# Toxin \(\zeta\) Reduces the ATP and Modulates the Uridine Diphosphate-N-acetylglucosamine Pool
## 3 Discussion  
This study provides new insights that allow us to unravel the mechanisms underlying the mode of action of the toxin \(\zeta\). In silico modeling predicts a large ATP binding pocket. Direct modeling of how ATP could be transferred to toxin \(\zeta\), deduced from various related proteins, failed to predict a preferred conformation, although any of them would be precluded by formation of the \(\zeta\epsilon_{2}\zeta\) complex, supporting a role of \(\epsilon_{2}\) as an inhibitor of ATP binding [22]. Further attempts to identify a preferred bound conformation for ATP using docking failed to single one out. We refined the solution structures of the ligands using QM to better understand their chemical properties. Based on extensive QM calculations, we propose here a previously unreported structure for ATP in solution. Further attempts to identify a preferred bound conformation for ATP using docking failed to single out a preferred one: all alternate locations identified for ATP maintain the same interactions with toxin \(\zeta\). Using  
Figure 6: Models of toxin \(\zeta\) active site with ATP\(\cdot\)Mg\({}^{2+}\) (yellow-orange) located in near and far conformations from UNAG-O3’ (green-orange). ATP-Py distances of 3.5 Å (**A**,**C**) and 7.6 Å (**B**,**D**) with UNAG-O3’. The water molecules and Na\({}^{+}\) ions (purple ball) trapped between them are shown. The Mg\({}^{2+}\) ion is depicted as a green ball. bound ligand structures, we determined relative affinities of ATP, ADP, UNAG and UNAG-P for toxin \(\zeta\), predicting a higher turnover rate for ADP and UNAG than for ATP and UNAG-P. This allowed us to elaborate predictions for in silico mutagenesis and assign putative roles to key amino acids. Since equimolar or significant phosphate transfer from ATP-Py to the O3'-UNAG was not observed (Figure S2b), and toxin \(\zeta\) is anticipated to be saturated with both ATP and UNAG, this suggests that the closest conformers may be underrepresented. This is in agreement with MD simulations, where ATP spent more time in a location far from UNAG. Our genetic and biochemical analyses do not favor an associative mechanism for ATP hydrolysis followed by phosphorylation of a fraction of UNAG [13; 14; 25]. Direct quantification of the fraction of UNAG-P produced under our in vitro conditions revealed that <7% of the total UNAG substrate might be phosphorylated. This is in remarkable agreement with in silico predictions. Therefore, we favor that toxin \(\zeta\), as a UNAG-dependent ATPase, regulates and modulates the ATP and the UNAG pools differently. We cannot discriminate whether toxin \(\zeta\) transfers the Py of ATP to a fraction of UNAG via a dissociative mechanism or by a two-step mechanism, but we can state that toxin \(\zeta\) is mainly an UNAG-dependent ATPase and with low efficiency an UNAG phosphorylase. Our results indicate that \(\zeta\) accomplishes its role by a combination of several mechanisms: first, it modifies the conformation of ligands in the bound state and during the reaction to a structure that is more favorable than the normal structure in solution; second the presence of water in an open active site further helps to reduce the activation energy, Na\({}^{+}\) weakens the P\({}_{\beta}\)-P\({}_{\gamma}\) bond attracting the dissociated P\({}_{\gamma}\); and finally the presence of the protein itself further reduces the activation energy, likely by stabilizing the transition state (Movie S1). In summary, in silico simulations favor a preferred role for toxin \(\zeta\) as ATPase over UNAG phosphorylation. This is consistent with higher affinity for ATP and UNAG-P, and with ATP tending to spend more time staying far away from UNAG, so that ATP hydrolysis is favored, rather than close, where it would phosphorylate UNAG. Our data are consistent with the observation that toxin \(\zeta\) expression, at or near physiological concentration, reduces the ATP pool, leading to an imbalance of ATP/GTP ratios, which subsequently dysregulated transcription of ~78 genes [13; 14].