Independent interaction of the acyltransferase HlyC with two maturation domains of the Escherichia coli toxin HlyA

The apparently unique fatty acylation mechanism that underlies activation (maturation) of Escherichia coli haemolysin and related toxins is further clarified by investigation of the interaction of protoxin with the specific acyltransferase HlyC. Using deleted protoxin variants and protoxin peptides as substrates in an in vitro maturation reaction dependent upon HlyC and acyl-acyl carrier protein, two independent HlyC recognition domains were identified on the 1024-residue protoxin, proA, and they were shown to span the two target lysine residues K₅₆₄ (KI) and K₆₉₀ (KII) that are fatty acylated. Each domain required 15–30 amino acids for basal recognition and 50–80 amino acids for wild-type acylation. The two domains (FAI and FAII) competed with each other in cis and in trans for HlyC. The affinity of FAI for HlyC is approximately four times greater than that of FAII resulting in an overall 80% acylation at KI and 20% acylation at KII in both whole toxin and peptide derivatives. No other proA sequences were required for toxin maturation, and excess Ca²⁺ prevented acylation of both lysines. The lack of primary sequence identity between FAI and FAll domains in proA and among corresponding sites on related protoxins currently precludes an explanation of the basis of HlyC recognition by proA.     

Insights into the catalytic mechanism of HlyC, the internal protein acyltransferase that activates Escherichia coli hemolysin toxin.

Hemolysin, a toxic protein secreted by pathogenic Escherichia coli, is converted from nontoxic prohemolysin, proHlyA, to toxic hemolysin, HlyA, by an internal protein acyltransferase, HlyC. Acyl-acyl carrier protein (ACP) is the essential acyl donor. The acyltransferase reaction proceeds through two partial reactions and entails formation of a reactive acyl-HlyC intermediate [Trent, M. S., Worsham, L. M., and Ernst-Fonberg, M. L. (1999) Biochemistry 38, 9541-9548]. The ping pong kinetic mechanism implied by these findings was validated using two different acyl-ACP substrates, thus verifying the independence of the previously demonstrated two partial reactions. Assessments of the stability of the acyl-HlyC intermediate revealed an increased stability at pH 8.6 compared to more acidic pHs. Mutations of a single conserved histidine residue essential for catalysis gave minimal activity when substituted with a tyrosine residue and no activity with a lysine residue. Unlike numerous other His23 mutants, however, the H23K enzyme showed significant acyl-HlyC formation although it was unable to transfer the acyl group from the proposed amide bond intermediate to proHlyA. These findings are compatible with transient formation of acyl-His23 during the course of HlyC catalysis. The effects of several other single site-directed mutations of conserved residues of HlyC on different portions of the reaction progress were examined using a 39 500 kDa fragment of proHlyA which was a more effective substrate than intact proHlyA.     

The bacterial antitoxin HipB establishes a ternary complex with operator DNA and phosphorylated toxin HipA to regulate bacterial persistence

Nearly all bacteria exhibit a type of phenotypic growth described as persistence that is thought to underlie antibiotic tolerance and recalcitrant chronic infections. The chromosomally encoded high-persistence (Hip) toxin–antitoxin proteins HipA_SO and HipB_SO from Shewanella oneidensis, a proteobacterium with unusual respiratory capacities, constitute a type II toxin–antitoxin protein module. Here we show that phosphorylated HipA_SO can engage in an unexpected ternary complex with HipB_SO and double-stranded operator DNA that is distinct from the prototypical counterpart complex from Escherichia coli. The structure of HipB_SO in complex with operator DNA reveals a flexible C-terminus that is sequestered by HipA_SO in the ternary complex, indicative of its role in binding HipA_SO to abolish its function in persistence. The structure of HipA_SO in complex with a non-hydrolyzable ATP analogue shows that HipA_SO autophosphorylation is coupled to an unusual conformational change of its phosphorylation loop. However, HipA_SO is unable to phosphorylate the translation factor Elongation factor Tu, contrary to previous reports, but in agreement with more recent findings. Our studies suggest that the phosphorylation state of HipA is an important factor in persistence and that the structural and mechanistic diversity of HipAB modules as regulatory factors in bacterial persistence is broader than previously thought.     

Toward understanding the mechanism of the complex cyclization reaction catalyzed by imidazole glycerolphosphate synthase: crystal structures of a ternary complex and the free enzyme

Imidazole glycerol phosphate synthase catalyzes formation of the imidazole ring in histidine biosynthesis. The enzyme is also a glutamine amidotransferase, which produces ammonia in a glutaminase active site and channels it through a 30-A internal tunnel to a cyclase active site. Glutaminase activity is impaired in the resting enzyme, and stimulated by substrate binding in the cyclase active site. The signaling mechanism was investigated in the crystal structure of a ternary complex in which the glutaminase active site was inactivated by a glutamine analogue and the unstable cyclase substrate was cryo-trapped in the active site. The orientation of N(1)-(5'-phosphoribulosyl)-formimino-5-aminoimidazole-4-carboxamide ribonucleotide in the cyclase active site implicates one side of the cyclase domain in signaling to the glutaminase domain. This side of the cyclase domain contains the interdomain hinge. Two interdomain hydrogen bonds, which do not exist in more open forms of the enzyme, are proposed as molecular signals. One hydrogen bond connects the cyclase domain to the substrate analogue in the glutaminase active site. The second hydrogen bond connects to a peptide that forms an oxyanion hole for stabilization of transient negative charge during glutamine hydrolysis. Peptide rearrangement induced by a fully closed domain interface is proposed to activate the glutaminase by unblocking the oxyanion hole. This interpretation is consistent with biochemical results [Myers, R. S., et al., (2003) Biochemistry 42, 7013-7022, the accompanying paper in this issue] and with structures of the free enzyme and a binary complex with a second glutamine analogue.     

HlyC, the internal protein acyltransferase that activates hemolysin toxin: roles of various conserved residues in enzymatic activity as probed by site-directed mutagenesis.

Hemolysin, a toxic protein produced by pathogenic Escherichia coli, is one of a family of homologous toxins and toxin-processing proteins produced by Gram-negative bacteria. HlyC, an internal protein acyltransferase, converts it from nontoxic prohemolysin to toxic hemolysin. Acyl-acyl carrier protein is the essential acyl donor. The acyltransferase reaction progresses through formation of a binary complex between acyl-ACP and HlyC to a reactive acyl-HlyC intermediate [Trent, M. S., Worsham, L. M., and Ernst-Fonberg, M. L. (1998) Biochemistry 37, 4644-4655]. The homologous acyltransferases of the family have a number of conserved amino acid residues that may be catalytically important. Experiments to illuminate the reaction mechanism were done. The formation of an acyl-enzyme intermediate suggested that the reaction likely proceeded through two partial reactions. The reversibility of the first partial reaction was shown by using separately subcloned, purified, and expressed substrates and enzyme. The effects of single site-directed mutations of conserved residues of HlyC on different portions of reaction progress (binary complex formation, acyl-enzyme formation, and enzyme activity, including kinetic parameters) were determined. Mutations of His23, the only residue essential for activity, formed normal binary complexes but were unable to form acyl-HlyC. The same was seen with S20A, a mutant with greatly impaired activity. Mutation of two conserved tyrosines separately to glycines results in greatly impaired binary complex and acyl-HlyC formation, but mutation of those residues to phenylalanines restored behavior to wild-type.     

Kinetic mechanism for the formation of the presynaptic complex of the bacterial recombinase RecA

RecA protein from Escherichia coli catalyzes DNA strand exchange during homologous recombination in a reaction that requires nucleoside triphosphate cofactor. In the first step of this reaction RecA protein polymerizes on single-stranded DNA to form a filament with a stoichiometry of three nucleotides/RecA monomer called the presynaptic complex. We have used fluorescence anisotropy of a fluorescein-labeled oligonucleotide to investigate presynaptic complex formation. RecA-ATPgammaS bound to oligonucleotide by a two-step process. Kinetic studies revealed an intermediate in the polymerization reaction that had greater mobility than the final product filament. The intermediate was transformed into the final product by a process that was independent of filament concentration and temperature, k = 0.3 +/- 0.1 min(-1). This process had the same rate as that reported for a step in the isomerization of presynaptic complex by ATPgammaS (Paulus, B. F., and Bryant, F. R. (1997) Biochemistry 36, 7832-7838). Judging from anisotropy measurements, the intermediate had hydrodynamic properties similar to a mixed filament containing RecA monomers with and without ATPgammaS. These results show that the presynaptic complex can assume conformations with different segmental mobilities that could play a role in homologous recombination.     

Self-organizing mechanism for the formation of ordered neural mappings

A model for the formation of ordered neural mappings in general, and of retinotectal connections, in particular is given. The main point came from the theory of noise induced transitions, i.e. order may be the result of the interplay between deterministic and random interactions. An activity-dependent self-organizing mechanism is presented in terms of modifiable synapses. Simulation experiments were done not only for the normal ontogenetic development but also for the plastic behaviour of the retinotopic connections.     

Proposed mechanism for the condensation reaction of citrate synthase: 1.9-A structure of the ternary complex with oxaloacetate and carboxymethyl coenzyme A

The crystal structure of the ternary complex citrate synthase-oxaloacetate-carboxymethyl coenzyme A has been solved to a resolution of 1.9 A and refined to a conventional crystallographic R factor of 0.185. The structure resembles a proposed transition state of the condensation reaction and suggests that the condensation reaction proceeds through a neutral enol rather than an enolate intermediate. A mechanism for the condensation reaction is proposed which involves the participation of three key catalytic groups (Asp 375, His 274, and His 320) in two distinct steps. The proposed mechanism invokes concerted general acid-base catalysis twice to explain both the energetics of the reaction and the experimentally observed inversion of stereochemistry at the attacking carbon atom.     

Structural basis of pore formation by the bacterial toxin pneumolysin

The bacterial toxin pneumolysin is released as a soluble monomer that kills target cells by assembling into large oligomeric rings and forming pores in cholesterol-containing membranes. Using cryo-EM and image processing, we have determined the structures of membrane-surface bound (prepore) and inserted-pore oligomer forms, providing a direct observation of the conformational transition into the pore form of a cholesterol-dependent cytolysin. In the pore structure, the domains of the monomer separate and double over into an arch, forming a wall sealing the bilayer around the pore. This transformation is accomplished by substantial refolding of two of the four protein domains along with deformation of the membrane. Extension of protein density into the bilayer supports earlier predictions that the protein inserts beta hairpins into the membrane. With an oligomer size of up to 44 subunits in the pore, this assembly creates a transmembrane channel 260 A in diameter lined by 176 beta strands.     

Molecular mechanism of N-terminal acetylation by the ternary NatC complex

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HlyC, the internal protein acyltransferase that activates hemolysin toxin: the role of conserved tyrosine and arginine residues in enzymatic activity as probed by chemical modification and site-directed mutagenesis.

Internal fatty acylation of proteins is a recognized means of modifying biological behavior. Escherichia coli hemolysin A (HlyA), a toxic protein, is transcribed as a nontoxic protein and made toxic by internal acylation of two lysine residue epsilon-amino groups; HlyC catalyzes the acyl transfer from acyl-acyl carrier protein (ACP), the obligate acyl donor. Conserved residues among the respective homologous C proteins that activate 13 different RTX (repeats in toxin) toxins of which HlyA is the prototype likely include some residues that are important in catalysis. Possible roles of two conserved tyrosines and two conserved arginines were investigated by noting the effects of chemical modifiers and site-directed mutagenesis. TNM modification of HlyC at pH 8.0 led to extensive inhibition that was prevented by the presence of the substrate myristoyl-ACP but not by the product, ACPSH. NAI had no effect. Y70G and Y150G greatly diminished enzyme activity, whereas mutations Y70F and Y150F exhibited wild-type activity. Modification of arginine residues with PG markedly lowered acyltransferase activity with moderate protection by both myristoyl-ACP and ACPSH. Under optimum conditions, four separate mutations of the two conserved arginine residues (R24A, R24K, R87A, and R87K) had little effect on acyltransferase activity.     

Endotoxin-lipoprotein complex formation as a factor in atherogenesis: associations with hyperlipidemia and with lecithin:cholesterol acyltransferase activity

A potential role of endotoxin–lipoprotein (bacterial lipopolysaccharide–lipoprotein, LPS–LP) complex formation as a pathogenic factor for atherosclerosis has not been studied yet. The aim of this study was to test the hypothesis that in endotoxinemia in humans hyperlipidemia associated with atherosclerosis development can favor an excessive LPS–LP complex formation, and endotoxin presented in blood can inhibit lecithin:cholesterol acyltransferase (LCAT), one of the key enzymes of reverse cholesterol transport. Endotoxin-binding capacity of lipoproteins (LP) in patients with normolipidemia and hyperlipidemia types IIa and IV was estimated from label incorporation into different LP fractions isolated by means of sequential ultracentrifugation following serum preincubation with Salmonella minnesota R595 ¹²⁵I-labeled LPS. The effect of varied concentrations of S. minnesota R595 LPS on LCAT activity was evaluated from the overall esterifying activity of serum using [1,2-³H₂]cholesterol-labeled substrate. The elevation of low density LP (LDL) and very low density LP (VLDL) contents in blood serum in hyperlipidemia types IIa and IV, respectively, resulted in significant elevation of LPS binding to these fractions. LPS added to the blood serum leads to the dose-dependent decrease in LCAT activity. The revealed phenomena of elevated LPS binding to atherogenic LP fractions in hypercholesterolemia and endotoxin-induced LCAT inhibition suggest the pathogenic role of LPS–LP complexes in atherogenesis.     

ATP hydrolysis-dependent formation of a dynamic ternary nucleoprotein complex with MutS and MutL.

Functional interactions of Escherichia coli MutS and MutL in mismatch repair are dependent on ATP. In this study, we show that MutS and MutL associate with immobilised DNA in a manner dependent on ATP hydrolysis and with an ATP concentration near the solution K m of the ATPase of MutS. After removal of MutS, MutL and ATP, much of the protein in this ternary complex is not stably associated, with MutL leaving the complex more rapidly than MutS. The rapid dissociation reveals a dynamic interaction with concurrent rapid association and dissociation of proteins from the DNA. Analysis by surface plasmon resonance showed that the DNA interacting with dynamically bound protein was more resistant to nuclease digestion than the DNA in MutS-DNA complexes. Non-hydrolysable analogs of ATP inhibit the formation of this dynamic complex, but permit formation of a second type of ternary complex with MutS and MutL stably bound to the immobilised DNA.     

A conformational mechanism for formation of a dead-end complex by the sarcoplasmic reticulum ATPase with thapsigargin.

Thapsigargin (TG), a plant sesquiterpene lactone extract, interacts tightly with the sarcoplasmic reticulum (SR) Ca2+ transport ATPase yielding a 1:1 stoichiometric complex. In addition to inhibiting steady state enzyme activity, TG can be shown to inhibit two individual partial reactions of the ATPase cycle (i.e. Ca2+ binding in the absence of ATP and enzyme phosphorylation by Pi in the absence of Ca2+) even when these reactions are studied separately without interdependence. As the two partial reactions occur at domains relatively distant from each other in the protein structure, it is apparent that the TG induced perturbation involves the entire enzyme. The rate of TG interaction with the ATPase, as estimated by the onset of functional inhibition and by the development of an intrinsic fluorescence signal, is relatively low in the presence of Ca2+. The interaction is much faster when Ca2+ is removed from the medium by the addition of EGTA or is dissociated from the enzyme by utilization of ATP. When the TG interaction with the ATPase is studied in the presence of Ca2+ as a function of temperature (15-35 degrees C) and pH (6.0-8.0), two distinct kinetic components are observed: a fast component which is prevalent at high temperature and low pH, and a slow component which is prevalent at low temperature and high pH. This pattern suggests that the enzyme resides in two states, whose relatively slow equilibration is temperature- and pH-dependent. As only one state is reactive to TG, the enzyme population residing in this state reacts immediately with TG. On the other hand, the enzyme population residing in the alternate state must undergo slow conversion to the reactive state before being affected by TG. It can be also demonstrated that in the presence of Ca2+ TG shifts the ATPase from a refractory state to a state which is able to form bidimensional crystalline arrays stabilized by decavanadate. It is concluded that TG reacts specifically with the ATPase conformation which is prevalent in the absence of Ca2+, thereby forming a catalytically inactive dead-end complex.     

Mechanism of the synergistic cytotoxicity between pentachlorophenol and copper-1,10-phenanthroline complex: the formation of a lipophilic ternary complex

When non- or sub-toxic levels of pentachlorophenol (PCP) and bis-(1, 10-phenanthroline)cupric complex, Cu(II)(OP)(2), were combined, a remarkable synergistic toxicity was observed as indicated by growth inhibition and bacterial inactivation. Similar synergistic cytotoxic effects were observed with other polychlorinated phenols and other positively charged cupric complexes. The synergism observed for these chemicals and similar reactive pairs of chemicals was found to be due to the formation of lipophilic ternary complexes which facilitated copper transport into the bacterial cells. The formation of ternary complexes of similar lipophilic character could be of relevance as a general mechanism of toxicity.     

A tentative mechanism of the ternary complex formation between phosphorylase kinase, glycogen phosphorylase b and glycogen

The kinetics of rabbit skeletal muscle phosphorylase kinase interaction with glycogen has been studied. At pH 6.8 the binding of phosphorylase kinase to glycogen proceeds only in the presence of Mg2+, whereas at pH 8.2 formation of the complex occurs even in the absence of Mg2+. On the other hand, the interaction of phosphorylase kinase with glycogen requires Ca2+ at both pH values. The initial rate of the complex formation is proportional to the enzyme and glycogen concentrations, suggesting the formation of the complex with stoichiometry 1:1 at the initial step of phosphorylase kinase binding by glycogen. According to the kinetic and sedimentation data, the substrate of the phosphorylase kinase reaction, glycogen phosphorylase b, favors the binding of phosphorylase kinase with glycogen. We suggest a model for the ordered binding of phosphorylase b and phosphorylase kinase to the glycogen particle that explains the increase in the tightness of phosphorylase kinase binding with glycogen in the presence of phosphorylase b.     

HlyC, the internal protein acyltransferase that activates hemolysin toxin: role of conserved histidine, serine, and cysteine residues in enzymatic activity as probed by chemical modification and site-directed mutagenesis

HlyC is an internal protein acyltransferase that activates hemolysin, a toxic protein produced by pathogenic Escherichia coli. Acyl-acyl carrier protein (ACP) is the essential acyl donor. Separately subcloned, expressed, and purified prohemolysin A (proHlyA), HlyC, and [1-14C]myristoyl-ACP have been used to study the conversion of proHlyA to HlyA [Trent, M. S., Worsham, L. M., and Ernst-Fonberg, M. L. (1998) Biochemistry 37, 4644-4655]. HlyC and hemolysin belong to a family of at least 13 toxins produced by Gram-negative bacteria. The homologous acyltransferases of the family show a number of conserved residues that are possible candidates for participation in acyl transfer. Specific chemical reagents and site-directed mutagenesis showed that neither the single conserved cysteine nor the three conserved serine residues were required for enzyme activity. Treatment with the reversible histidine-modifying diethyl pyrocarbonate (DEPC) inhibited acyltransferase activity, and acyltransferase activity was restored following hydroxylamine treatment. The substrate myristoyl-ACP protected HlyC from DEPC inhibition. These findings and spectral absorbance changes suggested that histidine, particularly a histidine proximal to the substrate binding site, was essential for enzyme activity. Site-directed mutageneses of the single conserved histidine residue, His23, to alanine, cysteine, or serine resulted in each instance in complete inactivation of the enzyme.