Asymmetric Cryo-EM Structure of Anthrax Toxin Protective Antigen Pore with Lethal Factor N-Terminal Domain.

Alexandra Machen,Narahari Akkaladevi,Mark Fisher,Rebecca Dillard,Edward Gogol,Tommi White,Caleb Trecazzi,Srayanta Mukherjee,Wonpil Im,Pierce O’Neil,Yifei Qi

doi:10.3390/toxins9100298

Abstract

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 LFN-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 LFN bound. At pH 7.5, the contributions from the three unstructured LFN 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.

Highlights

The lethality of anthrax, a zoonotic disease and bioterrorism agent, is due to the anthrax toxin.This tripartite toxin consists of a protective antigen (PA), lethal factor (LF; a mitogen-activated protein kinase kinase protease), and edema factor (EF; an adenylate cyclase) [1]
With the recent publication of the cryo-electron microscopy (cryo-EM) PA pore structure at pH 5.0 [17], the logical, but challenging, step in understanding anthrax toxin pore formation and translocation involves determining how bound LF influences the conformation of the PA pore
Our previous low-resolution LFN -PA-nanodisc structures were reconstructed from samples frozen on perforated carbon containing a thin carbon layer over holes [23]

Summary

Introduction

The lethality of anthrax, a zoonotic disease and bioterrorism agent, is due to the anthrax toxin. This tripartite toxin consists of a protective antigen (PA), lethal factor (LF; a mitogen-activated protein kinase kinase protease), and edema factor (EF; an adenylate cyclase) [1]. 83 kDa PA (PA83 ) binds to its target host cell receptor, either capillary morphogenesis protein 2 (CMG2). PA83 is cleaved by proteases, resulting in 20 kDa and. PA63 self-associates to form a heptameric PA prepore that can associate with up to three molecules of LF or EF [5]. Octameric PA prepores may assemble in solution, governed by LF or EF binding to PA63 monomers clipped in solution [6].

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Conclusion