Identification of Inhibitors for the DEDDh Family of Exonucleases and a Unique Inhibition Mechanism by Crystal Structure Analysis of CRN‑4 Bound with 2‑Morpholin-4-ylethanesulfonate (MES)

The DEDDh family of exonucleases plays essential roles in DNA and RNA metabolism in all kingdoms of life. Several viral and human DEDDh exonucleases can serve as antiviral drug targets due to their critical roles in virus replication. Here using RNase T and CRN-4 as the model systems, we identify potential inhibitors for DEDDh exonucleases. We further show that two of the inhibitors, ATA and PV6R, indeed inhibit the exonuclease activity of the viral protein NP exonuclease of Lassa fever virus in vitro. Moreover, we determine the crystal structure of CRN-4 in complex with MES that reveals a unique inhibition mechanism by inducing the general base His179 to shift out of the active site. Our results not only provide the structural basis for the inhibition mechanism but also suggest potential lead inhibitors for the DEDDh exonucleases that may pave the way for designing nuclease inhibitors for biochemical and biomedical applications

Structural basis for overhang excision and terminal unwinding of DNA duplexes by TREX1

<div><p>Three prime repair exonuclease 1 (TREX1) is an essential exonuclease in mammalian cells, and numerous in vivo and in vitro data evidenced its participation in immunity regulation and in genotoxicity remediation. In these very complicated cellular functions, the molecular mechanisms by which duplex DNA substrates are processed are mostly elusive because of the lack of structure information. Here, we report multiple crystal structures of TREX1 complexed with various substrates to provide the structure basis for overhang excision and terminal unwinding of DNA duplexes. The substrates were designed to mimic the intermediate structural DNAs involved in various repair pathways. The results showed that the Leu24-Pro25-Ser26 cluster of TREX1 served to cap the nonscissile 5′-end of the DNA for precise removal of the short 3′-overhang in L- and Y-structural DNA or to wedge into the double-stranded region for further digestion along the duplex. Biochemical assays were also conducted to demonstrate that TREX1 can indeed degrade double-stranded DNA (dsDNA) to a full extent. Overall, this study provided unprecedented knowledge at the molecular level on the enzymatic substrate processing involved in prevention of immune activation and in responses to genotoxic stresses. For example, Arg128, whose mutation in TREX1 was linked to a disease state, were shown to exhibit consistent interaction patterns with the nonscissile strand in all of the structures we solved. Such structure basis is expected to play an indispensable role in elucidating the functional activities of TREX1 at the cellular level and in vivo.</p></div

The TREX1-Y-structural dsDNA structure.

<p>(A) The nuclease activities of TREX1 in digesting Y-structural DNA. (B) An overview of the TREX1-Y-structural dsDNA structure. The last 2 phosphates in the 5′-end are displayed as orange balls and labeled as “P.” Arg128 interacted with the G4 and G5 bases via hydrogen bonding. (C) Schematic comparison of the TREX1-Y-structural dsDNA and the TREX1-L-structural dsDNA structures (1-nt-L-DNA). The cutting sites are labeled by scissors. dsDNA, double-stranded DNA; TREX1, three prime repair exonuclease 1.</p

X-ray data collection and refinement statistics for TREX1-DNA complexes.

<p>dI, deoxyinosine; dsDNA, double-stranded DNA; Mg²⁺, magnesium ion; r.m.s, root-mean-square; ssDNA, single-stranded DNA; TREX1, three prime repair exonuclease 1.</p

Structure comparison for the binding modes of TREX1 with various DNA substrates.

<p>(A) Superposition of the three structures of TREX1 in complexing with a duplex DNA with a long 3′-overhang (≥4-nt), including 4-nt-long 3′-overhang (4-nt-L-DNA) in the TREX1-L-structural dsDNA structure and the tight and loose conformations in the previous structures of TREX1-dsDNA complex (PDB accession code: 4YNQ). The colors and schematic diagrams of 3 duplex DNAs are displayed in the bottom panel. The right panel shows a close look for the difference between the scissile strands in the 3 duplex DNAs. The relative positions of the scissile strand of the duplex DNA in these 3 structures are different, only the positions of the last 2 nucleotides at the 3′-end of the scissile strand can fit well with each other. (B) Superposition of the three structures of TREX1 in complexing with a duplex DNA with a short 3′-overhang (<4-nt), including 1-nt-long 3′-overhang in the TREX1-L-structural dsDNA structure (1-nt-L-DNA), Y-structural DNA with 2-nt-long 3′-overhang in the TREX1-Y-structural dsDNA structure, and dI-containing dsDNA in the TREX1-dI-T-dsDNA structure. Comparison of these structures indicates the similarity of the binding regions between TREX1 and the 3′-end and 5′-end of the three types of dsDNA substrates. (C) Binding mode 1 is TREX1 with ssDNA substrates. (D) Binding mode 2 is TREX1 with duplexes of a long 3′-overhang, schematic representations in Boxes 1, 2, and 3. The PDB accession codes of these structures are 5YWT (4-nt-long L-structural dsDNA), 4YNQ (tight conformation), and 4YNQ (loose conformation). Binding mode 3 is TREX1 with a duplex of a short 3′-overhang, schematic representations in Boxes 4, 5, and 6. The PDB accession codes of these structures are 5YWT (1-nt-long L-structural dsDNA), 5YWS (2-nt-long Y-structural dsDNA), and 5YWU (dI-T-dsDNA). The scissile and nonscissile strands are colored in red and pink, respectively. α7 is the seventh α-helix (152–162 a.a). dI, deoxyinosine; dsDNA, double-stranded DNA; PDB, Protein Data Bank; ssDNA, single-stranded DNA; ssRNA, single-stranded RNA; TREX1, three prime repair exonuclease 1.</p

The TREX1-dI-T-dsDNA structure.

<p>(A) An overview of the TREX1-dI-T-dsDNA structure. The phosphate atoms of dsDNA in the TREX1-dI-T-dsDNA structure are shown as orange balls. The metal ions and active site residues in the TREX1-dI-ssDNA and TREX1-dI-T-dsDNA structures are colored in gray and blue, respectively. (B) Schematic representation for the Leu24-Pro25-Ser26 cluster wedging in the duplex end of dI-T-dsDNA. The scissile and nonscissile strands are colored in red and pink, respectively. dI, deoxyinosine; dsDNA, double-stranded DNA; ssDNA, single-stranded DNA; TREX1, three prime repair exonuclease 1.</p

The TREX1-dI-ssDNA structure.

<p>(A) Schematic representation of the DNA/RNA products generated by Endo V. (B) The nuclease activities of TREX1 in digesting bubbled DNAs containing a hypoxanthine base (also named dI), including dI-bubbled DNA and dI-bubbled DNA with 5′-overhang. The concentration of all substrates was 0.5 μM. (C) An overview of the TREX1-dI-ssDNA structure. The upper panel shows the dI-ssDNA molecule in the TREX1-dI-ssDNA structure. The omitted electron density map (black) is contoured at 2.0 σ. Scissile phosphate, Mg²⁺, and water molecules are shown in orange, blue, and light blue balls, respectively. The hydrogen bonds between DNA, TREX1, water, and Mg²⁺ are marked with blue dotted lines. dI, deoxyinosine; Endo V, endonuclease V; Mg²⁺, magnesium ion; ssDNA, single-stranded DNA; TREX1, three prime repair exonuclease 1.</p

The TREX1-L-structural dsDNA structure.

<p>(A) The nuclease activities of TREX1 in digesting duplex DNAs with 4-nt-long 3′-overhang. (B) The asymmetric unit in the crystal contained 1 TREX1 dimer and 2 ssDNA molecules. The parts shown with a transparent mode depict the symmetry of TREX1 and DNA. The DNA duplex was formed by the ssDNAs bound to 2 TREX1 molecules in separate dimers. The 2 3′-ends of this duplex were 1 nt and 4 nt long and formed an L-like structure at each 3′-terminal, and they are referred to as 1-nt-L-DNA and 4-nt-L-DNA, respectively. (C) The 5′-ends of the duplex region in 1-nt-L-DNA were blocked by Leu24-Pro25-Ser26 cluster. (D) Schematic representation of the 2 modes for TREX1 binding with 1-nt-L-DNA and 4-nt-L-DNA. dsDNA, double-stranded DNA; ssDNA, single-stranded DNA; TREX1, three prime repair exonuclease 1.</p

The terminal unwinding activity of TREX1 empowered by the Leu24-Pro25-Ser26 cluster.

<p>(A) Structure comparison of the loop region between the highly conserved β1 and β2 of TREX1, TREX2, and other classical DEDDh exonucleases. The PDB structures used in the structural alignment are mouse TREX1 (PDB accession code: 5YWU), human TREX2 (PDB accession code: 1Y97), <i>Caenorhabditis elegans</i> CRN-4 (PDB accession code: 3CG7), <i>E</i>. <i>coli</i> RNase T (PDB accession code: 4KA0), <i>E</i>. <i>coli</i> ExoX (PDB accession code: 4FZX), human ISG20 (PDB accession code: 31WLJ), and human 3′Hexo (PDB accession code: 4QOZ). In this region, only TREX1 and TREX2 contain a small helix that starts the Leu24-Pro25-Ser26 cluster. (B) The nuclease activities of TREX2 and RNase T in digesting duplex DNAs with 3′-overhang. The concentration of duplex DNA was 0.5 μM. TREX2 exhibited activities in digesting the duplex regions of DNA. RNase T at 500 nM only removed the 3′-overhang in duplex DNA and produced a duplex DNA with 1- or 0-nucleotide 3′-overhang. When the concentration of RNase T was increased by 20-fold to 10,000 nM, the double-strand structure of the substrates still persisted. (C) The nuclease activities of wild-type TREX1 in digesting ssDNA, dsDNA, and a PCR product (A linear 708 bp dsDNA). The concentration of ssDNA and dsDNA were 0.5 μM. The amount of the PCR product was 300 ng, and the concentration of EDTA was 5 mM. (D)(E)(F) The nuclease activities of 3 TREX1 mutants (L24W, S26W, and L24W/P25W/S26W) in digesting ssDNA and dsDNA substrates. dsDNA, double-stranded DNA; PDB, Protein Data Bank; ssDNA, single-stranded DNA; TREX1, three prime repair exonuclease 1.</p

Controlling The Activator Site To Tune Europium Valence in Oxyfluoride Phosphors

A new Eu³⁺-activated oxyfluoride phosphor Ca₁₂Al₁₄O₃₂F₂:Eu³⁺ (CAOF:Eu³⁺) was synthesized by a solid state reaction. Commonly red line emission was detected in the range of 570–700 nm. To achieve the requirement of illumination, this study revealed a crystal chemistry approach to reduce Eu ions from 3+ to 2+ in the lattice. Replacing Al³⁺–F^– by the appreciate dopant Si⁴⁺–O^2– is adopted to enlarge the activator site that enables Eu³⁺ to be reduced. The crystallization of samples was examined by powder X-ray diffraction (XRD) and high resolution transmission electron microscopy (HRTEM). Photoluminescence results indicated that as-synthesized phosphors Ca₁₂Al_{14‑<i>z</i>}Si_<i>z</i>O_32+<i>z</i>F_2–<i>z</i>:Eu (<i>z</i> = 0–0.5, CASOF:Eu) display an intense blue emission peaking at 440 nm that was produced by 4f–5d transition of Eu²⁺, along with the intrinsic emission of Eu³⁺ under UV excitation. Moreover, the effect of Si⁴⁺–O^2– substitution involved in the coordination environment of the activator site was investigated by further crystallographic data from Rietveld refinements. The ¹⁹F solid-state nuclear magnetic resonance (NMR) data were in agreement with refinement and photoluminescence results. Furthermore, the valence states of Eu in the samples were analyzed with the X-ray absorption near edge structure (XANES). The quantity of substituted Si⁴⁺–O^2– tunes chromaticity coordinates of Ca₁₂Al_{14–<i>z</i>}Si_<i>z</i>O_32+<i>z</i>F_2–<i>z</i>:Eu phosphors from (0.6101, 0.3513) for <i>z</i> = 0 to (0.1629, 0.0649) for <i>z</i> = 0.5, suggesting the potential for developing phosphors for white light emitting diodes (WLEDs). Using an activator that is valence tunable by controlling the size of the activator site represents a hitherto unreported structural motif for designing phosphors in phosphor converted light emitting diodes (pc-LEDs)