Solution Structures of Bacillus anthracis Protective Antigen Proteins Using Small Angle Neutron Scattering and Protective Antigen 63 Ion Channel Formation Kinetics.

Ariel Michelman-Ribeiro,Kenneth A Rubinson,Vitalii Silin,John J Kasianowicz

doi:10.3390/toxins13120888

Abstract

We are studying the structures of bacterial toxins that form ion channels and enable macromolecule transport across membranes. For example, the crystal structure of the Staphylococcus aureus α-hemolysin (α-HL) channel in its functional state was confirmed using neutron reflectometry (NR) with the protein reconstituted in membranes tethered to a solid support. This method, which provides sub-nanometer structural information, could also test putative structures of the Bacillus anthracis protective antigen 63 (PA63) channel, locate where B. anthracis lethal factor and edema factor toxins (LF and EF, respectively) bind to it, and determine how certain small molecules can inhibit the interaction of LF and EF with the channel. We report here the solution structures of channel-forming PA63 and its precursor PA83 (which does not form channels) obtained with small angle neutron scattering. At near neutral pH, PA83 is a monomer and PA63 a heptamer. The latter is compared to two cryo-electron microscopy structures. We also show that although the α-HL and PA63 channels have similar structural features, unlike α-HL, PA63 channel formation in lipid bilayer membranes ceases within minutes of protein addition, which currently precludes the use of NR for elucidating the interactions between PA63, LF, EF, and potential therapeutic agents.

Highlights

Ion channels and pore-forming toxins have been studied extensively because of their significance in the proper function and disease states of different tissues [1,2,3]
LF or EF associates with PA637, and the A-B complexes of LF-PA637 or EF-PA637 are removed from the cell membrane exterior by endocytotic vesicles, where PA637 channel formation is thought to occur after endosome acidification [9]
At pD 7.8, PA83 is a monomer and was modeled as a right parallelepiped with a volume ≈ 124 × 103 Å3, similar to the volume estimated from its molecular mass and a typical protein density value

Summary

Introduction

Ion channels and pore-forming toxins have been studied extensively because of their significance in the proper function and disease states of different tissues [1,2,3]. One class of channels is of particular interest because the pore-forming toxins work in concert with other proteins that are produced by their respective bacteria. One B-toxin (protective antigen 83, PA83) [4,5]. The latter binds to the exterior of cell membranes [6], is cleaved by a furin-like protease to PA63 [7], and one of the products (PA63) forms a heptameric pre-pore complex PA637 [8]. LF or EF associates with PA637 , and the A-B complexes of LF-PA637 or EF-PA637 are removed from the cell membrane exterior by endocytotic vesicles, where PA637 channel formation is thought to occur after endosome acidification [9]. PA637 is required for LF and EF to gain entry into the cytoplasm: LF inhibits mitogen-activated protein kinases (which leads to apoptosis), and EF leads to increased cyclic AMP concentration (which leads to cell edema) [10,11]

Methods

Results

Conclusion