Transient kinetic experiments demonstrate the existence of a unique catalytic enzyme form in the peptide-stimulated ATPase mechanism of Escherichia coli Lon protease

Lon is an oligomeric serine protease whose proteolytic activity is mediated by ATP hydrolysis. Although each monomeric subunit has an identical sequence, Lon contains two types of ATPase sites that hydrolyze ATP at drastically different rates. The catalytic low-affinity sites display pre-steady-state burst kinetics and hydrolyze ATP prior to peptide cleavage. The high-affinity sites are able to hydrolyze ATP at a very slow rate. By utilizing the differing Kd's, the high-affinity site can be blocked with unlabeled nucleotide while the activity at the low-affinity site is monitored. Little kinetic data are available that describe microscopic events along the reaction pathway of Lon. In this study we utilize MANT-ATP, a fluorescent analogue of ATP, to monitor the rate constants for binding of ATP as well as the release of ADP from Escherichia coli Lon protease. All of the adenine nucleotides tested bound to Lon on the order of 10(5) M(-1) s(-1), and the previously proposed conformational change associated with nucleotide binding was also detected. On the basis of the data obtained in this study we propose that the rate of ADP release is slightly different for the two ATPase sites. As the model peptide substrate [S2; YRGITCSGRQK(Bz)] [Thomas-Wohlever, J., and Lee, I. (2002) Biochemistry 41, 9418-9425] or the protein substrate casein affects only the steady-state ATPase activity of the low-affinity sites, we propose that Lon adopts a different form after its first turnover as an ATP-dependent protease. Based on the obtained rate constants, a revised kinetic model is presented for ATPase activity in Lon protease in both the absence and presence of the model peptide substrate (S2).     

Saturation and specificity of the Lon protease of Escherichia coli.

Lon is an ATP-dependent protease of Escherichia coli. The lon mutation has a pleiotropic phenotype: UV sensitivity, mucoidy, deficiency for lysogenization by bacteriophage lambda and P1, and lower efficiency in the degradation of abnormal proteins. All of these phenotypes are correlated with the loss of protease activity. Here we examine the effects of overproduction of one Lon substrate, SulA, and show that it protects two other substrates from degradation. To better understand this protection, we mutagenized the sulA gene and selected for mutants that have partially or totally lost their ability to saturate the Lon protease and thus can no longer protect another substrate. Some of the SulA mutants lost their ability to protect RcsA from degradation but could still protect the O thermosensitive mutant protein (Ots). All of the mutants retained their capacity to induce cell division inhibition. It was also found that deletion of the C-terminal end of SulA affected its activity but did not affect its susceptibility to Lon. We propose that Lon may have more than one specificity for peptide cleavage.     

The unique sites in SulA protein preferentially cleaved by ATP-dependent Lon protease from Escherichia coli.

SulA protein is known to be one of the physiological substrates of Lon protease, an ATP-dependent protease from Escherichia coli. In this study, we investigated the cleavage specificity of Lon protease toward SulA protein. The enzyme was shown to cleave approximately 27 peptide bonds in the presence of ATP. Among them, six peptide bonds were cleaved preferentially in the early stage of digestion, which represented an apparently unique cleavage sites with mainly Leu and Ser residues at the P1, and P1' positions, respectively, and one or two Gln residues in positions P2-P5. They were located in the central region and partly in the C-terminal region, both of which are known to be important for the function of SulA, such as inhibition of cell growth and interaction with Lon protease, respectively. The other cleavage sites did not represent such consensus sequences, though hydrophobic or noncharged residues appeared to be relatively preferred at the P1 sites. On the other hand, the cleavage in the absence of ATP was very much slower, especially in the central region, than in the presence of ATP. The central region was predicted to be rich in alpha helix and beta sheet structures, suggesting that the enzyme required ATP for disrupting such structures prior to cleavage. Taken together, SulA is thought to contain such unique cleavage sites in its functionally and structurally important regions whose preferential cleavage accelerates the ATP-dependent degradation of the protein by Lon protease.     

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.     

On high- and low-affinity agonist sites in GABAA receptors

GABAA receptors are activated via low-affinity binding sites for the agonists GABA or muscimol. Evidence has been provided that the amino acid residue alpha 1F64 located at the beta2(+)/alpha1(-) subunit interface forms part of this binding site. In radioactive ligand binding studies the agonist [3H]muscimol has been found to interact with the receptor via a high-affinity binding site. This site has been interpreted as a conformational variant of the low-affinity site. Alternatively, the high-affinity binding site has been located to the alpha1(+)/beta2(-) interface and the homologous residue to alpha 1F64, beta 2Y62 has been proposed to constitute an important part of this site. Here we investigated the effect of the point mutation alpha 1F64L and the homologous mutation beta 2Y62L on agonist and antagonist binding and functional properties in alpha 1 beta 2 gamma 2 GABAA receptors. While the mutation in the alpha1 subunit had drastic consequences on all studied properties, including desensitization, the mutation in the beta2 subunit had little consequence. Our observations are relevant for the relative location of high- and low-affinity agonist sites in GABAA receptors.     

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.     

Protein substrates and heat shock reduce the DNA-binding ability of Escherichia coli Lon protease

Interaction between the fusion protein MBP-Lon, formed by maltose-binding protein and Lon protease, and the plasmid pBR322 was studied to clarify the DNA-binding behavior of the Lon protease. Since the MBP-Lon fusion protein that was bound to the plasmid was strongly adsorbed by amylose resin, complex formation and dissociation were determined by quantifying the unadsorbed plasmid using agarose gel electrophoresis. The autolysis of MBP-Lon fusion protein was suppressed when the protein was bound to the plasmid. The plasmid was completely dissociated from MBP-Lon fusion protein by the addition of the protein substrates of Lon protease (i.e. -casein and denatured bovine serum albumin). In addition, at high temperatures, MBP-Lon fusion protein lost its plasmid-binding ability, although it fully retained ATP-dependent protease activity. These results suggest that Lon protease loses DNA-binding ability when cells are exposed to abnormal conditions and the amount of damaged proteins increases. On the other hand, DNA probably plays an important role in controlling the Lon protease activity in cells under normal conditions by entrapping the enzyme.     

Divalent cation binding to the high- and low-affinity sites on G-actin

Metal binding to skeletal muscle G-actin has been assessed by equilibrium dialysis using 45Ca2+ and by kinetic measurements of the increase in the fluorescence of N-acetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine-labeled actin. Two classes of cation binding sites were found on G-actin which could be separated on the basis of their Ca2+ affinity: a single high-affinity site with a Kd considerably less than 1 microM and three identical moderate-affinity binding sites with a Kd of 18 microM. The data for the Mg2+-induced fluorescence enhancement of actin labeled with N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine support a previously suggested mechanism [Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886] in which Ca2+ is replaced by Mg2+ at the moderate affinity site(s), followed by a slow actin isomerization. This isomerization occurs independently of Ca2+ release from the high-affinity site. The fluorescence data do not support a mechanism in which this isomerization is directly related to Ca2+ release from the high-affinity site. Fluorescence changes of labeled actin associated with adding metal chelators are complex and do not reflect the same change induced by Mg2+ addition. Fluorescence changes in the labeled actin have also been observed for the addition of Cd2+ or Mn2+ instead of Mg2+. It is proposed actin may undergo a host of subtle conformational changes dependent on the divalent cation bound. We have also developed a method by which progress curves of a given reaction can be analyzed by nonlinear regression fitting of kinetic simulations to experimental reaction time courses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Degradation in vitro of bacteriophage lambda N protein by Lon protease from Escherichia coli

Lon protease from Escherichia coli degraded lambda N protein in a reaction mixture consisting of the two homogeneous proteins, ATP, and MgCl2 in 50 mM Tris, Ph 8.0. Genetic and biochemical data had previously indicated that N protein is a substrate for Lon protease in vivo (Gottesman, S., Gottesman, M., Shaw, J. E., and Pearson, M. L. (1981) Cell 24, 225-233). Under conditions used for N protein degradation, several lambda and E. coli proteins, including native proteins, oxidatively modified proteins, and cloned fragments of native proteins, were not degraded by Lon protease. Degradation of N protein occurred with catalytic amounts of Lon protease and required the presence of ATP or an analog of ATP. This is the first demonstration of the selective degradation of a physiological substrate by Lon protease in vitro. The turnover number for N protein degradation was approximately 60 +/- 10 min-1 at pH 8.0 in 50 mM Tris/HCl, 25 mM MgCl2 and 4 mM ATP. By comparison the turnover number for oxidized insulin B chain was 20 min-1 under these conditions. Kinetic studies suggest that N protein (S0.5 = 13 +/- 5 microM) is intermediate between oxidized insulin B chain (S0.5 = 160 +/- 10 microM) and methylated casein (S0.5 = 2.5 +/- 1 microM) in affinity for Lon protease. N protein was extensively degraded by Lon protease with an average of approximately six bonds cleaved per molecule. In N protein, as well as in oxidized insulin B chain and glucagon, Lon protease preferentially cut at bonds at which the carboxy group was contributed by an amino acid with an aliphatic side chain (leucine or alanine). However, not all such bonds of the substrates were cleaved, indicating that sequence or conformational determinants beyond the cleavage site affect the ability of Lon protease to degrade a protein.     

A conserved domain in Escherichia coli Lon protease is involved in substrate discriminator activity

Lon protease of Escherichia coli regulates a diverse set of physiological responses including cell division, capsule production, plasmid stability, and phage replication. Little is known about the mechanism of substrate recognition by Lon. To examine the interaction of Lon with two of its substrates, RcsA and SulA, we generated point mutations in lon which affected its substrate specificity. The most informative lon mutant overproduced capsular polysaccharide (RcsA stabilized) yet was resistant to DNA-damaging agents (SulA degraded). Immunoblots revealed that RcsA protein persisted in this mutant whereas SulA protein was rapidly degraded. The mutant contains a single-base change within lon leading to a single amino acid change of glutamate 240 to lysine. E240 is conserved among all Lon isolates and resides in a charged domain that has a high probability of adopting a coiled-coil conformation. This conformation, implicated in mediating protein-protein interactions, appears to confer substrate discriminator activity on Lon. We propose a model suggesting that this coiled-coil domain represents the discriminator site of Lon.     

The Escherichia coli replication inhibitor CspD is subject to growth-regulated degradation by the Lon protease

Post-translational proteolysis-dependent regulation of critical cellular processes is a common feature in bacteria. The Escherichia coli Lon protease is involved in the control of the SOS response, acid tolerance and nutritional deprivation. Moreover, Lon plays a role in the regulation of toxin-antitoxin (TA) systems and thereby is linked to persister cell induction. Persister cells represent a small subpopulation that has reversibly switched to a dormant and non-dividing state without genomic alterations. Formation of persister cells permits viability upon nutritional depletion and severe environmental stresses. CspD is a replication inhibitor, which is induced in stationary phase or upon carbon starvation and increases the production of persister cells. It has remained unknown how CspD activity is counteracted when growth is resumed. Here we report that CspD is subject to proteolysis by the Lon protease both in vivo and in vitro. Turnover of CspD by Lon is strictly adjusted to the growth rate and growth phase of E. coli, reflecting the necessity to control CspD levels according to the physiological conditions.     

Regulatory role of C-terminal residues of SulA in its degradation by Lon protease in Escherichia coli

The SulA protein is a cell division inhibitor in Escherichia coli, and is specifically degraded by Lon protease. To study the recognition site of SulA for Lon, we prepared a mutant SulA protein lacking the C-terminal 8 amino acid residues (SA8). This deletion protein was accumulated and stabilized more than native SulA in lon(+) cells in vivo. Moreover, the deletion SulA fused to maltose binding protein was not degraded by Lon protease, and did not stimulate the ATPase or peptidase activity of Lon in vitro, probably due to the much reduced interaction with Lon. A BIAcore study showed that SA8 directly interacts with Lon. These results suggest that SA8 of SulA was recognized by Lon protease. The SA8 peptide, KIHSNLYH, specifically inhibited the degradation of native SulA by Lon protease in vitro, but not that of casein. A mutant SA8, KAHSNLYH, KIASNLYH, or KIHSNAYH, also inhibited the degradation of SulA, while such peptides as KIHSNLYA did not. These results show that SulA has the specified rows of C-terminal 8 residues recognized by Lon, leading to facilitated binding and subsequent cleavage by Lon protease both in vivo and in vitro.     

Genetic separation of high- and low-affinity transport systems for branched-chain amino acids in Escherichia coli K-12.

The Escherichia coli K-12 mutant strain AE4107 (livH::Mu) is defective in the high-affinity binding protein-mediated uptake system for L-leucine, L-valine, and L-isoleucine (LIV-I). We have used this strain to produce mutations in the residual LIV-II membrane-bound branched-chain amino acid uptake system. Mutants selected for their inability to utilize exogenous L-leucine were found to be defective in the LIV-II system and fell into two classes. One class, represented by strain AE410709 (livP9), showed a complete loss of saturable uptake for L-leucine, L-valine, and L-isoleucine up to 50 muM, and a second class, represented by strain AE4017012 (liv-12), showed a residual component of saturable leucine uptake with increased Km. These mutations, livP9 and liv-12, were closely linked and mapped in the 74 to 78 min region of the E. coli genetic map. Strains constructed so that they lacked both LIV-I and LIV-II transport systems excreted leucine. Strains of the genotype livH+ livP were found to have normal high-affinity binding protein-mediated transport (LIV-I and leucine specific), whereas the low-affinity (LIV-II) transport was completely missing. We concluded from these studies that the high-affinity binding protein-mediated transport systems (LIV-I and leucine specific) can operate independently of the membrane-bound LIV-II system.     

Regulatory role of cardiolipin in the activity of an ATP-dependent protease, Lon, from Escherichia coli

Lon is an ATP-dependent serine protease that plays a significant role in the quality control of proteins in cells, degrading misfolded proteins and certain short-lived regulatory proteins under stresses as such heat-shock and UV irradiation. It is known that some polymers containing phosphate groups regulate enzymatic activity by binding with Lon. We focused on the phospholipids of biological membrane components such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol and cardiolipin (CL), and examined whether or not liposomes containing these phospholipids regulate the enzymatic activity of Lon. CL-containing liposomes specifically inhibited both the proteolytic and ATPase activities of Lon in a dose-dependent manner. In addition, on pull-down assay, we found that CL-containing liposomes selectively bound to Lon. The interaction between CL-containing liposomes and Lon changed with the order of addition of Mg(2+)/ATP. When CL-containing liposomes were added after the addition of Mg(2+)/ATP to Lon, the binding of CL-containing liposomes to Lon was significantly decreased as compared with the reversed order. In fact, we found that CL-containing liposomes bound to Lon, resulting in inhibition of the enzymatic activity of Lon. These results suggest that Lon interacts with CL in biological membranes, which may regulate the functions of Lon as a protein-degrading centre in accordance with environmental changes inside cells.     

GroEL/GroES chaperone and Lon protease regulate expression of the Vibrio fischeri lux operon in Escherichia coli

Chaperone GroEL/GroES and Lon protease were shown to play a role in regulating the expression of the Vibrio fischeri lux operon cloned in Escherichia coli cells. The E. coli groE mutant carrying a plasmid with the full-length V. fischeri lux regulon showed a decreased bioluminescence. The bioluminescence intensity was high in E. coli cells with mutant lonA and the same plasmid. Bioluminescence induction curves lacked the lag period characteristic of lon ⁺ strains. Regulatory luxR of V. fischeri was cloned in pGEX-KG to produce the hybrid gene GST-luxR. The product of its expression, GST-LuxR, was isolated together with GroEL and Lon upon affinity chromatography on a column with glutathione-agarose, suggesting complexation of LuxR with these proteins. It was assumed that GroEL/GroES is involved in LuxR folding, while Lon protease degrades LuxR before its folding into an active globule or after denaturation.     

Domain structure and ATP-induced conformational changes in Escherichia coli protease Lon revealed by limited proteolysis and autolysis

Effector caspases-3, -6 and -7 are responsible for producing the morphological features associated with apoptosis, such as DNA fragmentation. The present study demonstrates that a member of a novel series of pyrrolo-1,5-benzoxazepines, PBOX-6, induces apoptosis in MCF-7 cells, which lack caspase-3. Apoptosis was accompanied by DNA fragmentation and the activation of caspase-7, but not caspases-3 and -6. Inhibition of caspase-7 activity reduced the extent of apoptosis induced, indicating that activation of caspase-7 is involved in the mechanism by which PBOX-6 induces apoptosis in MCF-7 cells. This study suggests that caspase-3 is not necessarily essential for DNA fragmentation and the morphological changes associated with apoptosis.     

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.     

Overproduction and purification of SulA fusion protein in Escherichia coli and its degradation by Lon protease in vitro

To overproduce extremely unstable SulA protein, which is the cell-division inhibitor of Escherichia coli, we fused the sulA gene to the maltose-binding protein (MBP) fusion vectors with or without the signal sequence (plasmids pMAL-p-SulA and pMAL-c-SulA respectively). The amount of the full-length fusion protein expressed from the plasmid pMAL-p-SulA (pre-MBP-SulA) in E. coli was much larger than that expressed from the plasmid pMAL-c-SulA (MBP-SulA). A major amount of the pre-MBP-SulA fusion protein was expressed in a soluble form and affinity-purified by amylose resin. Since site-specific cleavage of the fusion protein with factor Xa resulted in the precipitation of SulA protein, the pre-MBP-SulA fusion protein was used to study the degradation of SulA protein by E. coli Lon protease in vitro. It was found that only the SulA portion of the fusion protein was degraded by Lon protease in an ATP-dependent manner. This result provides direct evidence that Lon protease plays an important role in the rapid degradation of SulA protein in cells.