Structural Analysis of (p)ppGpp Reveals Its Versatile Binding Pattern for Diverse Types of Target Proteins

(p)ppGpp, highly phosphorylated guanosine, are global regulatory nucleotides that modulate several biochemical events in bacterial physiology ranging from core central dogma to various metabolic pathways. Conventionally, (p)ppGpp collectively refers to two nucleotides, ppGpp, and pppGpp in the literature. Initially, (p)ppGpp has been discovered as a transcription regulatory molecule as it binds to RNA polymerase and regulates transcriptional gene regulation. During the past decade, several other target proteins of (p)ppGpp have been discovered and as of now, more than 30 proteins have been reported to be regulated by the binding of these two signaling nucleotides. The regulation of diverse biochemical activities by (p)ppGpp requires fine-tuned molecular interactions with various classes of proteins so that it can moderate varied functions. Here we report a structural dynamics of (p)ppGpp in the unbound state using well-defined computational tools and its interactions with target proteins to understand the differential regulation by (p)ppGpp at the molecular level. We carried out replica exchange molecular dynamics simulation studies to enhance sampling of conformations during (p)ppGpp simulation. The detailed comparative analysis of torsion angle conformation of ribose sugar of unbound (p)ppGpp and bound states of (p)ppGpp was carried out. The structural dynamics shows that two linear phosphate chains provide plasticity to (p)ppGpp nucleotides for the binding to diverse proteins. Moreover, the intermolecular interactions between (p)ppGpp and target proteins were characterized through various physicochemical parameters including, hydrogen bonds, van der Waal’s interactions, aromatic stacking, and side chains of interacting residues of proteins. Surprisingly, we observed that interactions of (p)ppGpp to target protein have a consensus binding pattern for a particular functional class of enzymes. For example, the binding of (p)ppGpp to RNA polymerase is significantly different from the binding of (p)ppGpp to the proteins involved in the ribosome biogenesis pathway. Whereas, (p)ppGpp binding to enzymes involved in nucleotide metabolism facilitates the functional regulation through oligomerization. Analysis of these datasets revealed that guanine base-specific contacts are key determinants to discriminate functional class of protein. Altogether, our studies provide significant information to understand the differential interaction pattern of (p)ppGpp to its target and this information may be useful to design antibacterial compounds based on (p)ppGpp analogs.