Cold-Shock Domains—Abundance, Structure, Properties, and Nucleic-Acid Binding

The cold-shock domain has a deceptively simple architecture but supports a complex biology. It is conserved from bacteria to man and has representatives in all kingdoms of life. Bacterial cold-shock proteins consist of a single cold-shock domain and some, but not all are induced by cold shock. Cold-shock domains in human proteins are often associated with natively unfolded protein segments and more rarely with other folded domains. Cold-shock proteins and domains share a five-stranded all-antiparallel β-barrel structure and a conserved surface that binds single-stranded nucleic acids, predominantly by stacking interactions between nucleobases and aromatic protein sidechains. This conserved binding mode explains the cold-shock domains’ ability to associate with both DNA and RNA strands and their limited sequence selectivity. The promiscuous DNA and RNA binding provides a rationale for the ability of cold-shock domain-containing proteins to function in transcription regulation and DNA-damage repair as well as in regulating splicing, translation, mRNA stability and RNA sequestration.

Promiscuous DNA cleavage by HpyAII endonuclease is modulated by the HNH catalytic residues

Helicobacter pylori is a carcinogenic bacterium that is responsible for 5.5% of all human gastric cancers. H. pylori codes for an unusually large number of restriction–modification (R–M) systems and several of them are strain-specific and phase-variable. HpyAII is a novel Type IIs phase-variable restriction endonuclease present in 26695 strain of H. pylori. We show that HpyAII prefers two-site substrates over one-site substrates for maximal cleavage activity. HpyAII is less stringent in metal ion requirement and shows higher cleavage activity with Ni2+ over Mg2+. Mutational analysis of the putative residues of the HNH motif of HpyAII confirms that the protein has an active HNH site for the cleavage of DNA. However, mutation of the first Histidine residue of the HNH motif to Alanine does not abolish the enzymatic activity, but instead causes loss of fidelity compared with wildtype HpyAII. Previous studies have shown that mutation of the first Histidine residue of the HNH motif of all other known HNH motif motif-containing enzymes completely abolishes enzymatic activity. We found, in the case of HpyAII, mutation of an active site residue leads to the loss of endonuclease fidelity. The present study provides further insights into the evolution of restriction enzymes.

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.