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

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 ␣-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. Exotoxin A (PE)1 is one of the major virulence factors secreted by Pseudomonas aeruginosa. This toxin is able to kill a large range of mammalian cell lines by inhibiting their protein synthesis (1), and it is one of the favorite toxins to prepare immunotoxins that are promising agents for the treatment of cancers (2). PE is a single chain 66-kDa protein organized in three structural (3) and functional (4) domains successively involved in the intoxication process. First, domain I binds to the ␣ 2 -macroglobulin/low density lipoprotein receptor-related protein (5), enabling internalization via receptor-mediated endocytosis. Domain II will then mediate translocation into the cytosol of the entire toxin (6) or of a carboxyl-terminal fragment generated by furin proteolysis and encompassing domain III and most of domain II (1). Finally, domain III will catalyze the

Mechanism for HIV-1 Tat Insertion into the Endosome Membrane

“…Studies of the third helix of Antennapedia, a Drosophila homeodomain that is able to reach the cell cytosol from the outside, also identified a key Trp residue that is required for translocation and could not be replaced by Phe (45). We showed here that Tat uses a Trp-mediated regulated membrane insertion process, as described previously for other proteins such as annexin-V (32), protein kinase C-␣ (33), and PEA (24). In the case of PEA and Tat, the stimulus leading to Trp exposure and membrane insertion is acidification, and it was therefore interesting to examine whether a low-pH sensor system such as the one identified in PEA could also be found in Tat.…”

“…The resulting SUV had a diameter of 100 -200 nm. To prepare brominated vesicles, half of the PC was replaced by 1,2-(9,10-dibromo)stearoyl phosphatidylcholine (24). When indicated the 10-DN quencher was added at 10 mol % before drying the lipids (36).…”

“…Hence, Tat enters cells using a "conventional" endosomal translocating toxin strategy, just like diphtheria toxin, which is the best characterized example of this type of toxin (22). The study of the translocation process of endosometranslocating toxins was facilitated by their ability to insert into model membranes upon acidification (23,24). Whether Tat is also able to do so has yet to be demonstrated.…”

“…It is not clear how such a small protein as Tat, which is devoid of a hydrophobic ␣-helix (31), could undergo membrane insertion. Nevertheless, we previously showed that insertion of Pseudomonas exotoxin A (PEA) into the endosome membrane relies on a key tryptophan residue that becomes exposed at endosomal pH (pH 5.3-5.5) and then triggers membrane insertion (24). Other proteins such as annexin-V (32) and protein kinase C-␣ (33) also undergo such regulated Trp-mediated membrane insertion, although in that case the stimulus triggering insertion is a rise in Ca 2ϩ concentration and not acidification.…”

“…All known proteins whose membrane insertion is based on a Trp side chain have a molecular device that induces Trp exposure and thereby controls insertion (24,32,33). Because the Tat membrane insertion process only takes place within endosomes (15), if Tat actually belongs to this group of proteins it likely possesses a low-pH sensor able to trigger Trp exposure upon endosomal delivery.…”