Phylogenetic Studies and Inhibitor Design Targeting Protein Interacting Interface of Nucleoid-Associated Protein HU

The formations of nucleoprotein structures by promiscuous DNA binding proteins like HU are assisted with their protein protein interaction capability with other proteins. In E. coli Gal repressosome assembly, GalR piggybacks HU to the critical position on the DNA (hbs site) through a specific GalR-HU interaction using an interface at the bottom of alpha helical region, which we termed as HUpb interface. Similarly, MtbHU also interact with Topoisomerase I with the same interface to enhance its relaxation activity. In an earlier study, we determined the crystal structure of MtbHU, inhibited it using stilbene derivatives which inhibited the cell growth. It motivated us to understand the evolutionary and structural characteristics of the HUpb interface, which has not been investigated previously for HU or for any other NAPs. Our analyses found residue positions corresponding to MtbHU Thr11 to Gln20 form the interface while Ala23 serves the pocket lining residue. Due to ancestral mutations in the duplication event before the HU and IHF split, physicochemical properties of the interface vary among clades. Thus, this interface could engage different proteins in different HU oligomeric states in Proteobacteria. Protein-protein interfaces are usually a challenging target due to its flatter surface. In case of MtbHUpb interface, we observed that due to the presence of a partially hydrophobic pocket, small molecule scaffolds could fit into it, while the ligand can be further designed to interact with D17, which is the crucial residue for Topoisomerase I interaction. We used a two-step virtual screening protocol with known drug like molecules as starting set to an aim to re-purpose drugs. Our docking results showed compounds like Maltotetraose, Valrubicin, Iodixanol, Enalkiren, indinavir, Carfilzomib, oxytetracycline, quinalizarin, Raltitrexed, Epigallocatechin and their analogues exhibit high scoring binding at MtbHUpb interface. Our present report gives a model example of an evolutionary study of an interface of nucleoid associated protein, which is used to computationally design inhibitors. This strategy could be in general useful for designing inhibitors for various types of protein-protein interfaces using evolutionary guided design.