Activation of a covalent outer membrane phospholipase A dimer.

The activity of outer membrane phospholipase A (OMPLA) is regulated by reversible dimerization. However, native OMPLA reconstituted in phospholipid vesicles was found to be present as a dimer but nevertheless inactive. To investigate the importance of dimerization for control of OMPLA activity, a covalent OMPLA dimer was constructed and its properties were compared to native OMPLA both in a micellar detergent and after reconstitution in a phospholipid bilayer. Unlike native OMPLA, activity of the covalent OMPLA dimer was independent of type and concentration of detergent in micellar systems. In such systems, the covalent OMPLA dimer invariantly displayed high calcium affinity. In contrast, high calcium concentrations were required to activate a covalent OMPLA dimer when present in intact vesicles. Solubilization of the vesicles increased the affinity for calcium, suggesting that in an intact bilayer the dimer interface is not properly formed. This was supported by the observation that OMPLA variants having an impaired dimeric interface also lacked high affinity calcium binding. A covalent linkage was not able to restore high affinity calcium binding in these variants, demonstrating that a proper dimer interface is essential for optimal catalysis.     

Energetics of outer membrane phospholipase A (OMPLA) dimerization

Outer membrane phospholipase A (OMPLA) is a widely conserved transmembrane enzyme found in Gram-negative bacteria, and it is implicated in the virulence of a number of pathogenic organisms. The regulation of the protein's phospholipase activity is not well understood despite the existence of a number of high resolution structures. Previous biochemical studies have demonstrated that dimerization of OMPLA is a prerequisite for its phospholipase activity, and it has been shown in vitro that this dimerization is dependent on calcium and substrate binding. Therefore, to fully understand the regulation of OMPLA, it is necessary to understand the stability of the protein dimer and the extent to which it is influenced by its effector molecules. We have used sedimentation equilibrium analytical ultracentrifugation to dissect the energetics of Escherichia coli OMPLA dimerization in detergent micelles. We find that calcium contributes relatively little stability to the dimer, while interactions with the substrate acyl chain are the predominant force in stabilizing the dimeric conformation of the enzyme. The resulting thermodynamic cycle suggests that interactions between effector molecules are additive. These energetic measurements not only provide insight into the activation of OMPLA, but they also represent the first quantitative investigation of the association energetics of a transmembrane beta-barrel. This thermodynamic study allows us to begin to address the differences between protein-protein interfaces in transmembrane proteins with a helical fold to those of a beta-barrel fold and to more fully understand the forces involved in membrane protein interactions.     

Outer-membrane phospholipase A: known structure, unknown biological function

Outer-membrane phospholipase A (OMPLA) is one of the few enzymes present in the outer membrane of Gram-negative bacteria. The enzymatic activity of OMPLA is strictly regulated to prevent uncontrolled breakdown of the surrounding phospholipids. The activity of OMPLA can be induced by membrane perturbation and concurs with dimerization of the enzyme. The recently elucidated crystal structures of the inactive, monomeric and an inhibited dimeric form of the enzyme provide detailed structural insight into the functional properties of the enzyme. OMPLA is a serine hydrolase with a unique Asn-156-His-142-Ser-144 catalytic triad. Only in the dimeric state, complete substrate binding pockets and functional oxyanion holes are formed. A model is proposed for the activation of OMPLA in which membrane perturbation causes the formation of non-bilayer structures, resulting in the presentation of phospholipids to the active site of OMPLA and leading to the formation of the active dimeric species. Possible roles for OMPLA in maintaining the cell envelope integrity and in pathogenicity are discussed.     

Lipid chain selectivity by outer membrane phospholipase A

Outer membrane phospholipase A (OMPLA) is a unique, integral membrane enzyme found in Gram-negative bacteria and is an important virulence factor for pathogens such as Helicobacter pylori. This broad-specificity lipase degrades a variety of lipid substrates, and it plays a direct role in adjusting the composition and permeability of bacterial membranes under conditions of stress. Interestingly, OMPLA shows little preference for the lipid headgroup and, instead, the length of the hydrophobic acyl chain is the strongest determinant for substrate selection by OMPLA, with the enzyme strongly preferring substrates with chains equal to or longer than 14 carbon atoms. The question remains as to how a hydrophobic protein like OMPLA can achieve this specificity, particularly when the shorter chains can be accommodated in the binding pocket. Using a series of sulfonyl fluoride inhibitors with various lengths of acyl chain, we show here that the thermodynamics of substrate-induced OMPLA dimerization are guided by the acyl chain length, demonstrating that OMPLA uses a unique biophysical mechanism to select its phospholipid substrate.     

Outer membrane phospholipase A is dimeric in phospholipid bilayers: a cross-linking and fluorescence resonance energy transfer study.

In the cell, the activity of outer membrane phospholipase A (OMPLA) is strictly regulated to prevent uncontrolled breakdown of the membrane lipids. Previously, it has been shown that the enzymatic activity is modulated by reversible dimerization. The current studies were carried out to define the oligomeric state of OMPLA in a membrane and to investigate the activation process. Three single-cysteine variant proteins H26C, H234C, and S144C were produced and purified to homogeneity. Using maleimido-based homo-bifunctional cross-linking reagents, H26C could be efficiently cross-linked as assessed by SDS-PAGE, whereas S144C and H234C could not be cross-linked. These data suggest that residue 26 is located close to the dimer symmetry axis. H26C was specifically labeled with 5-({[(2-iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid and N,N'-dimethyl-N-(iodoacetyl)-N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)ethylenediamine as the fluorescent energy donor and acceptor, respectively, and dimerization was investigated using fluorescence resonance energy transfer (FRET). Quenching of the donor in the presence of the acceptor demonstrated the dimeric nature of OMPLA, in agreement with cross-linking data. The observed FRET effect was dependent on the cofactor calcium, and the presence of substrate, indicating the specificity of the dimerization process. The labeled protein was reconstituted in phospholipid vesicles. In bilayers, OMPLA exhibited low activity and was dimeric as assessed by FRET. Addition of detergent resulted in a 70-fold increase in activity, while the protein remained dimeric. The results are discussed in terms of the activation of dimeric OMPLA due to changes in the physical state of the bilayer which occur upon perturbation of the membrane integrity.     

Unusual catalytic triad of Escherichia coli outer membrane phospholipase A

Escherichia coli outer membrane phospholipase A (OMPLA) is an integral membrane enzyme. OMPLA is active as a homodimer and requires calcium as a cofactor. The crystal structures of the monomeric and the inhibited dimeric enzymes were recently determined [Snijder, H. J., et al. (1999) Nature 401, 717-721] and revealed that OMPLA monomers are folded into a 12-stranded antiparallel beta-barrel. The active site consists of previously identified essential residues Ser144 and His142 in an arrangement resembling the corresponding residues of a serine hydrolase catalytic triad. However, instead of an Asp or Glu that normally is present in the triad of serine hydrolases, a neutral asparagine (Asn156) was found in OMPLA. In this paper, the importance of the catalytic Asn156 is addressed by site-directed mutagenesis studies. All variants were purified at a 30 mg scale, and were shown to be properly folded using SDS-PAGE and circular dichroism spectroscopy. Using chemical cross-linking, it was shown that all variants were not affected in their calcium-dependent dimerization properties. The Asn156Asp variant exhibited a 2-fold lower activity than wild-type OMPLA at neutral pH. Interestingly, the activity of the variant is 1 order of magnitude higher than that of the wild type at pH >10. Modest residual activities (5 and 2.5%, respectively) were obtained for the Asn156Ala and Asn156Gln mutants, showing that the active site of OMPLA is more tolerant toward replacements of this third residue of the catalytic triad than other serine hydrolases, and that the serine and histidine residues are minimally required for catalysis. In the X-ray structure of dimeric OMPLA, the cofactor calcium is coordinating the putative oxyanion via two water molecules. We propose that this may lessen the importance for the asparagine in the catalytic triad of OMPLA.     

Quaternary structure predictions and estimation of mutational effects on the free energy of dimerization of the OMPLA protein

This study represents an extension to the outer membrane phospholipase A protein (OMPLA) of the docking-based protocols previously developed for quaternary structure predictions of transmembrane oligomeric proteins and for estimating mutational effects on the thermodynamics of protein–protein and protein–DNA association.Predictions of the likely architecture of OMPLA homo-dimers were carried out on 31 different forms of the monomer, 30 of which were variants of the unbound state. In all the test cases but the ones characterized by combined deletions of the 98–110 and 145–153 segments (L2 and L3, respectively), native-like complexes could be predicted, independent of the bound or unbound state of the structural model, of side chain conformation and presence or absence of amino acid deletions at the putative inter-monomer interface.The protocol for estimating mutational effects on the thermodynamics of protein–protein association proved effective as well. In fact, it was possible to estimate correctly the effects of five mutants on the free energy of dimerization of the sulfonylated form of OMPLA.The integrity of L2 and either one of the L1, L3 and L4 loops turned out to be more important than sulfonylation for the achievement of the native dimeric architecture. On the other hand, sulfonylation seems to be essential for a favorable dimerization energetics.     

Bacterial phospholipase A: structure and function of an integral membrane phospholipase

Within the large family of lipolytic enzymes, phospholipases constitute a very diverse subgroup with physiological functions such as digestion and signal transduction. Most phospholipases may associate with membranes at the lipid-water interface. However, in many Gram-negative bacteria, a phospholipase is present which is located integrally in the bacterial outer membrane. This phospholipase (outer membrane phospholipase A or OMPLA) is involved in transport across the bacterial outer membrane and has been implicated in bacterial virulence. OMPLA is calcium dependent and its activity is strictly regulated by reversible dimerisation. Recently the crystal structure of this integral membrane enzyme has been elucidated. In this review, we summarise the implications of these structural data for the understanding of the function and regulation of OMPLA, and discuss a mechanism for phospholipase dependent colicin release in Escherichia coli.     

Outer membrane phospholipase a dimer stability does not correlate to occluded surface area

Despite the key roles of oligomeric membrane proteins (MPs) in many known cellular pathways, the principles governing their oligomer stability are not well-understood. Previous work with the alpha-helical MPs bacteriorhodopsin (bR) and glycophorin A (GpA) shows that lost buried surface area linearly correlated with perturbations in protein stability. Although this is a significant discovery, the predictive power of this correlation is limited by the data. Because both bR and GpA have alpha-helical secondary structural motifs, it is unclear whether this correlation would be observed for MPs with a beta-barrel motif. We addressed this question by measuring the thermodynamic consequences of interfacial amino acid changes at the dimer interface of beta-barrel MP outer membrane phospholipase A (OMPLA). We created sequence variants to reduce the contact surface area of the OMPLA dimer interface by introducing single-alanine substitutions and used sedimentation equilibrium analytical ultracentrifugation to determine the dimerization free energies for these variants. The integrity of each variant was verified by two functional assays: specific activity and resistance to thermal denaturation, which showed that structural changes were restricted to the local environment. Using this information, we calculated the anticipated packing defects due to side chain deletion and compared this to the free energy perturbations for each residue. Contrary to the findings with bR and GpA, our study found no correlation between the contact surface area lost and the perturbations to OMPLA dimer stability. We conclude that van der Waals packing may not be a strong predictor of stability for all membrane proteins.     

Half-of-the-sites reactivity of outer-membrane phospholipase A against an active-site-directed inhibitor.

The reaction of a novel active-site-directed phospholipase A1 inhibitor with the outer-membrane phospholipase A (OMPLA) was investigated. The inhibitor 1-p-nitrophenyl-octylphosphonate-2-tridecylcarbamoyl-3-et hanesulfonyl -amino-3-deoxy-sn-glycerol irreversibly inactivated OMPLA. The inhibition reaction did not require the cofactor calcium or an unprotonated active-site His142. The inhibition of the enzyme solubilized in hexadecylphosphocholine micelles was characterized by a rapid (t1/2 = 20 min) and complete loss of enzymatic activity, concurrent with the covalent modification of 50% of the active-site serines, as judged from the amount of p-nitrophenolate (PNP) released. Modification of the remaining 50% occurred at a much lower rate, indicative of half-of-the-sites reactivity against the inhibitor of this dimeric enzyme. Inhibition of monomeric OMPLA solubilized in hexadecyl-N,N-dimethyl-1-ammonio-3-propanesulfonate resulted in an equimolar monophasic release of PNP, concurrent with the loss of enzymatic activity (t1/2 = 14 min). The half-of-the-sites reactivity is discussed in view of the dimeric nature of this enzyme.     

Induction of membrane permeability in Escherichia coli mediated by lysis protein of the ColE7 operon

Induction of the lysis protein of the ColE operon is known to be essential for colicin release. Thus far, the involvement of inner membrane in this unique protein exportation process has not been elucidated. In this work, fluorescent dyes were used to monitor the permeability change of both inner and outer membranes in response to induction of the lysis protein. We found that induction of permeability of the inner membrane appeared earlier than that of the outer membrane before the occurrence of the decline in culture turbidity. Interestingly, we also found that change of outer membrane permeability was alleviated in the outer membrane phospholipase A (OMPLA)-deficient mutant 135 min after induction. Thus, in this work, we show that permeability change of the inner membrane induced by the lysis protein is likely involved in the basal level of colicin release. A greater release of colicin coincided with the decline in culture turbidity and should be associated with the activation of OMPLA at the late stage of induction of the lysis protein.     

A molecular dynamics investigation of mono and dimeric states of the outer membrane enzyme OMPLA

OMPLA is a phospholipase found in the outer membranes of many Gram-negative bacteria. Enzyme activation requires calcium-induced dimerisation plus bilayer perturbation. As the conformation of OMPLA in the different crystal forms (monomer versus dimer; with/without bound Ca(2+)) is remarkably similar we have used multi-nanosecond molecular dynamics (MD) simulations to probe possible differences in conformational dynamics that may be related to enzyme activation. Simulations of calcium-free monomeric OMPLA, of the Ca(2+)-bound dimer, and of the Ca(2+)-bound dimer with a substrate analogue covalently linked to the active site serine have been performed, all with the protein embedded in a phospholipid (POPC) bilayer. All simulations were stable, but differences in the dynamic behaviour of the protein between the various states were observed. In particular, the stability of the active site and the hydrophobic substrate-binding cleft varied. Dimeric OMPLA is less flexible than monomeric OMPLA, especially around the active site. In the absence of bound substrate analogue, the hydrophobic substrate-binding cleft of dimeric OMPLA collapses. A model is proposed whereby the increased stability of the active site in dimeric OMPLA is a consequence of the local ordering of water around the nearby calcium ion. The observed collapse of the substrate-binding cleft may explain the experimentally observed occurrence of multiple dimer conformations of OMPLA, one of which is fully active while the other shows significantly reduced activity.     

Substrate interferes with dimerisation of outer membrane phospholipase A

Outer membrane phospholipase A (OMPLA) activity is regulated by reversible dimerisation with the dimer being the active species. Observed lag phases in activity indicated that dimerisation may be slow relative to turnover. A covalent OMPLA dimer indeed did not display lag phase behaviour. A model for OMPLA kinetics was proposed accounting for a slow dimerisation step. Preincubation conditions determined the initial amount of monomer and influenced both lag times and final activities. Under the conditions used, substrate concentrations higher than 50 mol% inhibited OMPLA activity and increased lag times. Our results may shed more light on mechanisms controlling OMPLA activity in vivo.     

High relative content of lysophospholipids of Helicobacter pylori mediates increased risk for ulcer disease

Helicobacter pylori phospholipase A (OMPLA) degrades bacterial membrane phospholipids to lysophospholipids. High levels of lysophospholipids are associated with higher hemolytic activity, increased release of urease and vacA and better adherence to epithelial cells in vitro. The phospholipase A gene (pldA) displays phase variation due to a slippage in a homopolymeric tract. The aim of this study was to determine if the relative amount of lysophospholipids in the cell wall is associated with ulcer disease, and to further investigate the significance of pldA phase variation. H. pylori isolates of 40 patients were examined. The relative lysophospholipid content of each isolate was determined and the pldA gene was sequenced. The study indicated that H. pylori can regulate its OMPLA activity by phase variation in the pldA gene or by protein level regulation among phase variants in the pldA 'ON' status. We found a significant difference between the relative amount of lysophospholipids of the ulcer group and the non-ulcer group (p=0.022). When the lysophospholipid/phospholipid ratios were compared with outcome, the OR for ulcer disease was 9.0 (95% CI 1.6-49.4; p=0.014). Isolates with a high OMPLA activity are significantly associated with patients with ulcer disease.     

Assembly of colicin A in the outer membrane of producing Escherichia coli cells requires both phospholipase A and one porin,but phospholipase A is sufficient for secretion

Three oligomeric forms of colicin A with apparent molecular masses of about 95 to 98 kDa were detected on sodium dodecyl sulfate (SDS)-polyacrylamide gels loaded with unheated samples from colicin A-producing cells of Escherichia coli. These heat-labile forms, called colicins Au, were visualized both on immunoblots probed with monoclonal antibodies against colicin A and by radiolabeling. Cell fractionation studies show that these forms of colicin A were localized in the outer membrane whether or not the producing cells contained the cal gene, which encodes the colicin A lysis protein responsible for colicin A release in the medium. Pulse-chase experiments indicated that their assembly into the outer membrane, as measured by their heat modifiable migration in SDS gels, was an efficient process. Colicins Au were produced in various null mutant strains, each devoid of one major outer membrane protein, except in a mutant devoid of both OmpC and OmpF porins. In cells devoid of outer membrane phospholipase A (OMPLA), colicin A was not expressed. Colicins Au were detected on immunoblots of induced cells probed with either polyclonal antibodies to OmpF or monoclonal antibodies to OMPLA, indicating that they were associated with both OmpF and OMPLA. Similar heat-labile forms were obtained with various colicin A derivatives, demonstrating that the C-terminal domain of colicin A, but not the hydrophobic hairpin present in this domain, was involved in their formation.     

Functional importance of calcium binding sites in outer membrane phospholipase A

Outer membrane phospholipase A (OMPLA) is an integral membrane enzyme that hydrolyses phospholipids requiring Ca(2+) as cofactor. In vitro studies have shown that OMPLA is only active as a dimer. The structures of monomeric and dimeric OMPLA provided possible clues to the activation process. In the inhibited dimeric species calcium ions are located at the dimer interface ideally suited to stabilise the oxyanion intermediates formed during catalysis. The side chain hydroxyl function of Ser152 is one of the ligands of this interfacial calcium. In the crystal structure of monomeric OMPLA the interfacial calcium site is lacking, but calcium was found to bind at a site involving the carboxylates of Asp149 and Asp184. In the current study the relevance of the identified calcium sites has been studied by site-directed mutagenesis. The Ser152Asn variant confirmed the importance of the interfacial calcium site for catalysis, and also demonstrated that this site is essentially involved in the dimerisation process. Replacements of the ligands in monomeric OMPLA, i.e. Asp149Asn, Asp149Ala and Asp184Asn, only showed minor effects on catalytic activity and dimerisation. A stronger effect observed for the variant Asp184Ala was explained by the proximity of Asp184 to the catalytically important Ser152 residue. We propose that Asp149 and Asp184 provide an electronegative funnel that may facilitate Ca(2+) transfer to the interfacial calcium site.     

Delineation of the translocation of colicin E7 across the inner membrane of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The lysis protein of the colicinogenic operon is essential for colicin release and its main function is to activate the outer membrane phospholipase A (OMPLA) for the traverse of colicin across the cell envelope. However, little is known about the involvement of the lysis protein in the translocation of colicin across the inner membrane into the periplasm. The introduction of specific point mutations into the lipobox or sorting signal sequence of the lysE7 gene resulted in the production of various forms of lysis proteins. Our experimental results indicated that cells with wild-type mature LysE7 protein exhibited higher efficiency of colicin E7 translocation across the inner membrane into the periplasm than those with premature LysE7 protein. Moreover, the degree of permeability of the inner membrane induced by the mature LysE7 protein was significantly increased as compared to the unmodified LysE7 precursor. These results suggest that the efficiency of colicin movement into the periplasm is correlated with the increase in inner membrane permeability induced by the LysE7 protein. Thus, we propose that mature LysE7 protein has two critical roles: firstly mediating the translocation of colicin E7 across the inner membrane into the periplasm, and secondly activating the OMPLA to allow colicin release.     

Functional importance of calcium binding sites in outer membrane phospholipase A

Outer membrane phospholipase A (OMPLA) is an integral membrane enzyme that hydrolyses phospholipids requiring Ca²⁺ as cofactor. In vitro studies have shown that OMPLA is only active as a dimer. The structures of monomeric and dimeric OMPLA provided possible clues to the activation process. In the inhibited dimeric species calcium ions are located at the dimer interface ideally suited to stabilise the oxyanion intermediates formed during catalysis. The side chain hydroxyl function of Ser152 is one of the ligands of this interfacial calcium. In the crystal structure of monomeric OMPLA the interfacial calcium site is lacking, but calcium was found to bind at a site involving the carboxylates of Asp149 and Asp184. In the current study the relevance of the identified calcium sites has been studied by site-directed mutagenesis. The Ser152Asn variant confirmed the importance of the interfacial calcium site for catalysis, and also demonstrated that this site is essentially involved in the dimerisation process. Replacements of the ligands in monomeric OMPLA, i.e. Asp149Asn, Asp149Ala and Asp184Asn, only showed minor effects on catalytic activity and dimerisation. A stronger effect observed for the variant Asp184Ala was explained by the proximity of Asp184 to the catalytically important Ser152 residue. We propose that Asp149 and Asp184 provide an electronegative funnel that may facilitate Ca²⁺ transfer to the interfacial calcium site.     

Interaction of Phospholipase A of the E. coli Outer Membrane with the Inhibitors of Eucaryotic Phospholipases A2 and Their Effect on the Ca2+-Induced Permeabilization of the Bacterial Membrane

Phospholipase A of the bacterial outer membrane (OMPLA) is a β-barrel membrane protein which is activated under various stress conditions. The current study examines interaction of inhibitors of eucaryotic phospholipases A₂—palmitoyl trifluoromethyl ketone (PACOCF₃) and aristolochic acid (AA)—with OMPLA and considers a possible involvement of the enzyme in the Ca²⁺-dependent permeabilization of the outer membrane of Escherichia coli. Using the method of molecular docking, it has been predicted that PACOCF₃ and AA bind to OMPLA at the same site and with the same affinity as the OMPLA inhibitors, hexadecanesulfonylfluoride and bromophenacyl bromide, and the substrate of the enzyme palmitoyl oleoyl phosphatidylethanolamine. It has also been shown that PACOCF₃, AA, and bromophenacyl bromide inhibit the Ca²⁺-induced temperature-dependent changes in the permeability of the bacterial membrane for the fluorescent probe propidium iodide and suppressed the transformation of E. coli cells with plasmid DNA induced by Ca²⁺ and heat shock. The cell viability was not affected by the eucaryotic phospholipases A₂ inhibitors. The study discusses a possible involvement of OMPLA in the mechanisms of bacterial transmembrane transport based on the permeabilization of the bacterial outer membrane.     

Structural investigations of the active-site mutant Asn156Ala of outer membrane phospholipase A: function of the Asn-His interaction in the catalytic triad

Outer membrane phospholipase A (OMPLA) from Escherichia coli is an integral-membrane enzyme with a unique His-Ser-Asn catalytic triad. In serine proteases and serine esterases usually an Asp occurs in the catalytic triad; its role has been the subject of much debate. Here the role of the uncharged asparagine in the active site of OMPLA is investigated by structural characterization of the Asn156Ala mutant. Asparagine 156 is not involved in maintaining the overall active-site configuration and does not contribute significantly to the thermal stability of OMPLA. The active-site histidine retains an active conformation in the mutant notwithstanding the loss of the hydrogen bond to the asparagine side chain. Instead, stabilization of the correct tautomeric form of the histidine can account for the observed decrease in activity of the Asn156Ala mutant.