## 6 Shifting the Paradigm and Conclusions  
As alluded to above for Vbp4 Da2, RBD1 of CDTb also contains a Ca2+ ion binding site that is hypothesized to play a role in CDT toxicity [11,12]. It was the crystal structure (PDB: 6 UWI) of CDTb that first indicated that RBD1 coordinates a Ca2+ ion [11]. The presence of this Ca2+ ion was confirmed by inductively coupled plasma mass spectrometry (ICP-MS) [11]. Nuclear magnetic resonance (NMR) of RBD1 then followed as a means to further probe RBD1 Ca2+-binding [11]. Two-dimensional [1H, 15N]-heteronuclear single quantum coherence spectroscopy (2D [1H, 15N]-HSQC) data for apo-RBD1 displayed severe line-broadening effects and a lack of residue dispersion consistent with a protein in a state undergoing conformational exchange in the millisecond to microsecond timescale [11]. In the presence of 6 mM Ca2+ (approximately three times the concentration found in the extracellular space), the 2D [1H, 15N]-HSQC spectral line-width values narrowed and significant residual dispersion congruent with a structured, folded protein emerged [11]. This relationship with Ca2+ is intriguing as it brings into question the biological role that such a dramatic Ca2+-induced conformational change plays in CDT toxicity. Structural and functional studies probing CDTb pore formation have revealed that the unfolded state of RBD1 is essential for \(\beta\)-barrel extension [13]. Specifically, atomicresolution cryo-EM structures of Ca\({}^{2+}\)-depleted CDTb and a Ca\({}^{2+}\)-binding mutant of CDTb (CDTb\({}^{\text{623A/D734A}}\)) exhibited extended \(\beta\)-barrel structures [13]. Furthermore, surface plasmon resonance (SPR) and electrochemical impedance spectroscopy (EIS) demonstrated how both Ca\({}^{2+}\)-depleted CDTb and CDTb\({}^{\text{623A/D734A}}\) were able to associate with a phospholipid biochip, while CDTb in the presence of Ca\({}^{2+}\) could not [13]. Together, these findings suggest that part of the endosomal pore-forming function of CDTb is driven by the Ca\({}^{2+}\)-depleted environment of the mature endosome. Further, the structure of CDTb revealed that in the preporc conformation, RBD1 and the \(\beta\)BD interface with one another through a hydrogen bond between S708 from RBD1 and Y345 from the \(\beta\)BD (Figures 2 and 6). A disruption of this H-bond may occur as RBD1 unfolds in the absence of Ca\({}^{2+}\), allowing the \(\beta\)BD to unfurl and assemble the \(\beta\)-barrel channel. This potential sensitivity to Ca\({}^{2+}\) may be present in other binary toxins. The Ca\({}^{2+}\)-binding site residues (N621, D623, Q644, S646, and D734) in CDTb are highly conserved among Ib and CSTb, with the exception of S646, which appears as a valine for Ib and an alanine for CSTb (Figure 2). Nevertheless, there is a serine directly downstream of the valine in Ib, and an arginine present directly downstream of the alanine in CSTb, both of which contain oxygen atoms that could be implicated in coordinating Ca\({}^{2+}\). These Ca\({}^{2+}\)-coordinating residues are not as well conserved in C2 II, BECb, PA, Vpb1, and Vpb4 (Figure 2). Despite this deviation from CDTb, many of these alternative residues contain the propensity to coordinate Ca\({}^{2+}\) with three of the five Ca\({}^{2+}\)-coordinating residues found in CDTb RBD1 having conserved substitutions in other binary toxin B subunits (Figure 2). The relevance of this Ca\({}^{2+}\) binding in CDTb, Ib, and CSTb is reinforced by the conservation of preporc-stabilizing residues S708 and Y345 (Figure 2). Figure 6: The interface of the \(\beta\)BD (violet; residues 298–401) and Ca\({}^{2+}\)-bound RBD1 (blue; residues 616–744) shown for a preporc CDTb protomer. Close-up view of the hydrogen bond interaction (yellow dotted line) between Y345 of \(\beta\)BD and S708 of RBD1 is highlighted in the inset. Domains are colored according to Figure 4. Structure modified from PDB: 6 UWI (\({}^{\text{5 ym}}\)CDTb).