Acid-triggered Membrane Insertion of Pseudomonas Exotoxin A Involves an Original Mechanism Based on pH-regulated Tryptophan Exposure

Jocelyn Méré,Juliette Morlon-Guyot,Anne Bonhoure,Laurent Chiche,Bruno Beaumelle

doi:10.1074/jbc.m412656200

Abstract

Exposure to low endosomal pH during internalization of Pseudomonas exotoxin A (PE) triggers membrane insertion of its translocation domain. This process is a prerequisite for PE translocation to the cytosol where it inactivates protein synthesis. Although hydrophobic helices enable membrane insertion of related bacterial toxins such as diphtheria toxin, the PE translocation domain is devoid of hydrophobic stretches and the structural features triggering acid-induced membrane insertion of PE are not known. Here we have identified a molecular device that enables PE membrane insertion. This process is promoted by exposure of a key tryptophan residue. At neutral pH, this Trp is buried in a hydrophobic pocket closed by the smallest alpha-helix of the translocation domain. Upon acidification, protonation of the Asp that is the N-cap residue of the helix leads to its destabilization, enabling Trp side chain insertion into the endosome membrane. This tryptophan-based membrane insertion system is surprisingly similar to the membrane-anchoring mechanism of human annexin-V and could be used by other proteins as well.

Highlights

Exotoxin A (PE)1 is one of the major virulence factors secreted by Pseudomonas aeruginosa
The translocation domains of related toxins such as diphtheria toxin or colicins show a characteristic three-layer structure with buried hydrophobic helices likely implicated in membrane insertion, Pseudomonas exotoxin A (PE) domain II is devoid of such helices and, more generally, of hydrophobic stretches [9]
We examined the three-dimensional structure of the translocation domain [3] in an attempt to identify the structural basis of the gain in PE translocation activity obtained by deletion of helix F

Summary

Introduction

Exotoxin A (PE) is one of the major virulence factors secreted by Pseudomonas aeruginosa. The translocation domains of related toxins such as diphtheria toxin or colicins show a characteristic three-layer structure with buried hydrophobic helices likely implicated in membrane insertion, PE domain II is devoid of such helices and, more generally, of hydrophobic stretches [9]. Deletion of any of the first five helices abrogates both translocation activity and toxicity, excising the smallest and last helix of the domain (helix F, 5 residues) increased the translocation rate by 60 –70% with a concomitant ϳ4-fold rise in toxicity [15]. This result indicated that this region has a key role in the translocation process. We found that Asp-358, which is the helix F N-cap residue, is the low pH sensor triggering helix F destabilization leading to Trp-305 membrane insertion and toxin translocation at low pH

Methods

Results

Conclusion