Monitoring anthrax toxin receptor dissociation from the protective antigen by NMR

Abstract

The binding of the Bacillus anthracis protective antigen (PA) to the host cell receptor is the first step toward the formation of the anthrax toxin, a tripartite set of proteins that include the enzymatic moieties edema factor (EF), and lethal factor (LF). PA is cleaved by a furin-like protease on the cell surface followed by the formation of a donut-shaped heptameric prepore. The prepore undergoes a major structural transition at acidic pH that results in the formation of a membrane spanning pore, an event which is dictated by interactions with the receptor and necessary for entry of EF and LF into the cell. We provide direct evidence using 1-dimensional 13 C-edited 1 H NMR that low pH induces dissociation of the Von-Willebrand factor A domain of the receptor capillary morphogenesis protein 2 (CMG2) from the prepore, but not the monomeric full length PA. Receptor dissociation is also observed using a carbon-13 labeled, 2-fluorohistidine labeled CMG2, consistent with studies showing that protonation of His-121 in CMG2 is not a mechanism for receptor release. Dissociation is likely caused by the structural transition upon formation of a pore from the prepore state rather than protonation of residues at the receptor PA or prepore interface.

Domain 4 of the anthrax protective antigen maintains structure and binding to the host receptor CMG2 at low pH

“…In vitro studies have shown that at this same pH, the receptor dissociates. 10,17 This implied to us that domain 4, in addition to domain 2, may also undergo a conformational change (unfolding) that results in the release the receptor. The crystal structure of domain 4 revealed a structure with amazing adherence to the topology of domain 4 within the context of the full-length PA protein.…”

“…A similar protocol was used for purification of CMG2 (also as a GST fusion). 17 Final purification of either domain 4 or CMG2 was done by applying protein to a Superdex-200 16/60 gel filtration column (GE Healthcare) equilibrated in PBS pH 7.4, 4 C. For experiments using the GST-domain 4 fusion, purification of the fusion was carried out in a similar manner as isolated domain 4, except that rather than adding thrombin, the protein was eluted with 50 mM Tris pH 8.0 containing 10 mM reduced glutathione. GST and GST-domain 4 were then dialyzed into 20 mM Tris pH 8.0.…”

“…Indeed, recent studies using immunoprecipitation have shown that both ATR/TEM8 and CMG2 dissociate from the prepore at pH values required for pore formation, 10 the latter corroborated with data from NMR studies. 17 Although not wellunderstood, we hypothesized that the large structural change from the prepore to the pore is likely to induce receptor dissociation, through a pH-induced unfolding of domain 4. In this study, we use X-ray crystallography to determine the structure of domain 4, and in addition we have investigated the structural properties of the isolated domain 4, whether it undergoes a structural change (unfolding) or loss of binding to CMG2 at pH values known to induce pore formation.…”

Biophysical characterization and immunization studies of dominant negative inhibitor (DNI), a candidate anthrax toxin subunit vaccine

“…This protein is known to bind tightly to PA (K D ~200 pM) 17 and gel-shift analysis using Native PAGE indicates that a 1:1 stoichiometry leads to a complete shift in the band that corresponds to PA to a higher MW. 13 Binding is in part due to the presence of an aspartic acid in PA (D683) in domain 4 which helps coordinate a magnesium ion on the surface of CMG2. 18 Also required for high affinity binding is a loop that is present in domain 2, the 2β3-2β4 loop, that sits in a groove on the surface of CMG2.…”

A Receptor-based Switch that Regulates Anthrax Toxin Pore Formation

“…Furthermore, it is not clear if the receptor remains attached following pore conversion and, if so, how it remains attached. Evidence supporting dissociation has come from co-immunoprecipitation experiments [23] and from previous NMR studies [24], [25]. On the other hand, evidence in favor of receptor attachment has come from other co-immunoprecipitation studies [19], [26], from NMR binding studies performed with a fragment (Domain 4) of PA [27], and from the finding that the presence of a receptor seems to influence voltage-dependent inactivation and small molecule inhibition properties of the newly formed pore [28].…”