Neutralization of the anthrax toxin by antibody-mediated stapling of its membrane-penetrating loop (original) (raw)
Related papers
Journal of Molecular Biology, 2009
The virulence of Bacillus anthracis is critically dependent on the cytotoxic components of the anthrax toxin, lethal factor (LF) and edema factor (EF). LF and EF gain entry into host cells through interactions with the protective antigen (PA), which binds to host cellular receptors such as CMG2. Antibodies that neutralize PA have been shown to confer protection in animal models and are undergoing intense clinical development. A murine monoclonal antibody, 14B7, has been reported to interact with domain 4 of PA (PAD4) and block its binding to CMG2. More recently, the 14B7 antibody was used as the platform for the selection of very high affinity single chain antibodies that have tremendous potential as a combination anthrax prophylactic and treatment. Here we report the high resolution X-ray structures of three high affinity single chain antibodies in the 14B7 family; 14B7 and two high affinity variants 1H and M18. In addition, we present the first neutralizing antibody-PA structure, M18 in complex with PAD4 at 3.8 Å resolution. These structures provide insights into the mechanism of neutralization and on the effect of various mutations on antibody affinity and enable a comparison between the binding of the M18 antibody and CMG2 with PAD4.
Toxins
The anthrax lethal toxin consists of protective antigen (PA) and lethal factor (LF). Understanding both the PA pore formation and LF translocation through the PA pore is crucial to mitigating and perhaps preventing anthrax disease. To better understand the interactions of the LF-PA engagement complex, the structure of the LF N-bound PA pore solubilized by a lipid nanodisc was examined using cryo-EM. CryoSPARC was used to rapidly sort particle populations of a heterogeneous sample preparation without imposing symmetry, resulting in a refined 17 Å PA pore structure with 3 LF N bound. At pH 7.5, the contributions from the three unstructured LF N lysine-rich tail regions do not occlude the Phe clamp opening. The open Phe clamp suggests that, in this translocation-compromised pH environment, the lysine-rich tails remain flexible and do not interact with the pore lumen region.
Structural Determinants for the Binding of Anthrax Lethal Factor to Oligomeric Protective Antigen
Journal of Biological Chemistry, 2006
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.
Journal of Biological Chemistry, 2003
A panel of variants with alanine substitutions in the small loop of anthrax toxin protective antigen domain 4 was created to determine individual amino acid residues critical for interactions with the cellular receptor and with a neutralizing monoclonal antibody, 14B7. Substituted protective antigen proteins were analyzed by cellular cytotoxicity assays, and their interactions with antibody were measured by plasmon surface resonance and analytical ultracentrifugation. Residue Asp 683 was the most critical for cell binding and toxicity, causing an ϳ1000-fold reduction in toxicity, but was not a large factor for interactions with 14B7. Substitutions in residues Tyr 681 , Asn 682 , and Pro 686 also reduced toxicity significantly, by 10 -100-fold. Of these, only Asn 682 and Pro 686 were also critical for interactions with 14B7. However, residues Lys 684 , Leu 685 , Leu 687 , and Tyr 688 were critical for 14B7 binding without greatly affecting toxicity. The K684A and L685A variants exhibited wild type levels of toxicity in cell culture assays; the L687A and Y688A variants were reduced only 1.5-and 5-fold, respectively.
A model of anthrax toxin lethal factor bound to protective antigen.
Anthrax toxin is made up of three proteins: the edema factor (EF), lethal factor (LF) enzymes, and the multifunctional protective antigen (PA). Proteolytically activated PA heptamerizes, binds the EF͞LF enzymes, and forms a pore that allows for EF͞LF passage into host cells. Using directed mutagenesis, we identified three LF-PA contact points defined by a specific disulfide crosslink and two pairs of complementary charge-reversal mutations. These contact points were consistent with the lowest energy LF-PA complex found by using Rosetta protein-protein docking. These results illustrate how biochemical and computational methods can be combined to produce reliable models of large complexes. The model shows that EF and LF bind through a highly electrostatic interface, with their flexible N-terminal region positioned at the entrance of the heptameric PA pore and thus poised to initiate translocation in an N-to C-terminal direction.
Proceedings of the National Academy of Sciences, 2005
Bacillus anthracis secretes three polypeptides: protective antigen (PA), lethal factor (LF), and edema factor (EF), which interact at the surface of mammalian cells to form toxic complexes. LF and EF are enzymes that target substrates within the cytosol; PA provides a heptameric pore to facilitate LF and EF transport into the cytosol. Other than administration of antibiotics shortly after exposure, there is currently no approved effective treatment for inhalational anthrax. Here we demonstrate an approach to disabling the toxin: high-affinity blockage of the PA pore by a rationally designed low-molecular weight compound that prevents LF and EF entry into cells. Guided by the sevenfold symmetry and predominantly negative charge of the PA pore, we synthesized small cyclic molecules of sevenfold symmetry, -cyclodextrins chemically modified to add seven positive charges. By channel reconstitution and high-resolution conductance recording, we show that per-6-(3aminopropylthio)--cyclodextrin interacts strongly with the PA pore lumen, blocking PA-induced transport at subnanomolar concentrations (in 0.1 M KCl). The compound protected RAW 264.7 mouse macrophages from cytotoxicity of anthrax lethal toxin ؍( PA ؉ LF). More importantly, it completely protected the highly susceptible Fischer F344 rats from lethal toxin. We anticipate that this approach will serve as the basis for a structure-directed drug discovery program to find new and effective treatments for anthrax.
Structure of anthrax lethal toxin prepore complex suggests a pathway for efficient cell entry
The Journal of general physiology, 2016
Anthrax toxin comprises three soluble proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). PA must be cleaved by host proteases before it oligomerizes and forms a prepore, to which LF and EF bind. After endocytosis of this tripartite complex, the prepore transforms into a narrow transmembrane pore that delivers unfolded LF and EF into the host cytosol. Here, we find that translocation of multiple 90-kD LF molecules is rapid and efficient. To probe the molecular basis of this translocation, we calculated a three-dimensional map of the fully loaded (PA63)7-(LF)3 prepore complex by cryo-electron microscopy (cryo-EM). The map shows three LFs bound in a similar way to one another, via their N-terminal domains, to the surface of the PA heptamer. The model also reveals contacts between the N- and C-terminal domains of adjacent LF molecules. We propose that this molecular arrangement plays an important role in the maintenance of translocation efficiency through the ...
Journal of Biological Chemistry, 2001
PA63, a proteolytically activated 63-kDa form of anthrax protective antigen (PA), forms heptameric oligomers and has the ability to bind and translocate the catalytic moieties, lethal factor (LF) and edema factor (EF) into the cytosol of mammalian cells. Acidic pH triggers oligomerization and membrane insertion by PA63. A disordered amphipathic loop in domain II of PA (2β2-2β3 loop) is involved in membrane insertion by PA63. Since conditions required for membrane insertion coincide with those for oligomerization of PA63 in mammalian cells, residues constituting the 2β2-2β3 loop were replaced with the residues of the amphipathic membrane-inserting loop of its homologue iota-b toxin secreted by Clostridium perfringens. It was hypothesized that such a molecule might assemble into hetero-heptameric structures with wild-type PA ultimately leading to the inhibition of cellular intoxication. The mutation blocked the ability of PA to mediate membrane insertion and translocation of LF into the cytosol but had no effect on proteolytic activation, oligomerization or binding LF. Moreover, an equimolar mixture of purified mutant PA (PA-I) and wild-type PA showed complete inhibition of toxin activity both in vitro on J774A.1 cells and in vivo in Fischer 344 rats thereby exhibiting a dominant negative effect. In addition, PA-I inhibited the channel forming ability of wild-type PA on the plasma membrane of CHO-K1 cells thereby indicating protein-protein interactions between the two proteins resulting in the formation of mixed oligomers with defective functional activity. Our findings provide a basis for understanding the mechanism of translocation and exploring the possibility of the use of this PA molecule as a therapeutic agent against anthrax toxin action in vivo. by guest on March 17, 2019 http://www.jbc.org/ Downloaded from Dominant Negative Mutant of PA Dominant Negative Mutant of PA
Assembly and Disassembly Kinetics of Anthrax Toxin Complexes †
Biochemistry, 2006
Proteolytic activation of the protective antigen (PA) component of anthrax toxin allows it to selfassociate into a ring-shaped homoheptamer, [PA 63 ] 7 , which can bind the enzymatic components, lethal factor (LF) and edema factor (EF). [PA 63 ] 7 is a pore-precursor (prepore), and under the low pH conditions of the endosome, it forms a transmembrane pore that allows LF and EF to enter the cytosol. We labeled PA with donor and acceptor fluorescent dyes and used Förster resonance energy transfer to measure the assembly and disassembly kinetics of the prepore complex in solution. The dissociation rate constant for [PA 63 ] 7 was 1 × 10 -6 s -1 (t 1/2 ∼7 days). In contrast, a ternary complex containing the PA-binding domain of LF (LF N ) bound to a PA 63 dimer composed of two nonoligomerizing mutants, dissociated rapidly (t 1/2 ∼1 minute). Thus, the substantial decrease in the rate of disassembly of [PA 63 ] 7 relative to the ternary complex is due to the cooperative interactions among neighboring subunits in the heptameric ring. Low concentrations of LF N promoted assembly of the prepore from proteolytically activated PA, whereas high concentrations inhibited assembly of both the prepore and the ternary complex. A self-assembly scheme of anthrax toxin complexes is proposed.