Structural and biochemical insights into the molecular mechanism of N-acetylglucosamine/N-Acetylmuramic acid kinase MurK from Clostridium acetobutylicum.

MurK is a MurNAc- and GlcNAc-specific amino sugar kinase, phosphorylates MurNAc and GlcNAc at the 6-hydroxyl group in an ATP-dependent manner, and contributes to the recovery of both amino sugars during the cell wall turnover in Clostridium acetobutylicum. Herein, we determined the crystal structures of MurK in complex with MurNAc, GlcNAc, and glucose, respectively. MurK represents the V-shaped fold, which is divided into a small N-terminal domain and a large C-terminal domain. The catalytic pocket is located within the deep cavity between the two domains of the MurK monomer. We mapped the significant enzyme-substrate interactions, identified key residues involved in the catalytic activity of MurK, and found that residues Asp77 and Arg78 from the ß4-α2-loop confer structural flexibilities to specifically accommodate GlcNAc and MurNAc, respectively. Moreover, structural comparison revealed that MurK adopts closed-active conformation induced by the N-acetyl moiety from GlcNAc/MurNAc, rather than closed-inactive conformation induced by glucose, to carry out its catalytic reaction. Taken together, our study provides structural and functional insights into the molecular mechanism of MurK for the phosphorylation of both MurNAc and GlcNAc, sugar substrate specificity, and conformational changes upon sugar substrate binding.

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.

Structural and biochemical insights into the interaction mechanism underlying HORMAD1 and its partner proteins.

The mammalian HORMA domain-containing protein 1 (HORMAD1) regulates DNA mismatch repair and homologous recombination (HR) repair in many cancers. Here, we show that the structure of human HORMAD1 adopts a self-closed conformation and displays an intra-molecular HORMA domain-closure motif interaction mode. Structural and biochemical data suggest that the interaction modes of the peptide motifs from HORMAD2 and MCM9 with HORMAD1 are highly similar to that of HORMAD1 own closure motif. The peptide motifs from diverse binding partners of HORMAD1 share a conserved Ser-Glu-Pro sequence. Additionally, structural comparison unveiled the HORMA-peptide motif interaction mode diversity among HORMA-containing proteins. Finally, cell-based assays revealed that this HORMA-closure motif interaction pattern contributes to DNA mismatch repair and is required for HORMAD1-dependent HR repair. Together, our results provide structural and biochemical insights into the common theme and functional plasticity of the HORMA domain-containing protein family, and also reveal a universal regulation mechanism for HORMAD1.