Effects of Introducing a Single Charged Residue into the Phenylalanine Clamp of Multimeric Anthrax Protective Antigen

Janowiak, Blythe E.; Fischer, Audrey; Collier, R. John

doi:10.1074/jbc.m109.093195

Cited by 19 publications

(21 citation statements)

References 36 publications

(60 reference statements)

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“…3), we asked whether these LF N residues bind directly to the Φ-clamp in the pore. We were not able to introduce a spin label directly into the Φ-clamp, as mutation of even a single F427 residue causes severe defects in LF N binding and translocation (30,31). However, it was shown in earlier work that residue S429, just 2 residues C-terminal to the Φ-clamp, can be mutated to Cys without significant effect on the function of PA (32).…”

mentioning

confidence: 96%

Interactions of anthrax lethal factor with protective antigen defined by site-directed spin labeling

Jennings-Antipov¹,

Song

Collier³

2011

Proc. Natl. Acad. Sci. U.S.A.

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The protective antigen (PA) moiety of anthrax toxin forms oligomeric pores that translocate the enzymatic moieties of the toxin -lethal factor (LF) and edema factor (EF)-across the endosomal membrane of mammalian cells. Here we describe site-directed spin-labeling studies that identify interactions of LF with the prepore and pore conformations of PA. Our results reveal a direct interaction between the extreme N terminus of LF (residues 2-5) and the Φ-clamp, a structure within the lumen of the pore that catalyzes translocation. Also, consistent with a recent crystallographic model, we find that, upon binding of the translocation substrate to PA, LF helix α1 separates from helices α2 and α3 and binds in the α-clamp of PA. These interactions, together with the binding of the globular part of the N-terminal domain of LF to domain 1′ of PA, indicate that LF interacts with the PA pore at three distinct sites. Our findings elucidate the state from which translocation of LF and EF proceeds through the PA pore.A nthrax toxin, in addition to its importance in regard to the pathogenesis of Bacillus anthracis, is one of the simplest and most tractable systems for studying protein translocation across membranes. The toxin is composed of two intracellularly acting enzymes-edema factor (EF) (1) and lethal factor (LF) (2)-and a receptor-binding (3, 4), pore-forming (5) protein, termed protective antigen (PA). These three proteins are secreted from the Bacillus anthracis as monomeric units. Monomeric PA 83 is activated by furin-family proteases (6) on the host cell surface and spontaneously assembles into homoheptamers, ½PA 63 7 (7), and homooctamers, ½PA 63 8 (8) (collectively termed prepores). ½PA 63 7 binds up to three, and ½PA 63 8 binds up to four, molecules of LF and/or EF (8, 9), forming noncovalent receptor-bound complexes at the cell surface. These complexes are taken into the cell through endocytosis (10) and trafficked to the endosome, where, upon exposure to low pH, the prepores insert into the membrane (5, 11, 12), forming pores through which LF and/or EF translocate to the cytosol (13).LF and EF bind to the surface of PA (on domain 1′) (14) through their homologous, ∼250-residue N-terminal domains (LF N and EF N , Fig. 1E) (15). Each has a positively charged, unstructured N terminus (16, 17) (∼20-30 residues), which is positioned at or near the entry of the lumen when the proteins bind to the prepore (18). PA pores have a negatively charged lumen (7) and form cation-selective channels in planar lipid bilayers (19). LF or LF N blocks current flow through these channels (20), whereas N-terminally truncated LF N mutants do not (21). These observations have led to the hypothesis that upon binding of LF N to domain 1′ of a pore, the N terminus is drawn into the lumen by electrostatic forces, initiating N-to C-terminal translocation. However, data have been lacking to indicate precisely how the unstructured LF or EF N terminus interacts with the lumen of the pore.The Phe427 residues of the PA prepore, located in the lume...

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confidence: 96%

Interactions of anthrax lethal factor with protective antigen defined by site-directed spin labeling

Jennings-Antipov¹,

Song

Collier³

2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Mutated forms of PA 63 had similar properties in the two systems; for example, presence of the F427D mutation in a single subunit of the PA 63 heptamer caused a 2.2-fold increase in single-pore conductance in the DIB system, identical to that seen in planar bilayers ( Fig. S2B) (21).…”

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confidence: 73%

“…Pore occlusion was essentially nil when WT PA 63 added to the capture droplet in step 2 was replaced by either the F427H single mutant (21) or the R178A/ K214E double mutant (23) (Fig. 4).…”

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confidence: 99%

Ultrasensitive detection of protein translocated through toxin pores in droplet-interface bilayers

Fischer

Holden

Pentelute

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Many bacterial toxins form proteinaceous pores that facilitate the translocation of soluble effector proteins across cellular membranes. With anthrax toxin this process may be monitored in real time by electrophysiology, where fluctuations in ionic current through these pores inserted in model membranes are used to infer the translocation of individual protein molecules. However, detecting the minute quantities of translocated proteins has been a challenge. Here, we describe use of the droplet-interface bilayer system to follow the movement of proteins across a model membrane separating two submicroliter aqueous droplets. We report the capture and subsequent direct detection of as few as 100 protein molecules that have translocated through anthrax toxin pores. The droplet-interface bilayer system offers new avenues of approach to the study of protein translocation.

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“…Interestingly, a single dominant-negative subunit with the key amino acid residues mutated was sufficient to significantly inhibit the translocase function of the PA 63 heptamer. More specifically, mutations of Phe427 (or ϕ-clamp) to almost any residue except Trp, Tyr, or Leu disrupted the PA transport function (99), whereas some other Phe427 mutations affected the conformational transition of the prepore to the pore (100). …”

Section: Pa Lf and Ef As Antitoxin Design Targetsmentioning

confidence: 99%

Designing inhibitors of anthrax toxin

Nestorovich

Bezrukov

2014

Expert Opinion on Drug Discovery

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Introduction Present-day rational drug design approaches are based on exploiting unique features of the target biomolecules, small- or macromolecule drug candidates, and physical forces that govern their interactions. The 2013 Nobel Prize in chemistry awarded “for the development of multiscale models for complex chemical systems” once again demonstrated the importance of the tailored drug discovery that reduces the role of the trial and error approach to a minimum. The “rational drug design” term is rather comprehensive as it includes all contemporary methods of drug discovery where serendipity and screening are substituted by the information-guided search for new and existing compounds. Successful implementation of these innovative drug discovery approaches is inevitably preceded by learning the physics, chemistry, and physiology of functioning of biological structures under normal and pathological conditions. Areas covered This article provides an overview of the recent rational drug design approaches to discover inhibitors of anthrax toxin. Some of the examples include small-molecule and peptide-based post-exposure therapeutic agents as well as several polyvalent compounds. The review also directs the reader to the vast literature on the recognized advances and future possibilities in the field. Expert opinion Existing options to combat anthrax toxin lethality are limited. With the only anthrax toxin inhibiting therapy (PA-targeting with a monoclonal antibody, raxibacumab) approved to treat inhalational anthrax, in our view, the situation is still insecure. The FDA’s animal rule for drug approval, which clears compounds without validated efficacy studies on humans, creates a high level of uncertainty, especially when a well-characterized animal model does not exist. Besides, unlike PA, which is known to be unstable, LF remains active in cells and in animal tissues for days. Therefore, the effectiveness of the post-exposure treatment of the individuals with anti-PA therapeutics can be time-dependent, requiring coordinated use of membrane permeable small-molecule inhibitors, which block the LF and EF enzymatic activity intracellularly. The desperate search for an ideal anthrax antitoxin allowed researchers to gain important knowledge of the basic principles of small-molecule interactions with their protein targets that could be easily transferred to other systems. At the same time, better identification and validation of anthrax toxin therapeutic targets at the molecular level, which include understanding of the physical forces underlying the target/drug interaction, as well as elucidation of the parameters determining the corresponding therapeutic windows, require further examination.

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Effects of Introducing a Single Charged Residue into the Phenylalanine Clamp of Multimeric Anthrax Protective Antigen

Cited by 19 publications

References 36 publications

Interactions of anthrax lethal factor with protective antigen defined by site-directed spin labeling

Interactions of anthrax lethal factor with protective antigen defined by site-directed spin labeling

Ultrasensitive detection of protein translocated through toxin pores in droplet-interface bilayers

Designing inhibitors of anthrax toxin

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