Structural model of human dUTPase in complex with a novel proteinaceous inhibitor

Human deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), essential for DNA integrity, acts as a survival factor for tumor cells and is a target for cancer chemotherapy. Here we report that the Staphylococcal repressor protein StlSaPIBov1 (Stl) forms strong complex with human dUTPase. Functional analysis reveals that this interaction results in significant reduction of both dUTPase enzymatic activity and DNA binding capability of Stl. We conducted structural studies to understand the mechanism of this mutual inhibition. Small-angle X-ray scattering (SAXS) complemented with hydrogen-deuterium exchange mass spectrometry (HDX-MS) data allowed us to obtain 3D structural models comprising a trimeric dUTPase complexed with separate Stl monomers. These models thus reveal that upon dUTPase-Stl complex formation the functional homodimer of Stl repressor dissociates, which abolishes the DNA binding ability of the protein. Active site forming dUTPase segments were directly identified to be involved in the dUTPase-Stl interaction by HDX-MS, explaining the loss of dUTPase activity upon complexation. Our results provide key novel structural insights that pave the way for further applications of the first potent proteinaceous inhibitor of human dUTPase

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Exploiting a Phage-Bacterium Interaction System as a Molecular Switch to Decipher Macromolecular Interactions in the Living Cell

Pathogenicity islands of Staphylococcus aureus are under the strong control of helper phages, where regulation is communicated at the gene expression level via a family of specific repressor proteins. The repressor proteins are crucial to phage-host interactions and, based on their protein characteristics, may also be exploited as versatile molecular tools. The Stl repressor from this protein family has been recently investigated and although the binding site of Stl on DNA was recently discovered, there is a lack of knowledge on the specific protein segments involved in this interaction. Here, we develop a generally applicable system to reveal the mechanism of the interaction between Stl and its cognate DNA within the cellular environment. Our unbiased approach combines random mutagenesis with high-throughput analysis based on the lac operon to create a well-characterized gene expression system. Our results clearly indicate that, in addition to a previously implicated helix-turn-helix segment, other protein moieties also play decisive roles in the DNA binding capability of Stl. Structural model-based investigations provided a detailed understanding of Stl:DNA complex formation. The robustness and reliability of our novel test system were confirmed by several mutated Stl constructs, as well as by demonstrating the interaction between Stl and dUTPase from the Staphylococcal ϕ11 phage. Our system may be applied to high-throughput studies of protein:DNA and protein:protein interactions

(A) Homology model of the Stl protein.

<p>Ribbon representation of the homology model of the <i>Staphylococcus aureus</i> pathogenicity island repressor Stl produced by Phyre2 Server [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139086#pone.0139086.ref038" target="_blank">38</a>]. Based on the homology model the protein is highly α helical (74%), and seems to be divided into two segments: the amino terminal segment colored cyan and the carboxy-terminal segment colored hotpink. According to Pfam and NCBI CDD the protein is predicted to contain a helix-turn-helix DNA binding motif. The position of the HTH predicted by NPS@ server is colored to dark blue [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139086#pone.0139086.ref044" target="_blank">44</a>]. <b>(B) SRCD spectrum of Stl and fitted curves by BeStSel and CONTIN algorithms.</b> Stl was measured at 2.1 mg/ml concentration in 50 mM Hepes, 200 mM NaCl, pH 7.5.</p

Secondary structure estimation from the synchrotron radiation CD spectrum of Stl and comparison to the homology model.

<p>^aThe secondary structure composition from the CD spectrum was estimated by the BeStSel and CONTIN algorithms. The two algorithms use different secondary structure components, however, the overall helix, β-sheet and turn+others contents are comparable.</p><p>^bThe secondary structure contents were also calculated for the Phyre2 homology model using the DSSP algorithm [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139086#pone.0139086.ref046" target="_blank">46</a>] and the BeStSel and CONTIN definitions [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139086#pone.0139086.ref047" target="_blank">47</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139086#pone.0139086.ref048" target="_blank">48</a>].</p><p>Secondary structure estimation from the synchrotron radiation CD spectrum of Stl and comparison to the homology model.</p

DNA binding domain of bacteriophage repressors.

<p><b>(A) Sequence alignment of the HTH motifs of bacteriophage repressors and Stl.</b> The number before each segment is the amino acid sequence number of the first residue in the sequence. Helices are with gray background, similar residues are in bold, box highlights residues interacting with DNA nucleobases. PDB ID of the proteins is indicated on the right side of the sequences. <b>(B) Experimentally determined structure of the DNA-bound CI bacteriophage repressor</b> DNA cartoons orange, protein cartoon: dark blue for HTHs, otherwise cyan. DNA bases and DNA interacting amino acid residues are stick representation with atomic coloring (protein carbon yellow, DNA carbon green, oxygen red, nitrogen blue, phosphorus orange.) The PDB ID of the structure is indicated. Stereo representation of all experimentally determined protein-DNA complex structures represented in the sequence alignment in this figure are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139086#pone.0139086.s004" target="_blank">S3</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139086#pone.0139086.s010" target="_blank">S9</a> Figs.</p

Electrophoretic mobility shift assay (EMSA) for testing the DNA binding ability of Stl constructs.

<p><b>(A)</b> Result of EMSA with the wild type Stl protein. Note that the DNA band is shifted upwards at 1 μM Stl concentration. <b>(B)</b> EMSA gel result of Stl-AA mutant protein. Note that even at relatively high (ie. 3 μM) concentration of the Stl-AA mutant construct, most DNA still appears at the lower position on the gel, indicating lack of binding to the mutant protein.</p

DNA and protein binding ability of Stl-CTD.

<p><b>(A) Electrophoretic mobility shift assay</b> was performed to investigate the DNA binding ability of Stl-CTD. Species and concentrations given in monomers are indicated on the figure. The band of the dsDNA is only shifted upwards if wild type Stl is added but there is no shift even at high concentrations Stl-CTD. <b>(B) Native gel electrophoresis</b> experiment was performed to investigate the dUTPase binding ability of Stl-CTD. Species and concentrations given in monomers are indicated. The mixture of Stl-CTD and Φ11 dUTPase shows up in distinct position comparing to the individual proteins, which clearly indicate the complex formation. <b>(C) Activity of the Φ11 dUTPase</b> was measured in the presence and absence of Stl-CTD. Each measurement was repeated three times. The quadratic binding equation was fit to the data resulted in the apparent K_i = 1.5 ± 0.5 nM. The total change in amplitude of the activity was 40%.</p

(A) Sequence alignment for the HTH segments of Stl-like repressors.

<p>Identical residues are in red (*), strongly similar (:) residues are in green, weakly similar residues are in blue (.). The protein investigated in the current study, termed as Stl throughput the text, is boxed (it is equivalent to the repressor of the SaPIbov1 pathogenicity island). <b>(B) Superimpositioned structural models of representative Stl-like repressors from different SaPIs.</b> Proteins are in cartoon representation. SaPIbov1 Stl is cyan, Stl-like repressors of SaPIbov3, SaPI1, and SsPI15305 pathogenicity islands are violetpurple, salmon, and green, respectively. Predicted HTH motifs of all proteins are colored dark blue. Stereo view of these structures is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139086#pone.0139086.s015" target="_blank">S14 Fig</a>.</p