Structural and functional insights into a novel two-component endolysin encoded by a single gene in Enterococcus faecalis phage.

Using bacteriophage-derived endolysins as an alternative strategy for fighting drug-resistant bacteria has recently been garnering renewed interest. However, their application is still hindered by their narrow spectra of activity. In our previous work, we demonstrated that the endolysin LysIME-EF1 possesses efficient bactericidal activity against multiple strains of Enterococcus faecalis (E. faecalis). Herein, we observed an 8 kDa fragment and hypothesized that it contributes to LysIME-EF1 lytic activity. To examine our hypothesis, we determined the structure of LysIME-EF1 at 1.75 Å resolution. LysIME-EF1 exhibits a unique architecture in which one full-length LysIME-EF1 forms a tetramer with three additional C-terminal cell-wall binding domains (CBDs) that correspond to the abovementioned 8 kDa fragment. Furthermore, we identified an internal ribosomal binding site (RBS) and alternative start codon within LysIME-EF1 gene, which are demonstrated to be responsible for the translation of the truncated CBD. To elucidate the molecular mechanism for the lytic activity of LysIME-EF1, we combined mutagenesis, lytic activity assays and in vivo animal infection experiments. The results confirmed that the additional LysIME-EF1 CBDs are important for LysIME-EF1 architecture and its lytic activity. To our knowledge, this is the first determined structure of multimeric endolysin encoded by a single gene in E. faecalis phages. As such, it may provide valuable insights into designing potent endolysins against the opportunistic pathogen E. faecalis

Cryo-EM structures of the human PA200 and PA200-20S complex reveal regulation of proteasome gate opening and two PA200 apertures.

Proteasomes are highly abundant and conserved protease complexes that eliminate unwanted proteins in the cells. As a single-chain ATP-independent nuclear proteasome activator, proteasome activator 200 (PA200) associates with 20S core particle to form proteasome complex that catalyzes polyubiquitin-independent degradation of acetylated histones, thus playing a pivotal role in DNA repair and spermatogenesis. Here, we present cryo-electron microscopy (cryo-EM) structures of the human PA200-20S complex and PA200 at 2.72 Å and 3.75 Å, respectively. PA200 exhibits a dome-like architecture that caps 20S and uses its C-terminal YYA (Tyr-Tyr-Ala) to induce the α-ring rearrangements and partial opening of the 20S gate. Our structural data also indicate that PA200 has two openings formed by numerous positively charged residues that respectively bind (5,6)-bisdiphosphoinositol tetrakisphosphate (5,6[PP]2-InsP4) and inositol hexakisphosphate (InsP6) and are likely to be the gates that lead unfolded proteins through PA200 and into the 20S. Besides, our structural analysis of PA200 found that the bromodomain (BRD)-like (BRDL) domain of PA200 shows considerable sequence variation in comparison to other human BRDs, as it contains only 82 residues because of a short ZA loop, and cannot be classified into any of the eight typical human BRD families. Taken together, the results obtained from this study provide important insights into human PA200-induced 20S gate opening for substrate degradation and the opportunities to explore the mechanism for its recognition of H4 histone in acetylation-mediated proteasomal degradation