Identification of small-molecule binding sites of a ubiquitin-conjugating enzyme-UBE2T through fragment-based screening.

UBE2T is an attractive target for drug development due to its linkage with several types of cancers. However, the druggability of ubiquitin-conjugating E2 (UBE2T) is low because of the lack of a deep and hydrophobic pocket capable of forming strong binding interactions with drug-like small molecules. Here, we performed fragment screening using 19 F-nuclear magnetic resonance (NMR) and validated the hits with 1 H-15 N-heteronuclear single quantum coherence (HSQC) experiment and X-ray crystallographic studies. The cocrystal structures obtained revealed the binding modes of the hit fragments and allowed for the characterization of the fragment-binding sites. Further screening of structural analogues resulted in the identification of a compound series with inhibitory effect on UBE2T activity. Our current study has identified two new binding pockets in UBE2T, which will be useful for the development of small molecules to regulate the function of this protein. In addition, the compounds identified in this study can serve as chemical starting points for the development of UBE2T modulators.

Backbone 1H, 15N and 13C resonance assignments for an E2 ubiquitin conjugating enzyme-UBE2T.

Ubiquitin-conjugating enzyme E2 T (UBE2T) plays important roles in ubiquitination of proteins through participation in transferring ubiquitin to its substrate. Due to its importance in protein modifications, UBE2T associates with diverse diseases and serves as an important target for drug discovery and development. The crystal structure of UBE2T has been determined and the structure reveals the lack of a druggable pocket for binding to small molecules for clinical applications. Despite the challenge, effort has been made to develop UBE2T inhibitors. We obtained UBE2T constructs with and without the C-terminal region which is flexible in solution. Herein, we report the backbone resonance assignments for human UBE2T without the C-terminal region. The backbone dynamics of UBE2T was also explored. The available assignments will be helpful for hit identification, determining ligand binding site and understanding the mechanism of action of UBE2T inhibitors.

RNase P enzymes: divergent scaffolds for a conserved biological reaction.

Ribonuclease P (RNase P) catalyzes the maturation of the 5' end of precursor-tRNAs (pre-tRNA) and is conserved in all domains of life. However, the composition of RNase P varies from bacteria to archaea and eukarya, making RNase P one of the most diverse enzymes characterized. Most known RNase P enzymes contain a large catalytic RNA subunit that associates with one to 10 proteins. Recently, a protein-only form of RNase P was discovered in mitochondria and chloroplasts of many higher eukaryotes. This proteinaceous RNase P (PRORP) represents a new class of metallonucleases. Here we discuss our recent crystal structure of PRORP1 from Arabidopsis thaliana and speculate on the reasons for the replacement of catalytic RNA by a protein catalyst. We conclude, based on an analysis of the catalytic efficiencies of ribonucleoprotein (RNP) and PRORP enzymes, that the need for greater catalytic efficiency is most likely not the driving force behind the replacement of the RNA with a protein catalyst. The emergence of a protein-based RNase P more likely reflects the increasing complexity of the biological system, including difficulties in importation into organelles and vulnerability of organellar RNAs to cleavage.

Mitochondrial ribonuclease P structure provides insight into the evolution of catalytic strategies for precursor-tRNA 5' processing.

Ribonuclease P (RNase P) catalyzes the maturation of the 5' end of tRNA precursors. Typically these enzymes are ribonucleoproteins with a conserved RNA component responsible for catalysis. However, protein-only RNase P (PRORP) enzymes process precursor tRNAs in human mitochondria and in all tRNA-using compartments of Arabidopsis thaliana. PRORP enzymes are nuclear encoded and conserved among many eukaryotes, having evolved recently as yeast mitochondrial genomes encode an RNase P RNA. Here we report the crystal structure of PRORP1 from A. thaliana at 1.75 Å resolution, revealing a prototypical metallonuclease domain tethered to a pentatricopeptide repeat (PPR) domain by a structural zinc-binding domain. The metallonuclease domain is a unique high-resolution structure of a Nedd4-BP1, YacP Nucleases (NYN) domain that is a member of the PIN domain-like fold superfamily, including the FLAP nuclease family. The structural similarity between PRORP1 and the FLAP nuclease family suggests that they evolved from a common ancestor. Biochemical data reveal that conserved aspartate residues in PRORP1 are important for catalytic activity and metal binding and that the PPR domain also enhances activity, likely through an interaction with pre-tRNA. These results provide a foundation for understanding tRNA maturation in organelles. Furthermore, these studies allow for a molecular-level comparison of the catalytic strategies used by the only known naturally evolved protein and RNA-based catalysts that perform the same biological function, pre-tRNA maturation, thereby providing insight into the differences between the prebiotic RNA world and the present protein-dominated world.