Sandbox Reserved 702

From Proteopedia

Revision as of 19:25, 5 January 2013

Template:Sandbox ESBS 2012

Anthrax edema factor

Introduction

The anthrax edema factor is an 89 kDa exotoxin produced by Bacillus anthracis, the bacterium that causes anthrax. It is an adenylate cyclase [ATP pyrophospate-lyase (cyclizing)]. Its enzyme classification number is EC 4.6.1.1. The edema factor increases intracellular cyclic AMP (cAMP) concentration in eukaryotic cells. In fact adenylyl cyclases catalyze the conversion of adenosine triphosphate (ATP) into cAMP and pyrophosphate. The edema factor is produced in an inactive form and calmodulin-activated. Bacillus anthracis also secretes other proteins, in particular the protective antigen (83 kDa) and the lethal factor (90 kDa). Edema factor, protective antigen and lethal factor can also be called factor I, II and III respectively.

The edema factor is delivered into host cells thanks to the protective antigen. When it is in the cell, the edema factor is activated by calmodulin and its enzymatic activity leads to a dramatic elevation of the cAMP range. Cyclic AMP is a second messenger that plays key roles in the signal transduction pathways and thus regulates diverse cellular responses. It binds to three families of signal transducers: cAMP-dependent protein kinases, cyclic nucleotide gated channels, and the guanine nucleotide exchange factor for Ras GTPase homologs Rap1 and Rap2.

Associated disease

Different forms of disease

Anthrax develops when the organism enters in contact with spores of Bacillus anthracis. Syptoms are depending on the contamination mode. The cutaneous contamination results from a contact of spores with an injury. It leads to an ulcer and to the formation of vesicles. In 80 % of the cases the wound heals without complications. However, sometimes an oedema can develop itsel and grow. In that case, anthrax can lead to the death of the patient.

A gastrointestinal contamination can result from the consumption of contaminated meat. This form of anthrax leads to ulcers, nauseas, diarrhoea and blood poisonning. It can also be lethal if it is not rapidly treated.

Finally, anthrax spores can cause a pulmonary infection by inhalation. The symptoms developed are similar to those of influenza and they evolve into breathing difficulties and hypotension. Blood poisonning and meningitis can also occur. Because of the severe symptoms, the pulmonary infection remains highly lethal. The mortality is caused by the combined effects of bacterial toxins (toxaemia) and baterial growth (becteremia).

Action of the toxin

The secreted proteins can produce two toxic actions. The protective antigen associated with the lethal factor forms the lehtal toxin wile associated with the edema factor it forms the edema toxin. The lethal toxin is involved in the bacterial virulence. The edema toxin plays a key role in anthrax pathogenesis by modulating functions necessary for immunity.

Injection of the lethal toxin causes death of rats, whereas the edema toxin causes oedema in the skin of guinea pigs.

Since the edema factor secreted by Bacillus anthracis leads to a massive cAMP formation, it affects intracellular signalling pathways. This factor may also play a key role in anthrax pathogenesis by disrupting the host cell's defence against bacterial infection. The toxin has effects on macrophages, dendritic cells, neutrophils, endothelial cells and on the antigen presentation of T cells. Fot instance, it inhibits the phagocytic activity of neutrophils and alters cytokine production of monocytes.

Entry of edema toxin in the host cell

The edema factor has a 30 kDa protective antigen-binding domain at its N-terminus. This domain exposes a richly negative-charged surface which easily interacts with the positively charged residues of the protective antigen. Edema factor's protective antigen-binding domain can be divided into two subdomains. The N-terminal domain is composed of three layers, α/β sandwich domain (four β-sheets β1 to β4, in sandwich between four α-helices α1 to α4). The C-terminal domain is composed of five helices. The protective antigen-binding domain contains five joining loops L1 to L5, and L5 has the key exposed residues that bind to the protective antigen. Residues in α6, α7 and in the joining loop between α7 and α8 at the C-terminal domain are also implied in the interaction.

The edema factor is delivered into host cells thanks to the protective antigen. Indeed, the protective antigen binds to cellular receptors (CMP2, capillary morphogenesis protein 2 or TEM8, tumor endothelial marker 8) and is cleaved at the sequence arginine-lysine-lysine-arginine by cell surface proteases. This proteolytic activation leads to the oligomerisation of a protective antigen heptamer. The heptamer is composed of the C-terminal 63 kDa fragment. One heptamer can bind three molecules of edema factor (or lethal factor). Such a complex gets into the cell by endocytosis and finally the protective antigen helps the translocation of the edema factor from late endosome into the cytoplasm. Once it is in the host cell, the edema factor becomes membrane-associated. It is not known whether it is due to its association with calmodulin or to its binding with other cellular elements.

Activation of the adenylyl cyclase activity by calmodulin

Domain organisation of the edema factor and of calmodulin

The edema factor has three domains: a protective antigen-binding domain (30 kDa at the N-terminus as seen before), a helical domain (17 kDa) and a catalytic core domain (43 kDa in the C-terminal 510 amino acid region). The catalytic core domain is itself composed of two domains called CA and CB. The helical domain and the catalytic core are linked by switch C. The adenylyl cyclase catalytic site is located at the interface of CA and CB. In the absence of calmodulin the enzyme remains inactive thanks to a disordered catalytic loop at the interaction site between the helical domain and the catalytic core domain.

Calmodulin has two globular domains, the N-terminal and C-terminal domains, that are connected by a flexible α-helix. Each one of these domains can bind two calcium ions thanks to two helix-loop-helix motifs. The binding of a calcium ion induces a conformational change: the domain goes from a hydrophilic "closed" conformation to an "open" state which exposes a hydrophobic pocket. This hydrophobic pocket plays an important role in the interaction of calmodulin with other molecules.

Molecular basis for the activation of edema factor by calmodulin

The helical domain of the edema factor interacts with the adenylyl cyclase domain and switch C in the absence of calmodulin. This locks the enzyme in an inactive state. The binding of calmodulin on four discrete regions dicrupts most of those interaction allowing the enzyme to switch in an active form. This takes place in two steps.

The calcium-free, closed N-terminal domain of calmodulin binds to the edema factor thanks to an interaction with its helical domain. This interaction is due to hydrogen bonds and a salt bridge between helices I and II of the N-terminal domain of calmodulin and helices L and M of the helical domain of the edema factor. When the N-terminal domain is noud to the helical domain, the calcium-loaded C-terminal domain in its open conformation inserts between the helical domain and the catalytic core. This allows a conformational change of switch C that will stabilize the catalytic loop (switch B) of the enzyme in an active state. A rigid-body rotation of CB relative to CA also occurs. This changes the pocket formed between these two domains and allows the interaction of the edema factor with the phospates of ATP. More precisely, basic lysines of the active site (K346, K353 and K372) are moved upwards. Indeed, the edema factor helical domain undergoes a 15 Å translation and a 30° rotation away from the catalytic core.

Two magnesium ions are coordinated by the catalytic site

Adenylyl cyclase activity of the edema factor requires two magnesium ions. One of them is coordinated by two aspartic residues (D491 and D493) and the histidine 577. The other one is coordianted by the non-esterified oxygens of α, β and γ phosphates of ATP and by aspartic D493. The distance between these two magnesium ions is about 4.5 Å. One ion deprotonates the 3'OH of ATP whereas the other one stabilizes the penta-covalent intermediate during the transition state.

Histidine 351 also plays a key role in the adenylyl cyclase reaction. Histidine is a crucial amino acid because its pKa (6.8) is close to pH of cytoplasm. It may intervene in proton-transfer reactions. Histidine 351 has a critical role in the catalytic activity of edema factor but is not a catalytic base. Since it is about 6.0 Å away from the 3'O of cAMP, its role is to allow a water molecule to enter between them. Histidine does not act as a general base (acceptor of 3'OH proton), but is facilitates an increase in the concentration of HO- ions in the proximity of the 3'OH group.

Mechanism of adenylyl cyclase of edema factor

The current model for the catalytic reaction of edema factor is the following: the reaction is mediated bu the nucleophilic attack of the 3' oxygen atom on the α-phosphate. Indeed, this oxygen in near the α-phosphate. The distance between the nucleophile 3'O and the α-phosphate atom os ATP is shorter than 3.5 Å. A 3'-endo conformtaion of the ribose and a direct coordination of the 3'O atom by the catalytic magnesium ion is the ideal geometry for the initiation of the nucleophilic attack of the 3'O on the α-phosphate.

The adenylyl cyclase reaction takes place in several steps: binding of ATP to the edema factor, enabling the deprotonation of 3'OH, stabilization of the penta-coordinated phosphorus intermediate and finally the release of cAMP and pyrophosphate. The enzyme binds its substrate, ATP and asparagine 583 interactss with it to restrict the rotation of the ribose. 3'OH is so hold in place for its nucleophilic attack durung the catalysis. Moreover, theprotonated histidine 351 stabilizes the HO- ion near the 3'OH group. Another model is that a neutral histidine deprotonates a water molecule and that the resulting HO- ion facilitates the deprotonation of 3'OH of ATP.

The magnesium ion that is coordinated by apsartates 491 and 493 and histidine 577 is localized near the 3'OH of ATP. Since it is positively charged, it stabilizes the negative charge of the 3'-oxyanion and thus facilitates the deprotonation of 3'OH. The action of this ion and of histidine 351 can be additive. To summarise, histidine 351 increases the local pH by attracting HO-, and the metal ion decreases the pKa of the 3'OH group. Maybe the magnesium ion also stabilizes the reaction intermediate by moving towards the non-bridging oxygen of α-phosphate durung the nucleophilic attack.

The second magnesium ion, that is coordinated with the oxygens of the phosphates of ATP, facilitates the bond breakage between α and β-phosphates. Indeed, it stabilizes the resulting negative charges. The stabilisation of the negative charged intermediate is also performed by different positive charged residues: arginine 329, lysine 353, lysine 372 and lysine 346.