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.     

Functional domains of Brevibacillus thermoruber lon protease for oligomerization and DNA binding: role of N-terminal and sensor and substrate discrimination domains

Lon protease is a multifunctional enzyme, and its functions include the degradation of damaged proteins and naturally short lived proteins, ATPase and chaperone-like activities, as well as DNA binding. A thermostable Lon protease from Brevibacillus thermoruber WR-249 (Bt-Lon) has been cloned and characterized with an N-terminal domain, a central ATPase domain that includes a sensor and substrate discrimination (SSD) domain, and a C-terminal protease domain. Here we present a detailed structure-function characterization of Bt-Lon, not only dissecting the individual roles of Bt-Lon domains in oligomerization, catalytic activities, chaperone-like activity, and DNA binding activity but also describing the nature of oligomerization. Seven truncated mutants of Bt-Lon were designed, expressed, and purified. Our results show that the N-terminal domain is essential for oligomerization. The truncation of the N-terminal domain resulted in the failure of oligomerization and led to the inactivation of proteolytic, ATPase, and chaperone-like activities but retained the DNA binding activity, suggesting that oligomerization of Bt-Lon is a prerequisite for its catalytic and chaperone-like activities. We further found that the SSD is involved in DNA binding based on gel mobility shift assays. On the other hand, the oligomerization of Bt-Lon proceeds through a dimer <--> tetramer <--> hexamer assembly model revealed by chemical cross-linking experiments. The results also showed that hydrophobic interactions may play important roles in the dimerization of Bt-Lon, and ionic interactions are mainly responsible for the assembly of hexamers.     

A Mutation in the N Domain of Escherichia coli Lon Stabilizes Dodecamers and Selectively Alters Degradation of Model Substrates

Escherichia coli Lon, an ATP-dependent AAA⁺ protease, recognizes and degrades many different substrates, including the RcsA and SulA regulatory proteins. More than a decade ago, the E240K mutation in the N domain of Lon was shown to prevent degradation of RcsA but not SulA in vivo. Here, we characterize the biochemical properties of the E240K mutant in vitro and present evidence that the effects of this mutation are complex. For example, Lon^E240K exists almost exclusively as a dodecamer, whereas wild-type Lon equilibrates between hexamers and dodecamers. Moreover, Lon^E240K displays degradation defects in vitro that do not correlate in any simple fashion with degron identity, substrate stability, or dodecamer formation. The Lon sequence segment near residue 240 is known to undergo nucleotide-dependent conformational changes, and our results suggest that this region may be important for coupling substrate binding with allosteric activation of Lon protease and ATPase activity.     

An Integrated Proteomic Approach Uncovers Novel Substrates and Functions of the Lon Protease in Escherichia coli

Controlling the cellular abundance and proper function of proteins by proteolysis is a universal process in all living organisms. In Escherichia coli, the ATP‐dependent Lon protease is crucial for protein quality control and regulatory processes. To understand how diverse substrates are selected and degraded, unbiased global approaches are needed. We employed a quantitative Super‐SILAC (stable isotope labeling with amino acids in cell culture) mass spectrometry approach and compared the proteomes of a lon mutant and a strain producing the protease to discover Lon‐dependent physiological functions. To identify Lon substrates, we took advantage of a Lon trapping variant, which is able to translocate substrates but unable to degrade them. Lon‐associated proteins were identified by label‐free LC‐MS/MS. The combination of both approaches revealed a total of 14 novel Lon substrates. Besides the identification of known pathways affected by Lon, for example, the superoxide stress response, our cumulative data suggests previously unrecognized fundamental functions of Lon in sulfur assimilation, nucleotide biosynthesis, amino acid and central energy metabolism.     

Recent developments in the mechanistic enzymology of the ATP-dependent Lon protease from Escherichia coli: highlights from kinetic studies

Lon protease, also known as protease La, is one of the simplest ATP-dependent proteases that plays vital roles in maintaining cellular functions by selectively eliminating misfolded, damaged and certain short-lived regulatory proteins. Although Lon is a homo-oligomer, each subunit of Lon contains both an ATPase and a protease active site. This relatively simple architecture compared to other hetero-oligomeric ATP-dependent proteases such as the proteasome makes Lon a useful paradigm for studying the mechanism of ATP-dependent proteolysis. In this article, we survey some recent developments in the mechanistic characterization of Lon with an emphasis on the utilization of pre-steady-state enzyme kinetic techniques to determine the timing of the ATPase and peptidase activities of the enzyme.     

Roles of the N domain of the AAA+ Lon protease in substrate recognition,allosteric regulation and chaperone activity

Degron binding regulates the activities of the AAA+ Lon protease in addition to targeting proteins for degradation. The sul20 degron from the cell‐division inhibitor SulA is shown here to bind to the N domain of Escherichia coli Lon, and the recognition site is identified by cross‐linking and scanning for mutations that prevent sul20‐peptide binding. These N‐domain mutations limit the rates of proteolysis of model sul20‐tagged substrates and ATP hydrolysis by an allosteric mechanism. Lon inactivation of SulA in vivo requires binding to the N domain and robust ATP hydrolysis but does not require degradation or translocation into the proteolytic chamber. Lon‐mediated relief of proteotoxic stress and protein aggregation in vivo can also occur without degradation but is not dependent on robust ATP hydrolysis. In combination, these results demonstrate that Lon can function as a protease or a chaperone and reveal that some of its ATP‐dependent biological activities do not require translocation.     

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.     

Crystal structure of the N domain of Lon protease from Mycobacterium avium complex

Lon protease is evolutionarily conserved in prokaryotes and eukaryotic organelles. The primary function of Lon is to selectively degrade abnormal and certain regulatory proteins to maintain the homeostasis in vivo. Lon mainly consists of three functional domains and the N‐terminal domain is required for the substrate selection and recognition. However, the precise contribution of the N‐terminal domain remains elusive. Here, we determined the crystal structure of the N‐terminal 192‐residue construct of Lon protease from Mycobacterium avium complex at 2.4 å resolution,and measured NMR‐relaxation parameters of backbones. This structure consists of two subdomains, the β‐strand rich N‐terminal subdomain and the five‐helix bundle of C‐terminal subdomain, connected by a flexible linker,and is similar to the overall structure of the N domain of Escherichia coli Lon even though their sequence identity is only 26%. The obtained NMR‐relaxation parameters reveal two stabilized loops involved in the structural packing of the compact N domain and a turn structure formation. The performed homology comparison suggests that structural and sequence variations in the N domain may be closely related to the substrate selectivity of Lon variants. Our results provide the structure and dynamics characterization of a new Lon N domain, and will help to define the precise contribution of the Lon N‐terminal domain to the substrate recognition.     

Co‐evolution of multipartite interactions between an extended tmRNA tag and a robust Lon protease in Mycoplasma

Messenger RNAs that lack in‐frame stop codons promote ribosome stalling and accumulation of aberrant and potentially harmful polypeptides. The SmpB‐tmRNA quality control system has evolved to solve problems associated with non‐stop mRNAs, by rescuing stalled ribosomes and directing the addition of a peptide tag to the C‐termini of the associated proteins, marking them for proteolysis. In Escherichia coli, the ClpXP system is the major contributor to disposal of tmRNA‐tagged proteins. We have shown that the AAA+ Lon protease can also degrade tmRNA‐tagged proteins, but with much lower efficiency. Here, we present a unique case of enhanced recognition and degradation of an extended Mycoplasma pneumoniae (MP) tmRNA tag by the MP‐Lon protease. We demonstrate that MP‐Lon can efficiently and selectively degrade MP‐tmRNA‐tagged proteins. Most significantly, our studies reveal that the larger (27 amino acids long) MP‐tmRNA tag contains multiple discrete signalling motifs for efficient recognition and rapid degradation by Lon. We propose that higher‐affinity multipartite interactions between MP‐Lon and the extended MP‐tmRNA tag have co‐evolved from pre‐existing weaker interactions, as exhibited by Lon in E. coli, to better fulfil the function of MP‐Lon as the sole soluble cytoplasmic protease responsible for the degradation of tmRNA‐tagged proteins.     

Regulated proteolysis of a transcription factor complex is critical to cell cycle progression in Caulobacter crescentus

Cell cycle transitions are often triggered by the proteolysis of key regulatory proteins. In Caulobacter crescentus, the G1‐S transition involves the degradation of an essential DNA‐binding response regulator, CtrA, by the ClpXP protease. Here, we show that another critical cell cycle regulator, SciP, is also degraded during the G1‐S transition, but by the Lon protease. SciP is a small protein that binds directly to CtrA and prevents it from activating target genes during G1. We demonstrate that SciP must be degraded during the G1‐S transition so that cells can properly activate CtrA‐dependent genes following DNA replication initiation and the reaccumulation of CtrA. These results indicate that like CtrA, SciP levels are tightly regulated during the Caulobacter cell cycle. In addition, we show that formation of a complex between CtrA and SciP at target promoters protects both proteins from their respective proteases. Degradation of either protein thus helps trigger the destruction of the other, facilitating a cooperative disassembly of the complex. Collectively, our results indicate that ClpXP and Lon each degrade an important cell cycle regulator, helping to trigger the onset of S phase and prepare cells for the subsequent programmes of gene expression critical to polar morphogenesis and cell division.     

Proteins interacting with mitochondrial ATP‐dependent Lon protease (MAP1) in Magnaporthe oryzae are involved in rice blast disease

The ATP‐dependent Lon protease is involved in many physiological processes. In bacteria, Lon regulates pathogenesis and, in yeast, Lon protects mitochondia from oxidative damage. However, little is known about Lon in fungal phytopathogens. MAP1, a homologue of Lon in Magnaporthe oryzae, was recently identified to be important for stress resistance and pathogenesis. Here, we focus on a novel pathogenic pathway mediated by MAP1. Based on an interaction system between rice and a tandem affinity purification (TAP)‐tagged MAP1 complementation strain, we identified 23 novel fungal proteins from infected leaves using a TAP approach with mass spectrometry, and confirmed that 14 of these proteins physically interact with MAP1 in vivo. Among these 14 proteins, 11 candidates, presumably localized to the mitochondria, were biochemically determined to be substrates of MAP1 hydrolysis. Deletion mutants were created and functionally analysed to further confirm the involvement of these proteins in pathogenesis. The results indicated that all mutants showed reduced conidiation and sensitivity to hydrogen peroxide. Appressorial formations were not affected, although conidia from certain mutants were morphologically altered. In addition, virulence was reduced in four mutants, enhanced (with lesions forming earlier) in two mutants and remained unchanged in one mutant. Together with the known virulence‐related proteins alternative oxidase and enoyl‐CoA hydratase, we propose that most of the Lon‐interacting proteins are involved in the pathogenic regulation pathway mediated by MAP1 in M. oryzae. Perturbation of this pathway may represent an effective approach for the inhibition of rice blast disease.     

Oligomeric structure of the ATP-dependent protease La (Lon) of Escherichia coli

Lon, also known as protease La, belongs to a class of ATP-dependent serine protease. It plays an essential role in degradation of abnormal proteins and of certain short-lived regulatory proteins, and is thought to possess a Ser-Lys catalytic dyad. To examine the structural organization of Lon, we performed an electron microscope analysis. The averaged images of Lon with end-on orientation revealed a six-membered, ring-shaped structure with a central cavity. The side-on view showed a two-layered structure with an equal distribution of mass across the equatorial plane of the complex. Since a Lon subunit possesses two large regions containing nucleotide binding and proteolytic domains, each layer of the Lon hexamer appears to consist of the side projections of one of the major domains arranged in a ring. Lon showed a strong tendency to form hexamers in the presence of Mg(2+), but dissociated into monomers and/or dimers in its absence. Moreover, Mg(2+)-dependent hexamer formation was independent of ATP. These results indicate that Lon has a hexameric ring-shaped structure with a central cavity, and that the establishment of this configuration requires Mg(2+), but not ATP.     

Lon recognition of the replication initiator DnaA requires a bipartite degron

DnaA initiates chromosome replication in bacteria. In Caulobacter crescentus, the Lon protease degrades DnaA to coordinate replication with nutrient availability and to halt the cell cycle during acute stress. Here, we characterize the mechanism of DnaA recognition by Lon. We find that the folded state of DnaA appears crucial for its degradation, in contrast to the well‐known role of Lon in degrading misfolded proteins. We fail to identify a single degradation motif (degron) sufficient for DnaA degradation, rather we show that both the ATPase domain and a species‐specific N‐terminal motif are important for productive Lon degradation of full‐length DnaA. Mutations in either of these determinants disrupt DnaA degradation in vitro and in vivo. However, analysis of truncation products reveals that appending other extensions to the ATPase domain is sufficient to trigger degradation, suggesting plasticity in Lon recognition. Our final working model is that Lon engages DnaA through at least two elements, one of which anchors DnaA to Lon and the other acting as an initiation site for degradation.     

Changes in specific protein degradation rates in Arabidopsis thaliana reveal multiple roles of Lon1 in mitochondrial protein homeostasis

Mitochondrial Lon1 loss impairs oxidative phosphorylation complexes and TCA enzymes and causes accumulation of specific mitochondrial proteins. Analysis of over 400 mitochondrial protein degradation rates using ¹⁵N labelling showed that 205 were significantly different between wild type (WT) and lon1‐1. Those proteins included ribosomal proteins, electron transport chain subunits and TCA enzymes. For respiratory complexes I and V, decreased protein abundance correlated with higher degradation rate of subunits in total mitochondrial extracts. After blue native separation, however, the assembled complexes had slow degradation, while smaller subcomplexes displayed rapid degradation in lon1‐1. In insoluble fractions, a number of TCA enzymes were more abundant but the proteins degraded slowly in lon1‐1. In soluble protein fractions, TCA enzymes were less abundant but degraded more rapidly. These observations are consistent with the reported roles of Lon1 as a chaperone aiding the proper folding of newly synthesized/imported proteins to stabilise them and as a protease to degrade mitochondrial protein aggregates. HSP70, prohibitin and enzymes of photorespiration accumulated in lon1‐1 and degraded slowly in all fractions, indicating an important role of Lon1 in their clearance from the proteome.     

The ClpPX protease is required for radioresistance and regulates cell division after gamma-irradiation in Deinococcus radiodurans

Protein degradation in bacteria is involved in diverse cellular responses to environmental stimuli and in removing potentially toxic damaged proteins or protein aggregates. ATP-dependent proteases play a key role in these processes. Here, we have individually inactivated all the ATP-dependent proteases belonging to the Clp or Lon families in Deinococcus radiodurans. The mutants were tested for survival after gamma-irradiation and for sensitivity to the tRNA analogue puromycin in order to assess the impact of each disruption on radioresistance, as well as on proteolysis of misfolded proteins. We found that inactivation of the ClpPX protease significantly decreased cell survival at elevated gamma-irradiation doses, while inactivation of Lon1 and Lon2 proteases reduced resistance to puromycin, suggesting that they play a role in eliminating damaged proteins. Mutants devoid of ClpPX protease displayed altered kinetics of DNA double-strand break repair and resumed cell division after an exceedingly long lag phase following completion of DNA repair. During this stasis period, most of the DeltaclpPX irradiated cells showed decondensed nucleoids and abnormal septa and some cells were devoid of DNA. We propose that the ClpPX protease is involved in the control of proper chromosome segregation and cell division in cells recovering from DNA damage.     

Functional mechanics of the ATP-dependent Lon protease- lessons from endogenous protein and synthetic peptide substrates

Lon, also known as the protease La, is a homo-oligomeric ATP-dependent protease, which is highly conserved in archaea, eubacteria and eukaryotic mitochondria and peroxisomes. Since its discovery, studies have shown that Lon activity is essential for cellular homeostasis, mediating protein quality control and metabolic regulation. This article highlights the discoveries made over the past decade demonstrating that Lon selectively degrades abnormal as well as certain regulatory proteins and thus plays significant roles in maintaining bacterial and mitochondrial function and integrity. In addition, Lon is required in certain pathogenic bacteria, for rendering pathogenicity and host infectivity. Recent research endeavors have been directed toward elucidating the reaction mechanism of the Lon protease by different biochemical and structural biological techniques. In this mini-review, the authors survey the diverse biological roles of Lon, and also place special emphasis on recent findings that clarify the mechanistic aspects of the Lon reaction cycle.     

Annexin XII E105K crystal structure: identification of a pH-dependent switch for mutant hexamerization

Annexins are a family of calcium- and phospholipid-binding proteins involved with numerous cellular processes including membrane fusion, ion channel activity, and heterocomplex formation with other proteins. The annexin XII (ANXB12) crystal structure presented evidence that calcium mediates the formation of a hexamer through a novel intermolecular calcium-binding site [Luecke et al. (1995) Nature 378, 512-515]. In an attempt to disrupt hexamerization, we mutated a conserved key ligand in the intermolecular calcium-binding site, Glu105, to lysine. Despite its occurrence in a new spacegroup, the 1.93 A resolution structure reveals a hexamer with the Lys105 epsilon-amino group nearly superimposable with the original intermolecular calcium position. Our analysis shows that the mutation is directly involved in stabilizing the hexamer. The local residues are reoriented to retain affinity between the two trimers via a pH-dependent switch residue, Glu76, which is now protonated, allowing it to form tandem hydrogen bonds with the backbone carbonyl and nitrogen atoms of Thr103 located across the trimer interface. The loss of the intermolecular calcium-binding site is recuperated by extensive hydrogen bonding favoring hexamer stabilization. The presence of this mutant structure provides further evidence for hexameric annexin XII, and possible in vivo roles are discussed.     

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.     

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.     

Age-related impairment of mitochondrial matrix aconitase and ATP-stimulated protease in rat liver and heart.

Mitochondrial matrix proteins are sensitive to oxidative inactivation, and oxidized proteins are known to accumulate during ageing. The Lon protease is believed to play an important role in the degradation of oxidized matrix proteins such as oxidized aconitase. We reported previously that an age-related accumulation of altered proteins occurs in the liver matrix of rats and that the ATP-stimulated proteolytic activity, referred as to Lon-like protease activity, decreases considerably in 27 month-old rats, whereas no concomitant changes in the levels of Lon protein expression occur in the liver. Here, we report that this decline is associated with a decrease in the activity of aconitase, an essential Krebs' cycle enzyme. Contrary to what we observed in the liver, the ATP-stimulated protease activity was found to remain constant in the heart mitochondrial matrix during ageing, and the levels of expression of the Lon protease increased in the older animals in comparison with the younger ones. Although the ATP-stimulated protease activity remained practically the same in older animals as in younger ones, a decrease in the level of aconitase activity was still observed. Altogether, these results indicate that matrix proteins, such as the critical enzymes aconitase and Lon protease, are inactivated with ageing and that the effects of ageing vary from one organ to another.