Sandbox Reserved 702: Difference between revisions

EF is produced in an inactive form. When it is in the cell, EF [[Adenylyl cyclase]] activity is induced by complexation with [[calmodulin]], so it is allosterically activated. Its enzymatic activity leads to a dramatic elevation of the cAMP range.

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. <ref> PMID: 6285339</ref>

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 <scene name='Sandbox_Reserved_702/Coloration_from_n_to_c_term/1'>C-terminal</scene> 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. <ref> PMID: 15719022</ref>  

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 <scene name='Sandbox_Reserved_702/Coloration_from_n_to_c_term/1'>C-terminal</scene> 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. <ref> PMID: 19560485</ref>

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 [[adenylate 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. <ref> PMID: 19560485</ref>

[[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. <ref> PMID: 15719022</ref>

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. <ref> PMID: 19560485</ref>

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. <ref> PMID: 19560485</ref> <ref> PMID: 15719022</ref>

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. <ref> PMID: 15719022</ref>

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 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. <ref> PMID: 15719022</ref>

The magnesium ion that is coordinated by <scene name='Sandbox_Reserved_702/Asp_and_his/2'>aspartates 491 and 493 and histidine 577</scene>  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 <scene name='Sandbox_Reserved_702/histidine_351/1'>Histidine 351</scene> 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. <ref> PMID: 15719022</ref>

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. <ref> PMID: 15719022</ref>

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.

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. <ref>Young, J. A., and R. J. Collier. 2007. Anthrax toxin: receptor-binding, internalization, pore formation, and translocation. Annu. Rev. Biochem. 76:243–265 </ref>