Isolation and Characterization of Fragments of the ATP-Dependent Protease Lon from Escherichia coli Obtained by Limited Proteolysis

Conditions of limited proteolysis of the protease Lon from Escherichia coli that provided the formation of fragments approximately corresponding to the enzyme domains were found for studying the domain functioning. A method of isolation of the domains was developed, and their functional characteristics were compared. The isolated proteolytic domain (LonP fragment) of the enzyme was shown to exhibit both peptidase and proteolytic activities; however, it cleaved large protein substrates at a significantly lower rate than the full-size protease Lon. On the other hand, the LonAP fragment, containing both the ATPase and the proteolytic domains, retained almost all of the enzymatic properties of the full-size protein. Both LonP and LonAP predominantly form dimers unlike the native protease Lon functioning as a tetramer. These results suggest that the N-terminal domain of protease Lon may play a considerable role in the process of the enzyme oligomerization.     

Limited proteolysis of E. coli ATP-dependent protease Lon - a unified view of the subunit architecture and characterization of isolated enzyme fragments

We carried out chymotryptic digestion of multimeric ATP-dependent Lon protease from Escherichia coli. Four regions sensitive to proteolytic digestion were located in the enzyme and several fragments corresponding to the individual structural domains of the enzyme or their combinations were isolated. It was shown that (i) unlike the known AAA(+) proteins, the ATPase fragment (A) of Lon has no ATPase activity in spite of its ability to bind nucleotides, and it is monomeric in solution regardless of the presence of any effectors; (ii) the monomeric proteolytic domain (P) does not display proteolytic activity; (iii) in contrast to the inactive counterparts, the AP fragment is an oligomer and exhibits both the ATPase and proteolytic activities. However, unlike the full-length Lon, its AP fragment oligomerizes into a dimer or a tetramer only, exhibits the properties of a non-processive protease, and undergoes self-degradation upon ATP hydrolysis. These results reveal the crucial role played by the non-catalytic N fragment of Lon (including its coiled-coil region), as well as the contribution of individual domains to creation of the quaternary structure of the full-length enzyme, empowering its function as a processive protease.     

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.     

Structural and functional characteristics of ATP-dependent Lon-proteinase from Escherichia coli

Enzymic and structural peculiarities of the ATP-dependent Lon protease from Escherichia coli and its mutant and modified forms were studied. Amino acid residues important for the function of proteolytic and ATPase sites and for the transmission of the interdomain signals of the activity coupling were found. It was shown that the protein substrates are hydrolyzed only by the full-size enzyme, whereas the isolated proteolytic domain displays a peptide-hydrolyzing activity.     

The peroxisomal Lon protease LonP2 in aging and disease: functions and comparisons with mitochondrial Lon protease LonP1

Peroxisomes are ubiquitous eukaryotic organelles with the primary role of breaking down very long‐ and branched‐chain fatty acids for subsequent β‐oxidation in the mitochondrion. Like mitochondria, peroxisomes are major sites for oxygen utilization and potential contributors to cellular oxidative stress. The accumulation of oxidatively damaged proteins, which often develop into inclusion bodies (of oxidized, aggregated, and cross‐linked proteins) within both mitochondria and peroxisomes, results in loss of organelle function that may contribute to the aging process. Both organelles possess an isoform of the Lon protease that is responsible for degrading proteins damaged by oxidation. While the importance of mitochondrial Lon (LonP1) in relation to oxidative stress and aging has been established, little is known regarding the role of LonP2 and aging‐related changes in the peroxisome. Recently, peroxisome dysfunction has been associated with aging‐related diseases indicating that peroxisome maintenance is a critical component of ‘healthy aging’. Although mitochondria and peroxisomes are both needed for fatty acid metabolism, little work has focused on understanding the relationship between these two organelles including how age‐dependent changes in one organelle may be detrimental for the other. Herein, we summarize findings that establish proteolytic degradation of damaged proteins by the Lon protease as a vital mechanism to maintain protein homeostasis within the peroxisome. Due to the metabolic coordination between peroxisomes and mitochondria, understanding the role of Lon in the aging peroxisome may help to elucidate cellular causes for both peroxisome and mitochondrial dysfunction.     

Slicing a protease: structural features of the ATP-dependent Lon proteases gleaned from investigations of isolated domains

ATP-dependent Lon proteases are multi-domain enzymes found in all living organisms. All Lon proteases contain an ATPase domain belonging to the AAA(+) superfamily of molecular machines and a proteolytic domain with a serine-lysine catalytic dyad. Lon proteases can be divided into two subfamilies, LonA and LonB, exemplified by the Escherichia coli and Archaeoglobus fulgidus paralogs, respectively. The LonA subfamily is defined by the presence of a large N-terminal domain, whereas the LonB subfamily has no such domain, but has a membrane-spanning domain that anchors the protein to the cytoplasmic side of the membrane. The two subfamilies also differ in their consensus sequences. Recent crystal structures for several individual domains and sub-fragments of Lon proteases have begun to illuminate similarities and differences in structure-function relationships between the two subfamilies. Differences in orientation of the active site residues in several isolated Lon protease domains point to possible roles for the AAA(+) domains and/or substrates in positioning the catalytic residues within the active site. Structures of the proteolytic domains have also indicated a possible hexameric arrangement of subunits in the native state of bacterial Lon proteases. The structure of a large segment of the N-terminal domain has revealed a folding motif present in other protein families of unknown function and should lead to new insights regarding ways in which Lon interacts with substrates or other cellular factors. These first glimpses of the structure of Lon are heralding an exciting new era of research on this ancient family of proteases.     

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 flggidus LonB protease (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 alpha-helical and proteolytic domains (alphaP). The deltaTM-AfLonTM-S590A enzyme form, obtained by site-directed mutagenesis of the catalytic Ser residue, is capable of recombination with the alphaP 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.     

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.     

Proteolysis Coupled to ATP Hydrolysis: Regulation of the Activity of Proteolytic Sites of Lon Protease from Escherichia coli

Regulation of activity of the proteolytic sites of Lon protease was studied. It was found that ATP–Mg has the properties of a noncompetitive activator of peptidase sites. The processive mechanism of the hydrolysis of protein substrates by Lon protease was experimentally confirmed under the conditions of ATP hydrolysis. It was shown that the oligomeric state of the enzyme is the necessary prerequisite for the processive proteolysis by native Lon protease. The study of the properties of the mixed mutant Lon-K362Q/S679A confirmed the existence of intra- and intersubunit pathways of signal transduction from the ATPase to proteolytic sites. The mutual influence of substrates of Lon protease was studied, and the existence of cooperative interactions between the peptidase sites in the oligomeric enzyme was suggested.     

Proteolysis coupled with ATP. Regulation of activity of proteolytic centers of Escherichia coli lon protease

Regulation of activity of the proteolytic sites of Lon protease was studied. It was found that ATP-Mg has the properties of a noncompetitive activator of peptidase sites. The processive mechanism of the hydrolysis of protein substrates by Lon protease was experimentally confirmed under the conditions of ATP hydrolysis. It was shown that the oligomeric state of the enzyme is the necessary prerequisite for the processive proteolysis by the native Lon protease. The study of the properties of the mixed mutant Lon-K362Q/S679A confirmed the existence of the intra- and intersubunit pathways of signal transduction from the ATPase to proteolytic sites. The mutual influence of substrates of Lon protease was studied, and the existence of cooperative interactions between the peptidase sites in the oligomeric enzyme was suggested.     

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.     

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.     

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.     

Coupling of Proteolysis to ATP Hydrolysis upon Escherichia coliLon Protease Functioning: II. Hydrolysis of ATP and Activity of the Enzyme Peptide Hydrolase Sites

The absence of direct correlation between the efficiency of functioning of ATPase and peptide hydrolase sites of Lon protease was revealed. It was shown that Lon protease is an allosteric enzyme, in which the catalytic activity of peptide hydrolase sites is provided by the binding of nucleotides, their magnesium complexes, and free magnesium ions in the enzyme ATPase sites. It was revealed that the ADP–Mg complex, an inhibitor of the native enzyme, is an activator of the Lon-K362Q (the Lon protease mutant in the ATPase site). Variants of functional contacts between different sites of the enzyme are considered. It was established that two ways of signal transduction from the ATPase sites to peptide hydrolase ones exist in the Lon protease oligomer--intra- and intersubunit ways. The enzyme ATPase sites are suggested to be located in the areas of the complementary surfaces of subunits. It is hypothesized that upon degradation of protein substrates by the E. coliLon protease in vivoATP hydrolysis acts as a factor of limitation of the enzyme degrading activity.     

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.     

Crystal structure of the AAA+ alpha domain of E. coli Lon protease at 1.9A resolution

The crystal structure of the small, mostly helical alpha domain of the AAA+ module of the Escherichia coli ATP-dependent protease Lon has been solved by single isomorphous replacement combined with anomalous scattering and refined at 1.9A resolution to a crystallographic R factor of 17.9%. This domain, comprising residues 491-584, was obtained by chymotrypsin digestion of the recombinant full-length protease. The alpha domain of Lon contains four alpha helices and two parallel strands and resembles similar domains found in a variety of ATPases and helicases, including the oligomeric proteases HslVU and ClpAP. The highly conserved "sensor-2" Arg residue is located at the beginning of the third helix. Detailed comparison with the structures of 11 similar domains established the putative location of the nucleotide-binding site in this first fragment of Lon for which a crystal structure has become available.     

Coupling of proteolysis and hydrolysis of ATP upon functioning of Lon proteinase of Escherichia coli. II. Hydrolysis of ATP and activity of peptide hydrolase sites of the enzyme

The absence of direct correlation between the efficiency of functioning of ATPase and peptidehydrolase sites of Lon protease was revealed. It was shown that Lon protease is an allosteric enzyme, in which the catalytic activity of peptidehydrolase sites is determined by the binding of nucleotides, their magnesium complexes, and free magnesium ions in the enzyme's ATPase sites. It was revealed that complex ADP-Mg, an inhibitor of the native enzyme, is an activator of the Lon-K362Q form of the Lon protease mutant in the ATPase site. Considered are variants of intersite functional contacts realizing in the enzyme. The existence of two ways of signal transduction was established from the ATPase sites to peptidehydrolase ones in the Lon protease oligomer--intra- and intersubunit ways. Location of the enzyme ATPase sites is suggested in the areas of the complementary surfaces of subunits. It is hypothesized that ATP hydrolysis upon degradation of protein substrates by the E. coli Lon protease in vivo acts as a factor of restriction of the enzyme's degrading activity.     

Correlation of an adenine-specific conformational change with the ATP-dependent peptidase activity of Escherichia coli Lon

Escherichia coli Lon, also known as protease La, is a serine protease that is activated by ATP and other purine or pyrimidine triphosphates. In this study, we examined the catalytic efficiency of peptide cleavage as well as intrinsic and peptide-stimulated nucleotide hydrolysis in the presence of hydrolyzable nucleoside triphosphates ATP, CTP, UTP, and GTP. We observed that the k(cat) of peptide cleavage decreases with the reduction in the nucleotide binding affinity of Lon in the following order: ATP > CTP > GTP approximately UTP. Compared to those of the other hydrolyzable nucleotide triphosphates, the ATPase activity of Lon is also the most sensitive to peptide stimulation. Collectively, our kinetic as well as tryptic digestion data suggest that both nucleotide binding and hydrolysis contribute to the peptidase turnover of Lon. The kinetic data that were obtained were further put into the context of the structural organization of Lon protease by probing the conformational change in Lon bound to the different nucleotides. Both adenine-containing nucleotides and CTP protect a 67 kDa fragment of Lon from tryptic digestion. Since this 67 kDa fragment contains the ATP binding pocket (also known as the alpha/beta domain), the substrate sensor and discriminatory (SSD) domain (also known as the alpha-helical domain), and the protease domain of Lon, we propose that the binding of ATP induces a conformational change in Lon that facilitates the coupling of nucleotide hydrolysis with peptide substrate delivery to the peptidase active site.     

Identification of a region in the N-terminus of Escherichia coli Lon that affects ATPase, substrate translocation and proteolytic activity

Lon, also known as protease La, is an AAA+ protease machine that contains the ATPase and proteolytic domain within each enzyme subunit. Three truncated Escherichia coli Lon (ELon) mutants were generated based on a previous limited tryptic digestion result and hydrogen-deuterium exchange mass spectrometry analyses performed in this study. Using methods developed for characterizing wild-type (WT) Lon, we compared the ATPase, ATP-dependent protein degradation and ATP-dependent peptidase activities. With the exception of not degrading a putative structured substrate known as CcrM (cell-cycle-regulated DNA methyltransferase), the mutant lacking the first 239 residues behaved like WT ELon. Comparing the activity data of WT and ELon mutants reveals that the first 239 residues are not needed for minimal enzyme catalysis. The mutants lacking the first 252 residues or residues 232-252 displayed compromised ATPase, protein degradation and ATP-dependent peptide translocation abilities but retained WT-like steady-state peptidase activity. The binding affinities of WT and Lon mutants were evaluated by determining the concentration of λ N (K(λN)) needed to achieve 50% maximal ATPase stimulation. Comparing the K(λN) values reveals that the region encompassing 232-252 of ELon could contribute to λ N binding, but the effect is modest. Taken together, results generated from this study reveal that the region constituting residues 240-252 of ELon is important for ATPase activity, substrate translocation and protein degradation.     

Functional domains of chicken gizzard myosin light chain kinase

The proteolytic susceptibility of chicken gizzard myosin light chain kinase, a calmodulin-dependent enzyme, has been utilized to define the relative location of the catalytic and regulatory domains of the enzyme. Myosin light chain kinase isolated from this source exhibits a Mr of 130,000 and is extremely sensitive to trypsin at 24 degrees C; however, the molecule is divided into susceptible and resistant domains such that proteolysis proceeds rapidly and at multiple sites in the sensitive regions even at 4 degrees C while the rest of the molecule remains relatively resistant to digestion. One of these sensitive areas is the calmodulin-binding domain. On the other hand, Staphylococcus aureus V8 protease digestion generates a calmodulin-binding fragment (Mr = 70,000) that retains Ca2+/calmodulin-dependent enzymatic activity and both of the phosphorylation sites recognized by cAMP-dependent protein kinase. In contrast, treatment with chymotrypsin produces a 95,000 Mr calmodulin-binding fragment that contains only the calmodulin-modulated phosphorylation site. Sequential proteolytic digestion studies demonstrated that the chymotryptic cleavage site responsible for the generation of this 95,000 Mr peptide is within 3,000 Mr of the V8 protease site which produces the 70,000 Mr fragment. Moreover, the non-calmodulin-modulated phosphorylation site must exist in this 3,000 Mr region. A calmodulin-Sepharose affinity adsorption protocol was developed for the digestion and used to isolate both the 70,000 and 95,000 Mr fragments for further study. Taken together, our results are compatible with a model for chicken gizzard myosin light chain kinase in which there is no overlap between the active site, the calmodulin-binding region, and the two sites phosphorylated by cAMP-dependent protein kinase with regard to their relative position in the primary sequence of the molecule.