## 3 The Complex Network of (p)ppGpp Signaling
### The (p)ppGpp Interactome
#### 3.4.2. Uracil Phosphoribosyltransferase  
As an important enzyme for transcription, uracil phosphoribosyltransferase (UPRT) catalyzes the reaction: uracil (U) + phosphoribosyl pyrophosphate (PRPP) \(\rightarrow\) uridine monophosphate (UMP) + pyrophosphate (PPi) [128]. The hydrolysis of the high-energy phosphodieser bond of the inorganic pyrophosphate (PPi) drives the otherwise reversible reaction to the right, resulting in the synthesis of UMP, a ubiquitous starting material for the cellular pyrimidine biosynthetic pathway. It was one of the major enzymes detected in the ppGpp affinity screening [116], suggesting that it may be inhibited in the stringent response, when replication and most of the transcription are halted as mentioned before, and therefore, nucleotide use is greatly reduced (Sections 3.1 and 3.3).  
To our knowledge, the 3D structure of _E. coli_ UPRT is currently not available; however, those of Toxoplasma gondii (PDB 1 BD3) and Mycobacterium tuberculosis (5E38) are known and are very similar [128; 129]. The amino acid sequences of all three are also highly similar, particularly in the C-terminal half. Of these, we chose the T. gondii enzyme because its structure has been solved not only in the apo form (PDB 1 BD3), but also in complex with uracil (PDB 1 BD4) (Figure 6A,B).  
We first ran CB-Dock2 on ppGpp-1 BD3 and ensured that the in silico structure is comparable to that of the actual crystal structure, 1 BD4 (Figure 6A,B). We note that UPRT is a complex enzyme that can exist in multiple conformations, and it belongs to the'morpehein' class of proteins that can form multiple different oligomers by changing the shape of the monomer [131]. In both T. gondii and M. tuberculosis, the UPRT structure is a symmetrical tetramer [128; 129]. In T. gondii UPRT, each monomeric unit binds a uracil in an identical position to produce 1 BD4 [128]. In contrast, the _E. coli_ enzyme has been reported to be a trimer [130]. In order to avoid the complication of diverse multimers, we remained focused on a single, monomeric polypeptide. Docking ppGpp onto 1 BD3 showed that unlike in ornithine decarboxylase (Section 3.4.1), it did not dock in the substrate (uracil)-binding pocket, but docked in a completely different area (Figure 6, compare between Panels B and C). In fact, it is possible that in 1 BD3, ppGpp binds to the interface region between two monomers, contacting both. A detailed analysis of the binding mode of ppGpp should await actual crystal structures. Nevertheless, these studies will be relevant  
Figure 6: In silico docking of ppGpp to UPRT. The crystal structures of the apo enzyme (**A**) and its complex with uracil, the substrate (**B**), are, respectively, PDB 1 BD3 and PDB 1 BD4, as shown. The in silico docked co-complex of 1 BD3 and ppGpp (**C**) show the significant difference in binding sites of the two ligands (both in red color). The pink segment, spanning a loop and a short helix, represent a region that was considered important for interaction with uracil [128; 129; 130]. Structure-blind docking was performed by CB-Dock2 (Supplementary material) as described earlier.  
for M. tuberculosis as well, as this important human pathogen also has a robust stringent response regulated by ppGpp, reviewed recently [132].  
Together, many elegant structural analyses of the past [31] and our in silico docking of two high-scoring ppGpp-binders [116] reveal the abundant variety of ppGpp targets and the binding patterns, explaining the large network of pathways triggered in the stringent response.