Binding of DNA-Intercalating Agents to Oxidized and Reduced Quinone Reductase 2

Quinone reductase 2 (NQO2) is an enzyme that might have intracellular signaling functions. NQO2 can exist in either an oxidized state or a reduced state, and binding of compounds to one or both of these states inhibits enzymatic activity and could also affect intracellular signaling. A wide range of planar aromatic compounds bind NQO2, and we have identified three DNA-intercalating agents [ethidium bromide, acridine orange (AO), and doxorubicin] as novel nanomolar inhibitors of NQO2. Ethidium and AO, which carry a positive charge in their aromatic ring systems, bound reduced NQO2 with an affinity 50-fold higher than that of oxidized NQO2, while doxorubicin bound only oxidized NQO2. Crystallographic analyses of oxidized NQO2 in complex with the inhibitors indicated that the inhibitors were situated deep in the active site. The aromatic faces were sandwiched between the isoalloxazine ring of FAD and the phenyl ring of F178, with their edges making direct contact with residues lining the active site. In reduced NQO2, ethidium and AO occupied a more peripheral position in the active site, allowing several water molecules to interact with the polar end of the negatively charged isoalloxazine ring. We also showed that AO inhibited NQO2 at a nontoxic concentration in cells while ethidium was less effective at inhibiting NQO2 in cells. Together, this study shows that reduced NQO2 has structural and electrostatic properties that yield a preference for binding of planar, aromatic, and positively charged molecules that can also function as DNA-intercalating agents

The HIP2~Ubiquitin Conjugate Forms a Non-Compact Monomeric Thioester during Di-Ubiquitin Synthesis

<div><p>Polyubiquitination is a post-translational event used to control the degradation of damaged or unwanted proteins by modifying the target protein with a chain of ubiquitin molecules. One potential mechanism for the assembly of polyubiquitin chains involves the dimerization of an E2 conjugating enzyme allowing conjugated ubiquitin molecules to be put into close proximity to assist reactivity. HIP2 (UBE2K) and Ubc1 (yeast homolog of UBE2K) are unique E2 conjugating enzymes that each contain a <i>C</i>-terminal UBA domain attached to their catalytic domains, and they have basal E3-independent polyubiquitination activity. Although the isolated enzymes are monomeric, polyubiquitin formation activity assays show that both can act as ubiquitin donors or ubiquitin acceptors when in the activated thioester conjugate suggesting dimerization of the E2-ubiquitin conjugates. Stable disulfide complexes, analytical ultracentrifugation and small angle x-ray scattering were used to show that the HIP2-Ub and Ubc1-Ub thioester complexes remain predominantly monomeric in solution. Models of the HIP2-Ub complex derived from SAXS data show the complex is not compact but instead forms an open or backbent conformation similar to UbcH5b~Ub or Ubc13~Ub where the UBA domain and covalently attached ubiquitin reside on opposite ends of the catalytic domain. Activity assays showed that full length HIP2 exhibited a five-fold increase in the formation rate of di-ubiquitin compared to a HIP2 lacking the UBA domain. This difference was not observed for Ubc1 and may be attributed to the closer proximity of the UBA domain in HIP2 to the catalytic core than for Ubc1.</p></div

Ubiquitin transfer from the HIP2~UbK48R thioester to the HIP2–UbCys disulfide.

<p>(A) Schematic diagram showing the reaction of fluorescently labeled HIP2~Ub^K48R (donor, pink) with the HIP2–Ub^Cys disulfide (acceptor, orange) to produce HIP2–Ub^Cys–Ub^K48R (HIP2–Ub₂). In these reactions only the donor Ub^K48R is fluorescently labeled with Alexa-680. (B) HIP2 enzyme reactions performed with Alexa-labeled ubiquitin or Ub^K48R were visualized with Coomassie stain (top) or fluorescence measured at 700 nm (bottom). Reaction components are listed above each gel as described in Materials and Methods. Lane 9 shows the addition of reducing agent (10 mM DTT and 10 mM TCEP) to liberate the di-ubiquitin species. Fluorescent di-ubiquitin reaction products (E2-Ub-Ub; Ub-Ub) are indicated by the filled black / green circles. Molecular weight standards and protein species are listed to the left and right of each gel respectively.</p

Ubiquitin and the HIP2-Ub thioester mimic act as acceptor molecules for di-ubiquitin formation.

<p>Coomassie stained (top) and fluorescent (bottom) images of Alexa-labeled HIP2~Ub^K48R thioester reactions. Thioester formation was performed for 30 min at 37°C using HIP2 and Alexa-labeled Ub^K48R as described in the Experimental Procedures. The HIP2~Ub^K48R thioester (lane 3) was incubated with HIP2–Ub^Cys, ubiquitin, or Ub^K48R for 1 h at 37°C. Reaction components are listed above each gel with a (+) or (-). Lane 1 shows the formation of the thioester and presence of residual E1 and HIP2 as indicated by reactivity with free Alexa dye. Lane 2 shows that E1 has been removed from all subsequent reactions. Reaction products are shown to the right of the gel. Red circles denote the Alexa-labeled Ub^K48R molecule in each reaction. Unknown products, observed only in fluorescence-imaged gels are indicated (#).</p

Assessment of HIP2–UbCys and Ubc1–UbCys oligomerization from SAXS data.

<p>Plots of calculated molecular weights for (A) HIP2–Ub^Cys (filled triangles) and (B) Ubc1–Ub^Cys (filled circles) as a function of protein concentration. The expected monomeric molecular weights for both E2-Ub conjugates (dotted lines) are displayed on each graph. (C, D) Calculated curves for a range of dimerization constants (<i>K</i>_d, μM) based on apparent molecular weight versus protein concentration. The curves are superimposed over the experimental data for (C) HIP2–Ub^Cys and (D) Ubc1–Ub^Cys.</p

Time course reactions for HIP2~Ub thioester reactivity.

<p>Fluorescent images of SDS gels for the reaction of Alexa-labeled HIP2~Ub^K48R (left) or HIP2Δ~Ub^K48R (center) with non-fluorescently labeled (<b>A</b>) ubiquitin, (<b>B</b>) HIP2–Ub^Cys, or (<b>C</b>) equimolar amounts of ubiquitin and HIP2–Ub^Cys. The initial lane in each reaction shows the Alexa-labeled Ub^K48R alone. Fluorescently-labeled HIP2~Ub^K48R and HIP2Δ~Ub^K48R were formed through a 20 min reaction at 37°C with HIP2 or HIP2Δ and Alexa-labeled Ub^K48R as described in the Experimental Procedures. Thioester formation was halted with EDTA (time = 0 min) and then reacted with either ubiquitin, HIP2–Ub^Cys or both and samples measured at 5, 10, 20, 30 and 40 min. Reducing agent (10 mM DTT and 10 mM TCEP) was added to the 40 min sample (+R). Red circles denote the Alexa-labeled Ub^K48R molecule in each reaction. Also shown are the measured fluorescence intensities for (<b>D</b>) Ub₂ formation from HIP2~Ub^K48R (●) or HIP2Δ~Ub^K48R (◯◻, (<b>E</b>) HIP2-Ub₂ formation for HIP2~Ub^K48R (■) or HIP2Δ~Ub^K48R (◻) and (<b>F</b>) competing reactions forming Ub₂ (●,◯) and HIP2-Ub2 (■,◻) from HIP2~Ub^K48R (●,■) or HIP2Δ~Ub^K48R (◯,◻) plotted as a function of time. Linear regression was used to fit each data set to approximate the initial product formation and the relative rates were determined by taking the ratio of the slopes. Each reaction was done in duplicate.</p

Sedimentation equilibrium results for HIP2-Ub and Ubc1-Ub covalent complexes.

<p>^a Molecular weight determined using global fits of 15k, 18k, 22k and 26k rpm to a single species model.</p><p>Sedimentation equilibrium results for HIP2-Ub and Ubc1-Ub covalent complexes.</p

Three-dimensional models of the HIP2-Ub conjugate based on SAXS scattering data.

<p>(<b>A</b>) Scattering data points for HIP2 (142 μM, black) along with the calculated scattering curve (red) derived from the monomeric HIP2 crystal structure coordinates (PDB 1YLA). The fit was optimized by manually modifying the position of the UBA domain (magenta) with respect to the catalytic domain (grey) in HIP2. Scattering data points for HIP2-Ub^Cys (96 μM, black) along with the calculated scattering curves (red) determined using the relative positions of ubiquitin (blue, cyan) conjugated to HIP2 (grey) based on the coordinates for (B) UbcH5b~Ub (PDB 3JW0) and (C) Ubc13~Ub (PDB 2GMI). (D) Comparison of scattering data (black) with a more globular structure where ubiquitin is found in the closed position such as that found in a truncated form of Ubc1 (PDB 1TTE). This arrangement produced a pronounced “dip” in the calculated curve compared to experimental data. Scattering curves were calculated using the program CRYSOL.</p

Crystal and Solution Structure Analysis of FhuD2 from <i>Staphylococcus aureus</i> in Multiple Unliganded Conformations and Bound to Ferrioxamine‑B

Iron acquisition is a central process for virtually all organisms. In <i>Staphylococcus aureus</i>, FhuD2 is a lipoprotein that is a high-affinity receptor for iron-bound hydroxamate siderophores. In this study, FhuD2 was crystallized bound to ferrioxamine-B (FXB), and also in its ligand-free state; the latter structures are the first for hydroxamate-binding receptors within this protein family. The structure of the FhuD2–FXB conformation shows that residues W197 and R199 from the C-terminal domain donate hydrogen bonds to the hydroxamate oxygens, and a ring of aromatic residues cradles the aliphatic arms connecting the hydroxamate moieties of the siderophore. The available ligand-bound structures of FhuD from <i>Escherichia coli</i> and YfiY from <i>Bacillus cereus</i> show that, despite a high degree of structural conservation, three protein families have evolved with critical siderophore binding residues on either the C-terminal domain (<i>S. aureus</i>), the N-terminal domain (<i>E. coli</i>), or both (<i>B. cereus</i>). Unliganded FhuD2 was crystallized in five conformations related by rigid body movements of the N- and C-terminal domains. Small-angle X-ray scattering (SAXS) indicates that the solution conformation of unliganded FhuD2 is more compact than the conformations observed in crystals. The ligand-induced conformational changes for FhuD2 in solution are relatively modest and depend on the identity of the siderophore. The crystallographic and SAXS results are used to discuss roles for the liganded and unliganded forms of FhuD2 in the siderophore transport mechanism

The mapping of chemical shifts on the crystal structure of the 14-3-3ζ /Cby complex.

<p>Due to the crowding of some peaks, the chemical shifts of some residues could not be confidently traced and were excluded from the analysis. (A) (Above) A monomer of the 14-3-3ζ /Cby crystal structure interface based on Cby residues (¹⁸SApSLSNLH²⁵). Residues R56, R127 and Y128 which contact the Cby phosphate group are coloured red, residues interfacing the peptide are coloured cyan, and 14-3-3ζ residues coloured magenta are found along the 14-3-3 dimer interface. (Below) The 14-3-3ζ dimer bound to Cby. (B) (Cby 7-mer), (C) (Cby 18-mer) and (D) (Cby S22P 18-mer). Residues with traceable assigned resonances are coloured on a blue—white—yellow gradient (0 ppm to 0.1 ppm) based on their combined chemical shift [Δω = ((Δδ¹H)² +(0.2*Δδ¹⁵N)²)^1/2] at a 3:1 peptide:protein ratio. Residues coloured in orange represent peaks that broadened out to disappearance upon addition of peptide. Residues R56, R127 and Y128 are coloured pink.</p