## 5 Structure and Function of Clostridial Binary Toxins
### CDTb/lb Heptamerization and Receptor Binding  
Given the sequence and structural similarities between CDTb and Ib, it follows that they share the lipolysis-stimulated lipoprotein receptor (LSR), a type-I single-pass transmembrane protein, characterized by an extracellular immunoglobulin-like domain and an extended intracellular segment, as their primary host cell surface receptor [43; 48; 49].  
Figure 4: Atomic-resolution structures of di-heptameric CDTb from _C. difficile_. The 3D structures of the CDTb di-heptamers are illustrated in \({}^{\mathrm{Sym}}\)CDTb and \({}^{\mathrm{Asym}}\)CDTb conformations with domains colored and labeled. Structures are modified from PDB: 6 UWT (\({}^{\mathrm{Sym}}\)CDTb) and 6 UWR (\({}^{\mathrm{Asym}}\)CDTb) [11]. The di-heptamer structures are highly stable, and can dissociate into ”active” heptameric structures upon dissociation of Ca\({}^{2+}\) from a newly described Ca\({}^{2+}\)-site within its RBD1 domain (residues 616–744). This figure was created with the program BioRender.  
Clostridial binary toxins also bind to CD44, a type-I cell surface glycoprotein typically associated with lipid rafts (cholesterol-rich regions of the cell membrane), to intoxate cells [50]. For example, it was observed that the Ib heptamer associates with lipid rafts and that CD44 was found occupying lipid rafts in Ib-treated Vero cells [51; 52; 53]. This toxin-host interface then triggers cellular uptake of the toxin via endocytosis. However, despite the surge in structural information for both CDT and iota toxin, the mechanisms by which endocytosis occurs for these toxins remain somewhat ambiguous. For example, there is uncertainty about whether proteolytic activation occurs prior to or after B subunit binding to the cell surface receptor. This uncertainty has fueled discourse on whether the B subunit binds to the cell as an inactivated monomer or as a heptamer (Figure 1) [10]. Moreover, studies indicate that LSR is not a component of lipid rafts and will instead localize to them after associating with CDTb [54]. Interestingly, experiments exploring the association between CDTb and LSR revealed CDTb, in the absence of CDTa, promotes cytotoxic effects such as cell rounding in cells expressing LSR [55]. Specifically, this is a consequence of pore formation, as the receptor-binding domains of CDTb alone were not sufficient in promoting cell rounding [55]. Furthermore, for HeLa cells that did not express LSR and for LSR knock-out CaCo-2 cells, CDTb-induced cell rounding was impeded [55]. Likewise, it has been observed that upon LSR association and heptamerization, Ib forms cytotoxic ion-permeable channels which facilitate the release of K\({}^{+}\) and Na\({}^{+}\) influx in Vero cells [43; 56; 57; 58]. Nevertheless, the formation of the heptamer, whether prior to or after coming into contact with LSR, is pivotal for CDT and iota toxin function. As such, efforts have been made to inhibit their oligomerization. Most notably, these efforts include the development of monoclonal antibodies (e.g., BINTOXB/9 and BINTOXB/22) which inhibit trypsin-induced CDTb oligomerization and subsequently CDT toxicity [59].