Two distinct modes of metal ion binding in the nuclease active site of a viral DNA-packaging terminase: insight into the two-metal-ion catalytic mechanism

Many dsDNA viruses encode DNA-packaging terminases, each containing a nuclease domain that resolves concatemeric DNA into genome-length units. Terminase nucleases resemble the RNase H-superfamily nucleotidyltransferases in folds, and share a two-metal-ion catalytic mechanism. Here we show that residue K428 of a bacteriophage terminase gp2 nuclease domain mediates binding of the metal cofactor Mg2+. A K428A mutation allows visualization, at high resolution, of a metal ion binding mode with a coupled-octahedral configuration at the active site, exhibiting an unusually short metal-metal distance of 2.42 A° . Such proximity of the two metal ions may play an essential role in catalysis by generating a highly positive electrostatic niche to enable formation of the negatively charged pentacovalent phosphate transition state, and provides the structural basis for distinguishing Mg2+ from Ca2+. Using a metal ion chelator -thujaplicinol as a molecular probe, we observed a second mode of metal ion binding at the active site, mimicking the DNA binding state. Arrangement of the active site residues differs drastically from those in RNase H-like nucleases, suggesting a drifting of the active site configuration during evolution. The two distinct metal ion binding modes unveiledmechanistic details of the two-metalion catalysis at atomic resolution

Characterization of the C-Terminal Nuclease Domain of Herpes Simplex Virus pUL15 as a Target of Nucleotidyltransferase Inhibitors

The natural product α-hydroxytropolones manicol and β-thujaplicinol inhibit replication of herpes simplex viruses 1 and 2 (HSV-1 and HSV-2, respectively) at nontoxic concentrations. Because these were originally developed as divalent metal-sequestering inhibitors of the ribonuclease H activity of HIV-1 reverse transcriptase, α-hydroxytropolones likely target related HSV proteins of the nucleotidyltransferase (NTase) superfamily, which share an “RNase H-like” fold. One potential candidate is pUL15, a component of the viral terminase molecular motor complex, whose C-terminal nuclease domain, pUL15C, has recently been crystallized. Crystallography also provided a working model for DNA occupancy of the nuclease active site, suggesting potential protein–nucleic acid contacts over a region of ∼14 bp. In this work, we extend crystallographic analysis by examining pUL15C-mediated hydrolysis of short, closely related DNA duplexes. In addition to defining a minimal substrate length, this strategy facilitated construction of a dual-probe fluorescence assay for rapid kinetic analysis of wild-type and mutant nucleases. On the basis of its proposed role in binding the phosphate backbone, studies with pUL15C variant Lys700Ala showed that this mutation affected neither binding of duplex DNA nor binding of small molecule to the active site but caused a 17-fold reduction in the turnover rate (kcat), possibly by slowing conversion of the enzyme–substrate complex to the enzyme–product complex and/or inhibiting dissociation from the hydrolysis product. Finally, with a view of pUL15-associated nuclease activity as an antiviral target, the dual-probe fluorescence assay, in combination with differential scanning fluorimetry, was used to demonstrate inhibition by several classes of small molecules that target divalent metal at the active site

Structure of a Bacterial Virus DNA-Injection Protein Complex Reveals a Decameric Assembly with a Constricted Molecular Channel.

The multi-layered cell envelope structure of Gram-negative bacteria represents significant physical and chemical barriers for short-tailed phages to inject phage DNA into the host cytoplasm. Here we show that a DNA-injection protein of bacteriophage Sf6, gp12, forms a 465-kDa, decameric assembly in vitro. The electron microscopic structure of the gp12 assembly shows a ~150-Å, mushroom-like architecture consisting of a crown domain and a tube-like domain, which embraces a 25-Å-wide channel that could precisely accommodate dsDNA. The constricted channel suggests that gp12 mediates rapid, uni-directional injection of phage DNA into host cells by providing a molecular conduit for DNA translocation. The assembly exhibits a 10-fold symmetry, which may be a common feature among DNA-injection proteins of P22-like phages and may suggest a symmetry mismatch with respect to the 6-fold symmetric phage tail. The gp12 monomer is highly flexible in solution, supporting a mechanism for translocation of the protein through the conduit of the phage tail toward the host cell envelope, where it assembles into a DNA-injection device

Fitting the phiX174 H protein central domain X-ray structure (blue; RCSB PDB code 4JPP) into the stem domain of the Sf6 gp12 EM map (gold).

<p>(A) The H protein structure is shown as ribbon diagram. The front halves of the H structure and the gp12 map were computationally removed for clarity. (B) A corss section of the gp12 map fitted with the H protein structure (molecular surface). (C) A view down the gp12 channel axis. The top half of the gp12 map was removed for clarity. The crown and stem domains of gp12 are indicated.</p

The gp12 assembles into a ring-like decamer.

<p>(A) Size exclusion chromatogram of the purified gp12 decamer. The peak corresponds to an estimated molecular weight of 471kDa, indicating a decamer (arrow). (B) Electron micrograph of the purified gp12 decamer negatively stained with uranyl acetate. Notice the dominant views are down the longitudinal axis of the gp12 decamer. Some side or tilted views are indicated with red arrows. (C) Electron micrograph of the frozen-hydrated gp12 decamer. Bar, 100 Å. (D) Class averages of the frozen-hydrated gp12 decamer. Enlarged views of two class averages are shown on the right. The 10-fold symmetry is clearly evident for both the crown (green arrowheads) and the stem domains (red arrows). The box size for class averages in the left panel is 309.8 Å.</p

3D reconstruction of the gp12 decameric assembly.

<p>(A) Class averages of the negatively stained gp12 decamer. The bottom row shows three class averages corresponding to stacked double decamers, demonstrating robustness of the 2D classification. The box size for class averages is 349.4 Å. (B) 3D reconstruction. Top left, front view. Top right, cutaway view. Bottom left, top view. Bottom right, tilted by 45° from the view on the left.</p

Schematic representation of translocation of the DNA-injection proteins and the assembly of the DNA-injection device in the host envelope.

<p>(A) The three DNA-injection proteins, gp12, gp11 and gp13, represented with an oval, triangle and trapezoid in blue, are packed in the phage particle. Note that the copy numbers of those proteins in the capsid and how they are arranged are not known. (B) upon attachment to the host cell, the DNA-injection proteins are translocated through the tail channel to the host cell envelope, where they assemble into an extended tube-like structure to allow delivery of phage DNA into the host cytoplasm. The arrangement of those proteins is not known and is drawn schematically. LPS, lipopolysaccharide; OM, outer membrane; PG, peptidoglycan; IM, inner membrane. The phage dsDNA is in green.</p