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Legend: Protein Ligand Solvent

The helical domain of the edema factor interacts with the adenylate 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. ^[19]

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. ^{[20] [21]}

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 The other one is coordinated 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. ^[22]

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. ^[23]

Mechanism of adenylyl cyclase of edema factor

The current model for the catalytic reaction of edema factor is the following: the reaction is mediated by 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 conformation 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. ^[24]

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 interacts with it to restrict the rotation of the ribose. 3'OH is so hold in place for its nucleophilic attack during the catalysis. Moreover, the protonated 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. ^[25]

The magnesium ion that is coordinated by 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 can be additive. To summarise, 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. ^[26]

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: ^[27]

The products, pysophosphate and cyclic AMP dissociate through different solvent accessible channels in a two-step process facilitated by product protonation. They are linked to the enzyme by electrostatic interactions with the magnesium ions. The binding of a water molecule to the ions is competitive with thier binding to reaction products and breaks the electrostatic links. Ligands are progressively solvated. In a first time the most important electrostatic interactions are preserved. This depends on the flexibility of the active site. Then the electrostatic interactions are broken and the products can diffuse into solvent. ^[28]

Prevention and treatment

It is possible to use classical therapeutic approaches to fight anthrax disease. Since the original vaccine trials by Louis Pasteur in 1881, an attenuated Stern strain is still successfully used as a vaccine in livestock. Many human vaccines based on PA have been developed in the 1960s.

Antibodies

A large number of treatments against anthrax toxin currently in development are antibodies. The majority of these antibodies target the receptor binding domain of PA, blocking binding of PA to cellular receptors. ^[29]

Efforts to make antibodies to EF have had varied levels of success.

Immunoglobulin G (IgG) have been produced, antibodies of moderate affinity to EF, one of which (9F5) was able to inhibit binding of EF to PA and prevent physiological effects of EF on Chinese hamster ovary (CHO) cells. ^[30]

Winterroth and colleagues described six antibodies of moderate affinity, including one IgM that could neutralize EF activity in CHO cells. This antibody had no significant protective effect against a Sterne strain infection in a mouse model but extended the mean time to death when combined with a subprotective dose of anti-PA antibody. ^[31]

Chen and colleagues developed chimpanzee antibodies that included one having very high affinity and that competes with calmodulin for binding to the helical domain of EF. This antibody was very effective at preventing ET-mediated edema in a mouse footpad model and provided significant protection in a systemic toxin challenge. ^[32]

Leysath et al. developed and characterized four anti-EF monoclonal antibodies (MAb).

MAb bind to epitopes on three different domains of EF: PA binding domain, catalytic CB domain, and helical domain. They showed that three of the four IgGs have neutralizing activity in vitro and in vivo, inhibiting production of cAMP. Antibody 7F10 has the highest efficacy and 3F2 is the first monoclonal antibody to EF whose epitope is on the catalytic CB domain. MAb are highly specific for EF and do not cross-react with LF or PA and can decrease edema levels and progression of disease in mouse models. These antibodies can serve as reagents in diagnostics assays, and can be useful reagents for further molecular studies of EF action. ^[33]

Non-nucleotide inhibitors

Schein et al. identified non-nucleotide inhibitors of EF. Inhibitors targeting sites for allosteric activators have recently been identified. ^[34] They chose to do a direct design based on analysis of the structure of the substrate binding site of the EF protein, rather than a design starting from modifying nucleotides related to the substrate itself. Comparison of the active site conformation in various crystal structures in the Protein database (PDB) revealed how the active site of the toxin differed from the mammalian adenyl cyclase enzymes. The docking of 3'-dATP to the crystal structure of EF (PDB structure: 1K90 ^[35] had low RMSD (root-mean-square deviations) between the predicted structure and the crystal structure, and thus, AutoDock 3.0 was reliable enough to predict the binding mode of the ligands to EF.

Although early treatment with antibiotics can greatly reduce the effects of bacterial infections, inhibitors of the toxins could play a therapeutic role in later stage infections, in preventing toxin induced diarrhea or even death. The fluorenone based inhibitors can be useful, but it could be better to combine them with LF inhibitors to treat late stage anthrax infections, where antibiotics might be unable to prevent death.

^[36]

Structural comparison of AC families and the development of selective EF inhibitor

There are at least six classes of adenylyl cyclase (the classification is based on their primary sequence). Five classes are found only in bacteria and the last one, class III, exists as well in prokaryotes as in eukaryotes. Class II adenylyl cyclase are secreted by pathogenic bacteria and the edema factor belongs to this class. The catalytic site of these several classes are different. This enables the fact that some molecules could inhibit the adenylyl cyclase toxins without inhibiting those of the class III. Inhibitors can be designed to interfere either with the binding of calmodulin or with the binding of the substrate. ^{[37] [38] [39]}

These inhibitors could be further developed as an anti-anthrax treatment, which will be administered with antibiotics. Among those, the most potent EF inhibitor is an approved drug, Adefovir. Adefovir can selectively inhibit the activity of EF both in the test tube and in cultured cells with no inhibition of the activity of endogenous host AC.

Adefovir is an acyclic nucleoside and it can treat chronic hepatitis B virus infection. Tenofovir, another acyclic nucleoside which is a drug against human immunodeficiency virus, also show high affinity to EF. ^[40]

About this Structure

1lvc is a 6 chain structure with sequence from Bacillus anthracis and Homo sapiens. Full crystallographic information is available from OCA.

See Also

• Adenylyl cyclase
• Anthrax edema factor
• Calmodulin

Reference

Contributors

Charlotte Kern - Aude Zimmermann ESBS