A loop network within the anthrax toxin pore positions the phenylalanine clamp in an active conformation.
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ABSTRACT:
SUBMITTER: Melnyk RA
PROVIDER: S-EPMC1479862 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The protective antigen component of anthrax toxin forms a homoheptameric pore in the endosomal membrane, creating a narrow passageway for the enzymatic components of the toxin to enter the cytosol. We found that, during conversion of the heptameric precursor to the pore, the seven phenylalanine-427 residues converged within the lumen, generating a radially symmetric heptad of solvent-exposed aromatic rings. This "phi-clamp" structure was required for protein translocation and comprised the major conductance-blocking site for hydrophobic drugs and model cations. We conclude that the phi clamp serves a chaperone-like function, interacting with hydrophobic sequences presented by the protein substrate as it unfolds during translocation.
| S-EPMC1815389 | biostudies-literature
Project description:Anthrax toxin is an intracellularly acting toxin where sufficient detail is known about the structure of its channel, allowing for molecular investigations of translocation. The toxin is composed of three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). The toxin's translocon, PA, translocates the large enzymes, LF and EF, across the endosomal membrane into the host cell's cytosol. Polypeptide clamps located throughout the PA channel catalyze the translocation of LF and EF. Here, we show that the central peptide clamp, the ϕ clamp, is a dynamic site that governs the overall peptide translocation pathway. Single-channel translocations of a 10-residue, guest-host peptide revealed that there were four states when peptide interacted with the channel. Two of the states had intermediate conductances of 10% and 50% of full conductance. With aromatic guest-host peptides, the 50% conducting intermediate oscillated with the fully blocked state. A Trp guest-host peptide was studied by manipulating its stereochemistry and prenucleating helix formation with a covalent linkage in the place of a hydrogen bond or hydrogen-bond surrogate (HBS). The Trp peptide synthesized with ʟ-amino acids translocated more efficiently than peptides synthesized with D- or alternating D,ʟ-amino acids. HBS stapled Trp peptide exhibited signs of steric hindrance and difficulty translocating. However, when mutant ϕ clamp (F427A) channels were tested, the HBS peptide translocated normally. Overall, peptide translocation is defined by dynamic interactions between the peptide and ϕ clamp. These dynamics require conformational flexibility, such that the peptide productively forms both extended-chain and helical states during translocation.
| S-EPMC5562545 | biostudies-other
Project description:Protective antigen (PA) is the anthrax toxin protein recognized by capillary morphogenesis gene 2 (CMG2), a transmembrane cellular receptor. Upon activation, seven ligand-receptor units self-assemble into a heptameric ring-like complex that becomes endocytozed by the host cell. A critical step in the subsequent intoxication process is the formation and insertion of a pore into the endosome membrane by PA. The pore conversion requires a change in binding between PA and its receptor in the acidified endosome environment. Molecular dynamics simulations totaling approximately 136 ns on systems of over 92,000 atoms were performed. The simulations revealed how the PA-CMG2 complex, stable at neutral conditions, becomes transformed at low pH upon protonation of His-121 and Glu-122, two conserved amino acids of the receptor. The protonation disrupts a salt bridge important for the binding stability and leads to the detachment of PA domain II, which weakens the stability of the PA-CMG2 complex significantly, and subsequently releases a PA segment needed for pore formation. The simulations also explain the great strength of the PA-CMG2 complex achieves through extraordinary coordination of a divalent cation.
| S-EPMC1432108 | biostudies-literature
Project description:Interaction between bacterial toxins and cellular surface receptors is an important component of the host-pathogen interaction. Anthrax toxin protective antigen (PA) binds to the cell surface receptor, enters the cell through receptor-mediated endocytosis, and forms a pore on the endosomal membrane that translocates toxin enzymes into the cytosol of the host cell. As the major receptor for anthrax toxin in vivo, anthrax toxin receptor 2 (ANTXR2) plays an essential role in anthrax toxin action by providing the toxin with a high-affinity binding anchor on the cell membrane and a path of entry into the host cell. ANTXR2 also acts as a molecular clamp by shifting the pH threshold of PA pore formation to a more acidic pH range, which prevents premature pore formation at neutral pH before the toxin reaches the designated intracellular location. Most recent studies have suggested that the disulfide bond in the immunoglobulin (Ig)-like domain of ANTXR2 plays an essential role in anthrax toxin action. Here we will review the roles of ANTXR2 in anthrax toxin action, with an emphasis on newly updated knowledge.
| S-EPMC4773787 | biostudies-literature
Project description:Membrane-embedded molecular machines are utilized to move water-soluble proteins across these barriers. Anthrax toxin forms one such machine through the self-assembly of its three component proteins--protective antigen (PA), lethal factor, and edema factor. Upon endocytosis into host cells, acidification of the endosome induces PA to form a membrane-inserted channel, which unfolds lethal factor and edema factor and translocates them into the host cytosol. Translocation is driven by the proton motive force, composed of the chemical potential, the proton gradient (?pH), and the membrane potential (??). A crystal structure of the lethal toxin core complex revealed an "? clamp" structure that binds to substrate helices nonspecifically. Here, we test the hypothesis that, through the recognition of unfolding helical structure, the ? clamp can accelerate the rate of translocation. We produced a synthetic PA mutant in which an ? helix was crosslinked into the ? clamp to block its function. This synthetic construct impairs translocation by raising a yet uncharacterized translocation barrier shown to be much less force dependent than the known unfolding barrier. We also report that the ? clamp more stably binds substrates that can form helices than those, such as polyproline, that cannot. Hence, the ? clamp recognizes substrates by a general shape-complementarity mechanism. Substrates that are incapable of forming compact secondary structure (due to the introduction of a polyproline track) are severely deficient for translocation. Therefore, the ? clamp and its recognition of helical structure in the translocating substrate play key roles in the molecular mechanism of protein translocation.
| S-EPMC5125381 | biostudies-literature
Project description:Cellular receptors can act as molecular switches, regulating the sensitivity of microbial proteins to conformational changes that promote cellular entry. The activities of these receptor-based switches are only partially understood. In this paper, we sought to understand the mechanism that underlies the activity of the ANTXR2 anthrax toxin receptor-based switch that binds to domains 2 and 4 of the protective antigen (PA) toxin subunit. Receptor-binding restricts structural changes within the heptameric PA prepore that are required for pore conversion to an acidic endosomal compartment. The transfer cross-saturation (TCS) NMR approach was used to monitor changes in the heptameric PA-receptor contacts at different steps during prepore-to-pore conversion. These studies demonstrated that receptor contact with PA domain 2 is weakened prior to pore conversion, defining a novel intermediate in this pathway. Importantly, ANTXR2 remained bound to PA domain 4 following pore conversion, suggesting that the bound receptor might influence the structure and/or function of the newly formed pore. These studies provide new insights into the function of a receptor-based molecular switch that controls anthrax toxin entry into cells.
| S-EPMC3234216 | biostudies-literature
Project description:Anthrax toxin, comprising protective antigen, lethal factor, and oedema factor, is the major virulence factor of Bacillus anthracis, an agent that causes high mortality in humans and animals. Protective antigen forms oligomeric prepores that undergo conversion to membrane-spanning pores by endosomal acidification, and these pores translocate the enzymes lethal factor and oedema factor into the cytosol of target cells. Protective antigen is not only a vaccine component and therapeutic target for anthrax infections but also an excellent model system for understanding the mechanism of protein translocation. On the basis of biochemical and electrophysiological results, researchers have proposed that a phi (Φ)-clamp composed of phenylalanine (Phe)427 residues of protective antigen catalyses protein translocation via a charge-state-dependent Brownian ratchet. Although atomic structures of protective antigen prepores are available, how protective antigen senses low pH, converts to active pore, and translocates lethal factor and oedema factor are not well defined without an atomic model of its pore. Here, by cryo-electron microscopy with direct electron counting, we determine the protective antigen pore structure at 2.9-Å resolution. The structure reveals the long-sought-after catalytic Φ-clamp and the membrane-spanning translocation channel, and supports the Brownian ratchet model for protein translocation. Comparisons of four structures reveal conformational changes in prepore to pore conversion that support a multi-step mechanism by which low pH is sensed and the membrane-spanning channel is formed.
| S-EPMC4519040 | biostudies-literature
Project description:Multimeric pores formed in the endosomal membrane by the Protective Antigen moiety of anthrax toxin translocate the enzymatic moieties of the toxin to the cytosolic compartment of mammalian cells. There is evidence that the side chains of the Phe(427) residues come into close proximity with one another in the lumen of the pore and form a structure, termed the Phe clamp, that catalyzes the translocation process. In this report we describe the effects of replacing Phe(427) in a single subunit of the predominantly heptameric pore with a basic or an acidic amino acid. Incorporating any charged residue at this position inhibited cytotoxicity >or=1,000-fold in our standard assay and caused strong inhibition of translocation in a planar phospholipid bilayer system. His and Glu were the most strongly inhibitory residues, ablating both cytotoxicity and translocation. Basic residues at position 427 prevented the Phe clamp from interacting with a translocation substrate to form a seal against the passage of ions and accelerated dissociation of the substrate from the pore. Acidic residues, in contrast, allowed the seal to form and the substrate to remain firmly bound, but blocked its passage, perhaps via electrostatic interactions with the positively charged N-terminal segment. Our findings are discussed in relation to the role of the Phe clamp in a Brownian ratchet model of translocation.
| S-EPMC2832964 | biostudies-literature
Project description:The anthrax toxin receptors, ANTXR1 and ANTXR2, act as molecular clamps to prevent the protective antigen (PA) toxin subunit from forming pores until exposure to low pH. PA forms pores at pH approximately 6.0 or below when it is bound to ANTXR1, but only at pH approximately 5.0 or below when it is bound to ANTXR2. Here, structure-based mutagenesis was used to identify non-conserved ANTXR2 residues responsible for this striking 1.0 pH unit difference in pH threshold. Residues conserved between ANTXR2 and ANTXR1 that influence the ANTXR2-associated pH threshold of pore formation were also identified. All of these residues contact either PA domain 2 or the neighboring edge of PA domain 4. These results provide genetic evidence for receptor release of these regions of PA as being necessary for the protein rearrangements that accompany anthrax toxin pore formation.
| S-EPMC1824706 | biostudies-literature
Project description:Proteolytically activated Protective Antigen (PA) moiety of anthrax toxin self-associates to form a heptameric ring-shaped oligomer (the prepore). Acidic pH within the endosome converts the prepore to a pore that serves as a passageway for the toxin's enzymatic moieties to cross the endosomal membrane. Prepore is stable in solution under mildly basic conditions, and lowering the pH promotes a conformational transition to an insoluble pore-like state. N-tetradecylphosphocholine (FOS14) was the only detergent among 110 tested that prevented aggregation without dissociating the multimer into its constituent subunits. FOS14 maintained the heptamers as monodisperse, insertion-competent 440-kDa particles, which formed channels in planar phospholipid bilayers with the same unitary conductance and ability to translocate a model substrate protein as channels formed in the absence of detergent. Electron paramagnetic resonance analysis detected pore-like conformational changes within PA on solubilization with FOS14, and electron micrograph images of FOS14-solubilized pore showed an extended, mushroom-shaped structure. Circular dichroïsm measurements revealed an increase in alpha helix and a decrease in beta structure in pore formation. Spectral changes caused by a deletion mutation support the hypothesis that the 2beta2-2beta3 loop transforms into the transmembrane segment of the beta-barrel stem of the pore. Changes caused by selected point mutations indicate that the transition to alpha structure is dependent on residues of the luminal 2beta11-2beta12 loop that are known to affect pore formation. Stabilizing the PA pore in solution with FOS14 may facilitate further structural analysis and a more detailed understanding of the folding pathway by which the pore is formed.
| S-EPMC2777363 | biostudies-literature