terminal truncation mutant (N-E226) were determined. We report that cationic but not anionic rcAAs stimulated Ms-Lon's peptidase activity but were themselves poor substrates for the enzyme. Peptidase stimulation by rcAAs correlated approximately with the degree of hydrophobicity of these polypeptides and reached levels >10-fold higher than observed previously for Ms-Lon stimulators such as alpha-casein. In contrast to alpha-casein, which stimulates Ms-Lon's peptidase activity by 40% and ATPase activity by 150%, rcAAs stimulated peptidase activity without concomitant stimulation of ATPase activity. Active site labeling experiments suggested that both rcAAs and ATP increased peptidase activity by increasing accessibility to the peptidase active site. Peptidase activity assays in the presence of both alpha-casein and rcAAs revealed that interactions of rcAAs and alpha-casein with Ms-Lon are extremely complex and not mutually exclusive. Specifically, (1) additions of low concentrations of alpha-casein (<50 microg/mL) caused a further stimulation of Ms-Lon's rcAA-stimulated peptidase activity; (2) additions of higher concentrations of alpha-casein inhibited Ms-Lon's rcAA-stimulated peptidase activity; (3) additions of all concentrations of alpha-casein inhibited N-E226's rcAA-stimulated peptidase activity. We conclude the Ms-Lon can interact with an rcAA, alpha-casein, and a substrate peptide simultaneously, and that formation of this quaternary complex requires the N-terminal domain of Ms-Lon. These data support models of Ms-Lon that include two allosteric polypeptide binding sites distinct from the catalytic peptidase site.     

Membrane protein degradation by AAA proteases in mitochondria: extraction of substrates from either membrane surface

Two AAA proteases, each with its catalytic site at the opposite membrane surface, mediate the ATP-dependent degradation of mitochondrial inner membrane proteins. We demonstrate here that a model substrate polypeptide containing hydrophilic domains at both sides of the membrane can be completely degraded by either of the AAA proteases, if solvent-exposed domains are in an unfolded state. A short protein tail protruding from the membrane surface is sufficient to allow the proteolytic attack of an AAA protease that facilitates domain unfolding at the opposite side. Our results provide a rationale for the membrane arrangement of AAA proteases in mitochondria and demonstrate that degradation of membrane proteins by AAA proteases involves an active extraction of transmembrane segments and transport of solvent-exposed domains across the membrane.     

β-Propeller repeats and a PDZ domain in the tricorn protease: predicted self-compartmentalisation and C-terminal polypeptide-binding strategies of substrate selection

Prokaryotic proteases demonstrate a variety of substrate-selection strategies that prevent uncontrolled protein degradation. Proteasomes and ClpXP-like proteases form oligomeric structures that exclude large substrates from central solvated chambers containing their active sites. Monomeric prolyl oligopeptidases have been shown to contain beta-propeller structures that similarly reduce access to their catalytic residues. By contrast, Tsp-like enzymes contain PDZ domains that are thought to specifically target C-terminal polypeptides. We have investigated the sequence of Thermoplasma acidophilum tricorn protease using recently-developed database search methods. The tricorn protease is known to associate into a 20 hexamer capsid enclosing an extremely large cavity that is 37 nm in diameter. It is unknown, however, how this enzyme selects its small oligopeptide substrates. Our results demonstrate the presence in tricorn protease of a PDZ domain and two predicted six-bladed beta-propeller domains. We suggest that the PDZ domain is involved in targeting non-polar C-terminal peptides, similar to those generated by the T. acidophilum proteasome, whereas the beta-propeller domains serve to exclude large substrates from the tricorn protease active site in a similar manner to that previously indicated for prolyl oligopeptidase.     

Role of α-helical domains in functioning of ATP-dependent Lon protease of Escherichia coli

Homooligomeric ATP-dependent LonA proteases are bifunctional enzymes belonging to the superfamily of AAA⁺ proteins. Their subunits are formed by five successively connected domains, i.e., N-terminal (N), α-helical (HI(CC)), nucleotide-binding (NB), the second α-helical (H), and proteolytic (P) domains. The presence of the inserted HI(CC) domain determines the uniqueness of LonA proteases among the AAA⁺ proteins. The role of the α-helical domains in the LonA protease functioning was studied with an example of E. coli Lon protease (Ec-Lon). The properties of the intact Ec-Lon and its mutant forms, i.e., Lon-R164A and Lon-R542A bearing the substituted arginine residues at the similar positions in the HI(CC) and H domains, were compared. The H domain was shown to play a crucial role in ATP hydrolysis and enzyme binding to the target protein. The HI(CC) domain is not decisive for the manifestation of the catalytic properties of the enzyme. However, it affects the functioning of Lon ATPase and peptidase sites and is involved in maintaining enzyme stability. The participation of the HI(CC) domain in the formation of three-dimensional structures of LonA proteases and/or their complexes with DNA is suggested.