Structural Determinants for the Binding of Anthrax Lethal Factor to Oligomeric Protective Antigen

Abstract

Anthrax lethal toxin assembles at the surface of mammalian cells when the lethal factor (LF) binds via its amino-terminal domain, LF N , to oligomeric forms of activated protective antigen (PA). LF⅐PA complexes are then trafficked to acidified endosomes, where PA forms heptameric pores in the bounding membrane and LF translocates through these pores to the cytosol. We used enhanced peptide amide hydrogen/deuterium exchange mass spectrometry and directed mutagenesis to define the surface on LF N that interacts with PA. A continuous surface encompassing one face of LF N became protected from deuterium exchange when LF N was bound to a PA dimer. Directed mutational analysis demonstrated that residues within this surface on LF N interact with Lys-197 on two PA subunits simultaneously, thereby showing that LF N spans the PA subunit:subunit interface and explaining why heptameric PA binds a maximum of three LF N molecules. Our results elucidate the structural basis for anthrax lethal toxin assembly and may be useful in developing drugs to block toxin action.Bacillus anthracis, the causative agent of anthrax, secretes three monomeric proteins that assemble at the host cell surface to form a series of toxic, non-covalent complexes. Toxin assembly begins when one of these proteins, termed protective antigen (PA 83 ) 4 (83 kDa), binds to either of two cell-surface receptors (1, 2) and a 20-kDa piece is proteolytically removed from the N terminus (3). The remaining receptorbound fragment, PA 63 (63 kDa), then spontaneously self-assembles into a ring-shaped homoheptamer (PA 63 ) 7 , termed the pre-pore (4). The latter can bind up to three copies of either, or both, of the two remaining toxin components, edema factor (EF) and lethal factor (LF) (5). The combination of LF and PA is sometimes called lethal toxin, and the combination of EF and LF, edema toxin. LF and EF bind competitively to oligomeric forms of PA 63 via their homologous N-terminal ϳ250-residue domains, termed LF N and EF N (6, 7). The resulting toxic complexes are then internalized into acidified compartments (8) where the prepore converts into a membrane-spanning pore (9), allowing EF and LF to translocate to the cytosol (10, 11). Once in the cytosol, LF, a Zn 2ϩ -dependent protease (12), and EF, a calmodulin-dependent adenylate cyclase (13), exert their toxic effects by modifying specific cytosolic targets.Describing how the three components of anthrax toxin recognize each other at the molecular level can elucidate the self-assembly process at the cell surface and how they dissociate prior to, or during, translocation in the endosome. Such information could also aid in designing inhibitors that block toxin action. Previously, conservation-guided mutagenesis was used to screen for residues on LF/EF and PA that mediate complex assembly (14 -19). Lacy et al. (14) identified a small rectangular patch of seven residues on the surface of LF N /EF N that were critical for interacting with PA. However, later mutagenesis studies revealed a larger footprint on PA t...

Lethal factor of anthrax toxin binds monomeric form of protective antigen

“…In contrast, we did not observe low mobility bands when two PA63 monomers were incubated with the D215K variant of either LF or LF-N (lane 3, Fig. 3A-3C), located on the predicted major binding site [7]. Our findings clearly demonstrated that only the major binding site mutation, D215A, abolishes the LF/PA binding.…”

“…A binding mode that places the N terminus of LF over the pore lumen of the PA heptamer was proposed based on a computer docking simulation, specific disulfide cross-linking, and charge reversal mutations [11]. Although the authors suggest that LF binding requires PA dimerization, their results indicate that mainly residues from one PA63 molecule contribute to LF binding [7].…”

“…Similarly, counterparts in LF-N, which include seven residues Asp182, Asp187, Leu188, Tyr223, His229, Leu235, and Tyr236, were determined to form a patch on one face of the 3D structure of LF [12]. Later, based on results of enhanced peptide amide hydrogen/deuterium exchange mass spectrometry in combination with site directed mutagenesis, the researchers located an enlarged surface area of the binding patch and showed that two 40-Å apart residues of LF, Glu168, and Asp215, interact with the same basic residue in PA, Lys197, presumably on adjacent subunits of the PA63 heptamer [7]. A binding mode that places the N terminus of LF over the pore lumen of the PA heptamer was proposed based on a computer docking simulation, specific disulfide cross-linking, and charge reversal mutations [11].…”

“…Previously, it was reported that mutations of the residues Glu168 and Asp215 in LF disrupt its interaction with PA [7]. With the assumption that the LF binds only to a PA63 dimer, mutation of any of the two binding sites would abolish binding of LF to PA. To test such a hypothesis, we prepared binding-defective mutants E168A and D215K in the context of both LF and LF-N.…”

An approach to characterizing single‐subunit mutations in multimeric prepores and pores of anthrax protective antigen

“…16 A good understanding of this recognition has come from mutation-based mapping, energy minimization, and hydrogen-deuterium exchange measurements. [29][30][31] Under acidic conditions, free LF N transitions to a molten globule state, 10 which may have a weaker affinity for the binding sites on the surface of domain 1 0 . However, evidence from planar bilayer experiments indicates that the disordered $30-residue N-terminal segment of LF N interacts with the Phe clamp under acidic conditions, at which this segment is positively charged.…”