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.     

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.     

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.     

Arg149 is involved in switching the low affinity, open state of the binding protein AfProX into its high affinity, closed state

The substrate binding protein AfProX from the Archaeoglobus fulgidus ProU ATP binding cassette transporter is highly selective for the compatible solutes glycine betaine (GB) and proline betaine, which confer thermoprotection to this hyperthermophilic archaeon. A detailed mutational analysis of the substrate binding site revealed the contribution of individual amino acids for ligand binding. Replacement of Arg149 by an Ala residue displayed the largest impact on substrate binding. The structure of a mutant AfProX protein (substitution of Tyr111 with Ala) in complex with GB was solved in the open liganded conformation to gain further insight into ligand binding. In this crystal structure, GB is bound differently compared to the GB closed liganded structure of the wild-type AfProX protein. We found that a network of amino acid side chains communicates the presence of GB toward Arg149, which increases ligand affinity and induces domain closure of AfProX. These results were corroborated by molecular dynamics studies and support the view that Arg149 finalizes the high-affinity state of the AfProX substrate binding protein.     

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.     

Isolation and characterization of fragments of 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 plays a considerable role in the process of the enzyme oligomerization.     

Crystal structure of Cas1 from Archaeoglobus fulgidus and characterization of its nucleolytic activity

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and CRISPR-associated (Cas) proteins are involved in bacterial acquired immunity against incoming hazardous genetic materials. Cas1 is ubiquitous in CRISPR-containing microorganisms and supposed to recognize and cleave a foreign nucleic acid, and integrate the cleaved fragment into host genome using a yet unidentified mechanism. However, all the reported Cas1s did not show the nucleolytic activity, which makes its role still obscure. The elucidated crystal structure of Cas1 from Archaeoglobus fulgidus (AfCas1) shows a butterfly-like dimeric structure. The Asp out of three confirmed nucleolytic residues of Glu, His, and Asp in other Cas1s is replaced with Glu in AfCas1. Further, insertion of five residues into one of two loops, which are close to the catalytic center of and disordered in other Cas1 structures, partially covers the active site of AfCas1. Nonetheless, in vitro assays show that its nucleic acid-binding activity was not impaired against the tested single-stranded (ss) DNA, various forms of double-stranded (ds) DNA, or ssRNA with a hydrolyzing activity against ssRNA and dsDNA in a metal ion-dependent way. These results support the proposed Cas1’s function at the early step of this bacterial immune system.     

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.     

Involvement of the <Emphasis Type="Italic">N</Emphasis>-terminal region and its characteristic coiled-coil fragment in the function and structure maintenance of <Emphasis Type="Italic">E. coli</Emphasis> LonA protease

The truncated form of E. coli LonA protease (EcLon) lacking the N-terminal fragment 1–172 (Lon173) and the variant with deleted coiled-coil (CC) fragment 173–283 (dCC-Lon, a deletion form) are produced and characterized to study the role of the N-terminal region in the functioning of this protease. A comparative analysis of the properties of full-length EcLon protease, dCC-Lon, and Lon173 as well as an earlier produced form with retained C-terminal region (235–280) of CC fragment, Lon235, is performed. As is shown, fragment 1–280 plays an important role in both formation of the ATPase site and maintenance of a stable EcLon protease conformation. Fragment 107–172 is of a paramount importance for implementation of the processive mechanism of ATP-dependent proteolysis.     

Crystal structures of archaemetzincin reveal a moldable substrate-binding site

Background

Archaemetzincins are metalloproteases occurring in archaea and some mammalia. They are distinct from all the other metzincins by their extended active site consensus sequence HEXXHXXGXXHCX₄CXMX₁₇CXXC featuring four conserved cysteine residues. Very little is known about their biological importance and structure-function relationships.

Principal Findings

Here we present three crystal structures of the archaemetzincin AfAmzA (Uniprot {"type":"entrez-protein","attrs":{"text":"O29917","term_id":"74514303","term_text":"O29917"}}O29917) from Archaeoglobus fulgidus, revealing a metzincin architecture featuring a zinc finger-like structural element involving the conserved cysteines of the consensus motif. The active sites in all three structures are occluded to different extents rendering the enzymes proteolytically inactive against a large variety of tested substrates. Owing to the different ligand binding there are significant differences in active site architecture, revealing a large flexibility of the loops covering the active site cleft.

Conclusions

The crystal structures of AfAmzA provide the structural basis for the lack of activity in standard proteolytic assays and imply a triggered activity onset upon opening of the active site cleft.     

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.     

Serine 363 of a Hydrophobic Region of Archaeal Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from Archaeoglobus fulgidus and Thermococcus kodakaraensis Affects CO2/O2 Substrate Specificity and Oxygen Sensitivity

Archaeal ribulose 1, 5-bisphospate carboxylase/oxygenase (RubisCO) is differentiated from other RubisCO enzymes and is classified as a form III enzyme, as opposed to the form I and form II RubisCOs typical of chemoautotrophic bacteria and prokaryotic and eukaryotic phototrophs. The form III enzyme from archaea is particularly interesting as several of these proteins exhibit unusual and reversible sensitivity to molecular oxygen, including the enzyme from Archaeoglobus fulgidus. Previous studies with A. fulgidus RbcL2 had shown the importance of Met-295 in oxygen sensitivity and pointed towards the potential significance of another residue (Ser-363) found in a hydrophobic pocket that is conserved in all RubisCO proteins. In the current study, further structure/function studies have been performed focusing on Ser-363 of A. fulgidus RbcL2; various changes in this and other residues of the hydrophobic pocket point to and definitively establish the importance of Ser-363 with respect to interactions with oxygen. In addition, previous findings had indicated discrepant CO₂/O₂ specificity determinations of the Thermococcus kodakaraensis RubisCO, a close homolog of A. fulgidus RbcL2. It is shown here that the T. kodakaraensis enzyme exhibits a similar substrate specificity as the A. fulgidus enzyme and is also oxygen sensitive, with equivalent residues involved in oxygen interactions.     

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.     

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.     

Anaerobic oxidation of long-chain n-alkanes by the hyperthermophilic sulfate-reducing archaeon,Archaeoglobus fulgidus

The thermophilic sulfate-reducing archaeon Archaeoglobus fulgidus strain VC-16 (DSM 4304), which is known to oxidize fatty acids and n-alkenes, was shown to oxidize saturated hydrocarbons (n-alkanes in the range C₁₀–C₂₁) with thiosulfate or sulfate as a terminal electron acceptor. The amount of n-hexadecane degradation observed was in stoichiometric agreement with the theoretically expected amount of thiosulfate reduction. One of the pathways used by anaerobic microorganisms to activate alkanes is addition to fumarate that involves alkylsuccinate synthase as a key enzyme. A search for genes encoding homologous enzymes in A. fulgidus identified the pflD gene (locus-tag AF1449) that was previously annotated as a pyruvate formate lyase. A phylogenetic analysis revealed that this gene is of bacterial origin and was likely acquired by A. fulgidus from a bacterial donor through a horizontal gene transfer. Based on three-dimensional modeling of the corresponding protein and molecular dynamic simulations, we hypothesize an alkylsuccinate synthase activity for this gene product. The pflD gene expression was upregulated during the growth of A. fulgidus on an n-alkane (C₁₆) compared with growth on a fatty acid. Our results suggest that anaerobic alkane degradation in A. fulgidus may involve the gene pflD in alkane activation through addition to fumarate. These findings highlight the possible importance of hydrocarbon oxidation at high temperatures by A. fulgidus in hydrothermal vents and the deep biosphere.     

Effect of the Deletion of the (173–280) Fragment of the Inserted α-Helical Domain on the Functional Properties of АТР-Dependent Lon Protease from <Emphasis Type="Italic">E. coli</Emphasis>

The effect of the coiled-coil (CC) region of the α-helical inserted domain of Escherichia coli Lon protease (Ec-Lon) on the functional activity of the enzyme has been characterized. A recombinant form des-CC(G5)-Lon in which the deleted CC fragment is replaced by a pentaglycine peptide has been obtained and investigated. It has been shown that the CC region is involved in the recognition of the nucleotide nature by the enzyme and the interaction of the enzyme with the protein substrate. It has been also established that the CC region is necessary for the formation and functioning of the ATPase and peptidase active centers, the occurrence of allosteric interactions between them, and for the implementation of proteolysis by a unique processive mechanism.     

Structure of the Alkalohyperthermophilic Archaeoglobus fulgidus Lipase Contains a Unique C-Terminal Domain Essential for Long-Chain Substrate Binding

Several crystal structures of AFL, a novel lipase from the archaeon Archaeoglobus fulgidus, complexed with various ligands, have been determined at about 1.8 Å resolution. This enzyme has optimal activity in the temperature range of 70-90 °C and pH 10-11. AFL consists of an N-terminal α/β-hydrolase fold domain, a small lid domain, and a C-terminal β-barrel domain. The N-terminal catalytic domain consists of a 6-stranded β-sheet flanked by seven α-helices, four on one side and three on the other side. The C-terminal lipid binding domain consists of a β-sheet of 14 strands and a substrate covering motif on top of the highly hydrophobic substrate binding site. The catalytic triad residues (Ser136, Asp163, and His210) and the residues forming the oxyanion hole (Leu31 and Met137) are in positions similar to those of other lipases. Long-chain lipid is located across the two domains in the AFL-substrate complex. Structural comparison of the catalytic domain of AFL with a homologous lipase from Bacillus subtilis reveals an opposite substrate binding orientation in the two enzymes. AFL has a higher preference toward long-chain substrates whose binding site is provided by a hydrophobic tunnel in the C-terminal domain. The unusually large interacting surface area between the two domains may contribute to thermostability of the enzyme. Two amino acids, Asp61 and Lys101, are identified as hinge residues regulating movement of the lid domain. The hydrogen-bonding pattern associated with these two residues is pH dependent, which may account for the optimal enzyme activity at high pH. Further engineering of this novel lipase with high temperature and alkaline stability will find its use in industrial applications.     

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.     

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.