Molecular mechanism of human ISG20L2 for the ITS1 cleavage in the processing of 18S precursor ribosomal RNA.

The exonuclease ISG20L2 has been initially characterized for its role in the mammalian 5.8S rRNA 3' end maturation, specifically in the cleavage of ITS2 of 12S precursor ribosomal RNA (pre-rRNA). Here, we show that human ISG20L2 is also involved in 18S pre-rRNA maturation through removing the ITS1 region, and contributes to ribosomal biogenesis and cell proliferation. Furthermore, we determined the crystal structure of the ISG20L2 nuclease domain at 2.9 Å resolution. It exhibits the typical αßα fold of the DEDD 3'-5' exonuclease with a catalytic pocket located in the hollow near the center. The catalytic residues Asp183, Glu185, Asp267, His322 and Asp327 constitute the DEDDh motif in ISG20L2. The active pocket represents conformational flexibility in the absence of an RNA substrate. Using structural superposition and mutagenesis assay, we mapped RNA substrate binding residues in ISG20L2. Finally, cellular assays revealed that ISG20L2 is aberrantly up-regulated in colon adenocarcinoma and promotes colon cancer cell proliferation through regulating ribosome biogenesis. Together, these results reveal that ISG20L2 is a new enzymatic member for 18S pre-rRNA maturation, provide insights into the mechanism of ISG20L2 underlying pre-rRNA processing, and suggest that ISG20L2 is a potential therapeutic target for colon adenocarcinoma.

Molecular basis for the MacroD1-mediated hydrolysis of ADP-ribosylation.

MacroD1 is an enzyme that hydrolyzes protein mono-ADP-ribosylation. However, the key catalytic residues of MacroD1 in these biochemical reactions remain elusive. Here, we present the crystal structure of MacroD1 in a complex with ADP-ribose (ADPR). The ß5-α10-loop functions as a switch loop to mediate substrate recognition and right orientation. The conserved Phe272 in the ß5-α10-loop plays a crucial role in the orientation of ADPR distal ribose, and a conserved hydrogen-bond network contributes significantly to hold and orient the catalytic water12, which mediates ADPR hydrolysis. Moreover, we found that MacroD1 was recruited to the sites of DNA damage via recognition of ADP-ribosylation at DNA lesions. The MacroD1-mediated ADPR hydrolysis is essential for DNA damage repair. Taken together, our study provides structural and functional insights into the molecular mechanism of MacroD1-mediated ADPR hydrolysis and its role in DNA damage repair.

Variation of effluent organic matter (EfOM) during anaerobic/anoxic/oxic (A2O) wastewater treatment processes.

Here, we studied seasonal variation of effluent organic matter (EfOM), based on molecular weight distribution and fluorescent components, during the traditional anaerobic/anoxic/oxic (A2O) wastewater treatment processes. Microbial community structure and effect of temperature on some isolated pure strains were analyzed to explain the related mechanism. Results showed that the anaerobic process played a key role in EfOM removal by removing building blocks, low molecular weight (LMW) neutrals, biopolymers, and protein-related substances (C4 and C5), thus determining the fate of EfOM during the A2O processes. On the other hand, humic substances, LMW neutrals, large molecular-sized hydrophobic humic-like compounds (C3), and aromatic proteins (C4) were generated during the anoxic process in summer and winter. Proteobacteria (Gamma-, Beta-, and Alpha-proteobacteria) and Bacteroidetes constituted over 50% of the sludge community. Temperature was found to be positively correlated with the generation of soluble microbial products (SMP) based on the performance of the mixture of isolated Herbaspirillum sp. (Beta-proteobacteria) and Pseudomonas sp. (Gamma-proteobacteria). Through comprehensive analysis of the co-action of Proteobacteria and temperature, we proposed the Synergetic Effect of Temperature and Proteobacteria as a possible mechanism of the seasonal variation of EfOM. These findings are important for understanding the fate of EfOM during the wastewater treatment processes and therefore be helpful for better EfOM control.

Author Correction: Molecular basis for the inhibition of the methyl-lysine binding function of 53BP1 by TIRR.

The original version of this Article contained an error in the author affiliations. Xiaochun Yu was incorrectly associated with College of Life Sciences, Hebei University, Baoding 071000 Hebei, China.This has now been corrected in both the PDF and HTML versions of the Article.

Molecular basis for the inhibition of the methyl-lysine binding function of 53BP1 by TIRR.

53BP1 performs essential functions in DNA double-strand break (DSB) repair and it was recently reported that Tudor interacting repair regulator (TIRR) negatively regulates 53BP1 during DSB repair. Here, we present the crystal structure of the 53BP1 tandem Tudor domain (TTD) in complex with TIRR. Our results show that three loops from TIRR interact with 53BP1 TTD and mask the methylated lysine-binding pocket in TTD. Thus, TIRR competes with histone H4K20 methylation for 53BP1 binding. We map key interaction residues in 53BP1 TTD and TIRR, whose mutation abolishes complex formation. Moreover, TIRR suppresses the relocation of 53BP1 to DNA lesions and 53BP1-dependent DNA damage repair. Finally, despite the high-sequence homology between TIRR and NUDT16, NUDT16 does not directly interact with 53BP1 due to the absence of key residues required for binding. Taken together, our study provides insights into the molecular mechanism underlying TIRR-mediated suppression of 53BP1-dependent DNA damage repair.

Structure-function analyses reveal the mechanism of the ARH3-dependent hydrolysis of ADP-ribosylation.

ADP-ribosylation of proteins plays key roles in multiple biological processes, including DNA damage repair. Recent evidence suggests that serine is an important acceptor for ADP-ribosylation, and that serine ADP-ribosylation is hydrolyzed by ADP-ribosylhydrolase 3 (ARH3 or ADPRHL2). However, the structural details in ARH3-mediated hydrolysis remain elusive. Here, we determined the structure of ARH3 in a complex with ADP-ribose (ADPR). Our analyses revealed a group of acidic residues in ARH3 that keep two Mg2+ ions at the catalytic center for hydrolysis of Ser-linked ADP-ribosyl group. In particular, dynamic conformational changes involving Glu41 were observed in the catalytic center. Our observations suggest that Mg2+ ions together with Glu41 and water351 are likely to mediate the cleavage of the glycosidic bond in the serine-ADPR substrate. Moreover, we found that ADPR is buried in a groove and forms multiple hydrogen bonds with the main chain and side chains of ARH3 residues. On the basis of these structural findings, we used site-directed mutagenesis to examine the functional roles of key residues in the catalytic pocket of ARH3 in mediating the hydrolysis of ADP-ribosyl from serine and DNA damage repair. Moreover, we noted that ADPR recognition is essential for the recruitment of ARH3 to DNA lesions. Taken together, our study provides structural and functional insights into the molecular mechanism by which ARH3 hydrolyzes the ADP-ribosyl group from serine and contributes to DNA damage repair.

Crystal structure and biochemical features of dye-decolorizing peroxidase YfeX from Escherichia coli O157 Asp143 and Arg232 play divergent roles toward different substrates.

YfeX from Escherichia coli O157 is a bacterial dye-decolorizing peroxidase that represents both dye-decoloring activity and typical peroxidase activity. We reported the crystal structure of YfeX bound to heme at 2.09 Å resolution. The YfeX monomer resembles a ferredoxin-like fold and contains two domains. The three conserved residues surrounding the heme group are His215, Asp143 and Arg232. His215 functions as the proximal axial ligand of the heme iron atom. Biochemical data show that the catalytic significance of the conserved Asp143 and Arg232 depends on the substrate types and that YfeX may adopt various catalytic mechanisms toward divergent substrates. In addition, it is observed that an access tunnel spans from the protein molecular surface to the heme distal region, it serves as the passageway for the entrance and binding of the H2O2.

Crystal Structure of Murein-Tripeptide Amidase MpaA from Escherichia coli O157 at 2.6 Å Resolution.

Peptidoglycan (PG) is an essential component of the cell wall, and undergoes reconstruction by various PG hydrolases during cell growth, development and division. The murein- tripeptide (Mtp) amidase MpaA belongs to PG hydrolase family and is responsible for cleaving the γ-D-Glumeso- Dap amide bond in the Mtp released during PG turnover. The current paper reports the crystal structure of MpaA from Escherichia coli (E. coli) O157 at 2.6 Å resolution. The asymmetric unit consists of two protein molecules and each monomer represents the common α/ß fold of metallocarboxypeptidases (MCP). The Tyr133-Asp143 loop appears to mediate the entrance and binding of the substrate into the active groove. A structural comparison of MpaA with its homologue from Vibrio harveyi showed that MpaA has narrower active pocket entrance with a smaller surface opening, which is determined by the Val204-Thr211 loop. The reported structure provides a starting point for the molecular mechanism of MpaA in a significant human pathogen.

Crystal structure and molecular mechanism of an aspartate/glutamate racemase from Escherichia coli O157.

EcL-DER, the aspartate/glutamate racemase from the pathogen Escherichia coli O157, exhibits racemase activity for l-aspartate and l-glutamate. This study reports the crystal structures of apo-EcL-DER, the EcL-DER-l-aspartate and the EcL-DER-d-aspartate complexes. The EcL-DER structure contains two domains, forming pseudo-mirror symmetry in the active site. A unique catalytic pair consisting of Thr(83) and Cys(197) exists in the active site. The characteristic conformations of l-Asp and d-Asp in the active site provide a straight structural evidence for the racemization mechanism of EcL-DER. In addition, the diversity of catalytic pairs implies that PLP-independent amino acid racemases adopt various catalytic mechanisms and are classified into different subgroups.