Structure of a dominant negative mutant reveals a stalled intermediate state of anthrax protective antigen pore maturation

Abstract

Anthrax is a severe bacterial infection caused by Bacillus anthracis, which produces a tripartite toxin that includes protective antigen (PA), lethal factor (LF) and edema factor (EF). A series of dominant-negative mutations have been previously identified that prevent the heptameric PA prepore from forming the pH-induced, membrane spanning beta-barrel pore that is required for translocation of EF and LF to the cytoplasm of the infected cell. Here we show that the dominant negative D425A mutation stalls the formation of the pore at a reversible intermediate maturation state, which exhibits many of the structural aspects of the pore but fails to form the phi(ɸ)-clamp and beta-barrel structure needed for full pore maturation. Overall, this structure reveals that ɸ-clamp and beta-barrel pore formation are later steps in the pathway to pore formation, thereby providing a regulatory mechanism to prevent premature translocation of EF and LF.

Neutralization of the anthrax toxin by antibody-mediated stapling of its membrane penetrating loop

“…Similarly, in three out of seven subunits of the asymmetric PA complex (PDB ID 6UJI), the D2L2 loops reside in the inter-subunit cleft. However, the D2L2 loops in the other four subunits are unresolved [25]. In all crystal structures of monomeric PA, the D2L2 loops are absent as well [8,[18][19][20][21][22]24,27]; this is also true for the recently published cryo-EM structure of the prepore-LF complex that otherwise has an excellent resolution [9].…”

“…cAb29 is an effective neutralizer of anthrax toxin, which caught our attention for its ability to block the prepore to pore transition. We assumed that cAb29's function could be explained either by interfering with the pH-sensing of the prepore, as suggested by earlier research [15,25], or by interacting with PA's membrane penetrating loop, thereby directly preventing its movement.…”

Neutralization of the anthrax toxin by antibody-mediated stapling of its membrane-penetrating loop

“…Similarly, in three of the seven subunits of the asymmetric PA complex (PDB entry 6uji) the D2L2 loops reside in the inter-subunit cleft. However, the D2L2 loops in the other four subunits are unresolved (Scott et al, 2020). In all crystal structures of monomeric PA, the D2L2 loops are also absent (Feld et al, 2012;Rajapaksha et al, 2012;Kintzer et al, 2012;Chadegani et al, 2014;Ganesan et al, 2016); this is also true for the recently published cryo-EM structure of the prepore-LF complex, which otherwise has an excellent resolution (Antoni et al, 2020).…”

“…research (Scott et al, 2020;Mechaly et al, 2012), or by interacting with the membrane-penetrating loop of PA, thereby directly preventing its movement.…”