## 5 Structure and Function of Clostridial Binary Toxins
### Mechanisms of Endosomal Export for CDTa/Ia  
A critical regulatory feature in translocation for CDTa/Ia is the \(\varphi\)-gate or \(\varphi\)-clamp, which is positioned in a ring at the entrance of the pore created by CDTb/Ib (Figures 2 and 5) [8,14,15]. In CDTb, the \(\varphi\)-clamp consists of a phenylalanine (F455) from heptamerization domain 2 (HD2), contributed by each CDTb protomer [8,11,15]. In Ib, this phenylalanine (F454) is donated by each Ib protomer from the D2c domain [14]. As the \(\beta\)-barrel extension is triggered, the \(\varphi\)-clamp enters a conformational constricted \"closed\" state [11,12]. In CDTb, the closed \(\varphi\)-clamp of the extended \(\beta\)-barrel forms a passageway with a diameter of 3 A, whereas the non-\(\beta\)-barrel, or \"open\" state pore diameter is 12.5 A [11]. A similar narrowing is observed for Ib with a closed \(\varphi\)-clamp diameter of 6 A; however, structural data of prepore Ib are lacking [14]. The putative mechanism suggests that the acidic environment of mature endosomes facilitates the N-terminal threading of CDTa/Ia through the \(\varphi\)-clamp followed by extended-chain Brownian ratchet model-mediated translocation through the \(\beta\)-barrel [14,15]. Aided by pH-mediated 'flipping' of NSX-loop conformations, the \(\varphi\)-clamp is a critical constriction point required for the unfolding and passage of the enzymatic component through the \(\beta\)-barrel [14,15]. The fifth and final constriction occurs at residue H314 (CDTb) and H313 (Ib), located in the \(\beta\)-barrel stem just below the \(\varphi\)-clamp (Figure 5) [14,15]. Upon channeling of the enzymatic subunit through the \(\beta\)-barrel stem, chaperones such as heat-shock proteins 70 (Hsp70) and 90 (Hsp90), and peptidyl-prolyl cis-/trans-isomerases (PPIases) such as cyclophilin A, are proposed to then aid CDTa/Ia in entering the cytosol [49,62,63,64,65]. The current model suggests these host cell factors facilitate the transmembrane transport of the enzymatic payload through interactions with the ADP-ribosyltransferase domain of the enzyme [66,66]. Furthermore, this chaperone intervention is necessary as the enzymatic A subunits, after having passed through the sterically constrictive B subunits, will be at least partially unfolded and require refolding [63]. Moreover, inhibition of these chaperones and PPIases has been performed and the results from these studies have presented a novel mechanism of action to consider as well as a new pharmacological strategy against CDT-producing strains of _C. difficile_[65,67, 9].  
As was discussed, CDTb and Ib endosomal pore formation is partially dependent upon endosomal acidification [8,43,68]. Interestingly, this acidification is accompanied by the transport of Ca2+ ions out of the endosomal space and into the cytosol via Ca2+-permeable channels, resulting in a significant drop in free Ca2+ ion concentration after endosome formation [69,70]. While the importance of the constriction motifs discussed and the acidic pH of mature endosomes in the function of CDTb/Ib cannot be understated, an additional factor impacting toxin translocation centered upon Ca2+ ion concentration has emerged and is deserving of exploration.