Mechanism of diphtheria toxin catalytic domain delivery to the eukaryotic cell cytosol and the cellular factors that directly participate in the process

Abstract

Research on diphtheria and anthrax toxins over the past three decades has culminated in a detailed understanding of their structure function relationships (e.g., catalytic (C), transmembrane (T), and receptor binding (R) domains), as well as the identification of their eukaryotic cell surface receptor, an understanding of the molecular events leading to the receptor-mediated internalization of the toxin into an endosomal compartment, and the pH triggered conformational changes required for pore formation in the vesicle membrane. Recently, a major research effort has been focused on the development of a detailed understanding of the molecular interactions between each of these toxins and eukaryotic cell factors that play an essential role in the efficient translocation of their respective catalytic domains through the trans-endosomal vesicle membrane pore and delivery into the cell cytosol. In this review, I shall focus on recent findings that have led to a more detailed understanding of the mechanism by which the diphtheria toxin catalytic domain is delivered to the eukaryotic cell cytosol. While much work remains, it is becoming increasingly clear that the entry process is facilitated by specific interactions with a number of cellular factors in an ordered sequential fashion. In addition, since diphtheria, anthrax lethal factor and anthrax edema factor all carry multiple coatomer I complex binding motifs and COPI complex has been shown to play an essential role in entry process, it is likely that the initial steps in catalytic domain entry of these divergent toxins follow a common mechanism.

Keywords: catalytic domain entry; coatomer complex I; diphtheria toxin.

Figure 1

Schematic depiction of the mechanism of diphtheria toxin entry into eukaryotic cell cytosol. (1) Diphtheria toxin binds to its cell surface receptor and is (2) internalized in clathrin coated pits into early endosomal vesicles. Upon acidification of the endosomal lumen, (3) the transmembrane domain of the toxin undergoes a spontaneous dynamic reorganization and inserts into the membrane forming a pore through which (4) the C-domain is delivered to the cytosol. The delivery of the C-domain is facilitated by at least COPI complex, thioredoxin reductase and Hsp90. Once refolded into an active conformation, the C-domain catalyzes the ADP-ribosylation of elongation factor 2. Diphtheria toxin: red = catalytic domain; green = transmembrane domain; yellow = native receptor binding domain.

Figure 2

Schematic representation of the insertion of the diphtheria toxin transmembrane domain into the endosomal vesicle membrane which results in the formation of a transmembrane pore. Following furin mediated “nicking” of the toxin after Arg194 and denaturation of the catalytic domain, the N-terminal portion of the transmembrane domain with its disulfide bond linked C-terminal end of the catalytic domain appears to be threaded into the pore. Upon emergence of one or more of the KXKXX motifs on the cytosolic side of the vesicle membrane, COPI complex binds to these sequences and facilitates the translocation of the catalytic domain.

Figure 3

Partial amino acid sequences of N-terminal regions of the diphtheria toxin transmembrane domain, anthrax lethal factor, and anthrax edema factor showing the positions of their respective T1 or T1-like motifs and the clustering of multiple KXKXX COPI binding motifs on each protein.