# Toxin \(\zeta\) Reduces the ATP and Modulates the Uridine Diphosphate-N-acetylglucosamine Pool
## 2 Results  
### Toxin \(\zeta\) Has a Large ATP Site Pocket  
Crystallographic data are available for the apo \(\zeta\epsilon_{2}\zeta\) complex (1 GVN) and for this complex bound to UNAG (3Q8X) [19; 22; 23]. In both structures, the C-terminal residues of the \(\zeta\) polypeptide chains cannot be seen in the electron density maps, implying a high degree of flexibility. To understand the properties of the _S. pyogenes_ toxin \(\zeta\), especially its unassigned C-terminal region, and its ATP bound form, we performed a computational analysis. The model for full-length toxin \(\zeta\) was built using iterative threading as implemented in the I-TASSER suite [28]. According to this model, the missing C-terminal region might fold in the face opposite to the antitoxin \(\epsilon_{2}\) binding site, and the putative ATP binding site, and therefore it would be unlikely to interfere with toxin \(\zeta\) activity. This is consistent with the observation that a PezT variant lacking the unassigned C-terminal end is an active toxin [23].  
The ATP binding site in the \(\zeta\) toxin was initially assigned based on similarity to genuine phosphotransferases [19; 22]. Pocket predictions using 3v [29] showed only two putative pockets: the one corresponding to the known UNAG binding site, and a larger one, where the N-terminal end of the \(\epsilon_{2}\) antitoxin binds in the crystal structure [19; 22]. This is the proposed binding location for ATP (Figure 1A,B). The I-TASSER modeled toxin \(\zeta\) structures were superposed with the known structures of related phosphotransferases as chloramphenicol phosphotransferase (1 QHX) [30], O-phosphosyl-tRNA kinase (3 AM1) [31] and polynucleotide kinase (4 GP7) [32] using DALI and UCSF Chimera [33; 34], and optimized using OpenBabel [35] and GROMACS [35]. These studies gave inconclusive results: the ATP pocket has a very open conformation and it is significantly larger than ATP, allowing for wide choice in the placement of ATP-P\(\gamma\) relative to the C3\({}^{\prime}\)-OH group of the N-acetylglucosamine moiety of UNAG (UNAG-O3\({}^{\prime}\)), which ranged from 3.5-8.5 A (Figure 1A,B, Supplemental Movie S1). Thus, we decided to use molecular docking to search for better conformations.  
Prior to molecular docking, the ligands were optimized using semi-empirical Quantum Mechanics (SQM) and Quantum Molecular Dynamics (QMD) with Gabedit [36] and OpenMopac [37] conducting a conformational search in vacuo and in solution. Interestingly, ATP tended to adopt a conformation between L-shaped (with the triphosphate tail rising near perpendicularly to the nucleotide ring), and a more compact, bent \"scorpion-like\" structure (Movie S1). Docking with Autodock Vina [38] showed a distance between ATP-P\(\gamma\) and UNAG-O3\({}^{\prime}\) of 3-8 A, and the conformers produced by DOCK6 [38]  
Figure 1: The UNAG and ATP pockets. The two main pockets identified by 3v superimposed to \(\zeta\), UNAG and the predicted binding site for ATP-Mg\({}^{2+}\). Toxin \(\zeta\) active sites with ATP (purple) located in extreme positions in the binding pocket, either far (-8.5 Å, (**A**)) or close (-3.5 Å, (**B**)) to the O3\({}^{\prime}\) of UNAG (cyan). The Mg\({}^{2+}\) ion is denoted as a green ball.  
showed a distance of 3.5-6.5 A. Further refinement using molecular dynamics (MD) simulations in GROMACS [39] showed that ATP tends to stay less time in a configuration near (~3.5 A, ~7.4% of the time) than far (~4.5 A, 92.6% of the time) from UNAG, with several intervening water molecules, which tend to be temporarily trapped close to the Py of ATP. About 75.5% of the time, the distance between ATP-Py and UNAG-O3' was > 5 A apart (Movie S1). Representative conformers were selected to compare ligand-protein affinities using Xscore [40] and DrugScoreDSX [41]. Affinity predictions showed a greater affinity for ATP and UNAG-P than for ADP and UNAG (Table 1).