Enzymes participating in the synthesis of purines are promising targets for new pharmaceuticals, especially in Helicobacter pylori which relies solely on purine salvage pathway (PSP), which is poorly investigated. In the proposed project, we will examine enzymes of PSP in greater detail, particularly their potential stable or transient protein-protein interactions (PPIs) in the cell. In the first avenue of the project we will develop protocol for investigation of PPIs in H. pylori cells based on BioID methodology which is suitable for detecting even weak and transient interactions. Afterwards, enzymes of H. pylori PSP will be purified and characterized (using an array of available biophysical methods), and interactions between them investigated in vitro and in silico. Key residues identified by computational methods (docking, MD simulations) will be mutated, mutant enzymes characterized and their inability to form interactions verified (by AUC, ITC, nES GEMMA and other methods). We aim to discover whether investigated enzymes form interactions similar to those of de novo purine synthesis enzymes (organized in so-called purinosome), and whether these interactions or enzymes themselves can present potential pharmaceuticals targets. In the second avenue of the project, we will focus on PPIs between subunits of oligomeric enzymes in H. pylori PSP, in order to discover which residues are crucial for communication between monomers (so-called ''hub residues''). It is known that oligomeric organization of enzymes is closely related to their allosteric regulation, however not much is known about exact routes and mechanism of the allostery. Therefore, we will continue our work on the relational database of protein 3D-structures and MD simulations, on development of tools for mining this database and finally on in vitro confirmation of predicted ''hub residues'' in enzymes of PSP. This innovative approach will surely contribute to the understanding of allostery phenomenon.