Domain Flexibility Modulates the Heterogeneous Assembly Mechanism of Anthrax Toxin Protective Antigen

Abstract

Individually, the three protein components of anthrax toxin are nontoxic, but when they assemble, they form active holotoxin complexes. The role of the protective antigen (PA) component of the toxin is to deliver the two other enzyme components, lethal factor (LF) and edema factor (EF), across the plasma membrane and into the cytoplasm of target cells. PA is produced as a proprotein, which must be proteolytically activated, and generally cell-surface activation is mediated by a furin-family protease. Activated PA can then assemble into one of two non-interconverting oligomers, a homoheptamer and homooctamer, which have unique properties. Herein we describe molecular determinants that influence the stoichiometry of PA in toxin complexes. By tethering PA Domain 4 to Domain 2 with two different length cross-links, we can control the relative proportions of PA heptamers and octamers. The longer cross-link favors octamer formation, whereas the shorter one favors formation of the heptamer. X-ray crystal structures of PA (up to 1.45 Å resolution), including these cross-linked PA constructs, reveal that a hinge-like movement of Domain 4 correlates with the relative preference for each oligomeric architecture. Furthermore, we report the conformation of the flexible loop containing the furin-cleavage site and show that for efficient processing, the furin site cannot be moved ~5 or 6 residues within the loop. We propose that there are different orientations of Domain 4 relative to the main body of PA that favor the formation of either the heptamer or the octamer.

Engineering Anthrax Toxin Variants That Exclusively Form Octamers and Their Application to Targeting Tumors

“…The staphylococcal ␣-hemolysin has been shown to form heptamers but under certain conditions will also form hexameric rings (25). Anthrax toxin was long thought to form heptamers exclusively (9), but the work by Krantz and colleagues (10,11,26,27) has shown that functional octamers can also be formed, a conclusion that is confirmed and extended by these studies. This suggests a cautionary approach when considering the oligomeric states of other anthrax-like toxins, such as clostridial C2 toxin and iota toxin (28), and even all other pore-forming toxins, to include consideration that alternative oligomeric forms might be present in certain situations.…”

“…Comparison of the octamer and heptamer crystal structures revealed that there are two orientations of PA domain 4 (the receptor-binding domain) that alternate in the octamer to accommodate the new geometry. Constraining the location of PA domain 4 using different linkers connected to the remainder of the protein altered the proportion of octamers and heptamers (11).…”

Anthrax toxin protective antigen integrates poly-γ- d -glutamate and pH signals to sense the optimal environment for channel formation

“…3 B and C). The per-residue deviations of 4EE2 relative to 3TEW (30) (Fig. S4) are largest in D2 and D4 (>1 Å).…”

“…3B). These loops include the furin-proteolysis site (residues 162-174) [also modeled in the 1.45-Å PA structure 3TEW (30)] and some of the β-barrel residues, 276-287 and 343-350 (17).…”

“…3C), 3TEX, 3TEY, and 3TEZ (30), and 3Q8B and 3MHZ (20). We compared 4EE2 and 3TEW, as the latter is the highest-resolution structure of the native D2-D4 interface.…”