## 3 The Complex Network of (p)ppGpp Signaling
### The (p)ppGpp Interactome
#### 3.4.1. SpeC, a Constitutive Ornithineine Decarboxylase  
One of the hits in the ppGpp-affinity Mass-Spec described above is SpeC, a 'constitutive' ornithine decarboxylase (ODC) in _E. coli_ (UniProtKB P21169) [118]. However, its three-dimensional structure is not available. The structure of the D-ornithine decarboxylase of Salmonella [119] (SpeF; UniProtKB Q8 ZNC4), also a Gram-negative bacterium and phylogenetically very close to _E. coli_[118,119], is known (PDB 6N2H), but it also decarboxylulates D-lysine, and uses both amino acids in D-configuration rather than the usual L form [120]. At a length of 465 amino acids, it is also much shorter than _E. coli_ SpeC (711 amino acids). The structure of the other major ODC (SpeF) of _E. coli_, which belongs to the 'inducible' class of ODC, has recently been solved, but in complex with stalled ribosomes [121]. On the other hand, the apo structure of Salmonella SpeF is available for the single polypeptide (PDB 6N2H). Lastly, the structure of a 445 aa long ODC form Trypanosoma brucei (XP_011780677.1, PDB 1 NJJ) in complex with D-ornithine is available, which is a dimer, but each monomer has a molecule of D-ornithine embedded in it [122]. Its primary structure is highly similar to that of the Salmonella enzyme (Supplementary material), and, thus, we reasoned that the two enzymes may share similar catalytic domains and structures. For the best approximation in the docking analysis, we first docked the structure of ornithine, the substrate, into the available Salmonella SpeF structure (PDB 6N2H), and we found that the resultant structure was comparable to those of the known co-crystal structure of ornithine and the T. brucei enzyme monomer (PDB 1 NJJ) (Figure 4). In both cases, ornithine fitted into a bowl-like cavity, similarly located in the 3D structure of the two proteins. Some of the earliest biochemical studies of ODC showed that both GTP and ppGpp regulate ODC enzymatic activity [123,124]. Subsequently, a more detailed analysis of partially purified _E. coli_ ODC confirmed these results [125], and also showed that both ppGpp and GTP are competitive with respect to ornithine, but GDP or cyclic 3',5'-AMP (cAMP) had no detectable effect. However, it should be noted that the authors did not know whether the measured activity was due to the constitutive enzyme (SpeC) or the inducible one (SpeF) [125]. Starting with the same ODC structure (PDB 6N2H), we conducted in silico docking of four different ligands to it: ornithine, ppGpp, guanosine 5'-triphosphate (GTP or ppG), guanosine 5'-diphosphate (GDP or ppG), and cAMP. As shown by the same scale of presentation (Figure 5), all ligands bound inside or near the same cavity; interestingly,  
Figure 4: Structural comparison between ornithine docked into _S. typhimurium_ ornithine decarboxylase (ODC) and the crystal structure of ornithine-_T. brucei_ ODC complex, using CB-Dock2 (Supplementary material). The surface topology view of each structure, presented in PyMol (cyan color), shows the ornithine molecule (in red) in a crevice at approximately similar locations in the overall structure. however, ppGpp and GTP were positioned very close to the ornithine-docking site, whereas GDP and cAMP were considerably farther, with cAMP being the farthest. In fact, the cAMP molecule is so far away that it was deep inside a small tunnel to the far right corner of the cavity (Figure 5E,E') and was barely visible in this angle of presentation; to make it easily visible, we made a second presentation, in which the complex is rotated downward. For better viewing of the interior of the complexes and the structural segments, the same molecules are also presented in a ribbon diagram, where the comparative distances are clearly discernible (Figure 5A-E). These results may explain why ppGpp and GTP in the previous study competed with ornithine for interaction with ODC, while GDP and cAMP showed no detectable effect [125].