The JNK1 kinase interacts with NEIL1.

<p>(A) Western blot analysis of endogenous JNK1 present in HEK293T WCE and in NEIL1-FLAG immunoprecipitates. (B) Far-Western analysis to confirm the interaction between NEIL1 and JNK1. Top panel, Coomassie stained SDS-PAGE gel of NEIL1 and its phosphomimetic and ablating mutants (50 pmoles). Bottom panel, membrane after transfer, denaturation, slow refolding, binding to JNK1 from HEK293 WCE, and probing with an anti-JNK1 antibody. BSA was used as a negative control.</p

Binding of NEIL1 and mutant enzymes to DNA.

<p>(A) Representative phosphor-autodiogram for binding of NEIL1-WT to a 35-mer furan-containing DNA where the substrate (10 pM) was incubated with increasing (0–600 nM) amounts of enzyme. Complex formation is indicated by the presence of shifted bands C1 and C2. (B) Graphical fitting of the EMSA data indicated above using GraphPad Prism 6. The data were fit to the one-site specific binding equation. The K_d values for WT and the NEIL1 mutants are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157860#pone.0157860.t001" target="_blank">Table 1</a> and are representative of experiments performed in duplicate.</p

Sites of phosphorylation within the NEIL1 DNA glycosylase.

<p>(A) Domain map of NEIL1 indicating the position of known sites of phosphorylation. The residues S207, S306, and S61 identified in this study are shown in blue and the Y263 and S269 sites previously identified [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157860#pone.0157860.ref039" target="_blank">39</a>] are indicated in black. (B) SDS-PAGE gel of SBP-tagged NEIL1 after affinity pull-down from HEK293T cell-extracts overexpressing NEIL1. The gel was stained with Coomassie blue and the NEIL1-SBP band was cut from the gel and digested with trypsin for identification of phosphorylated peptides via LC-MS/MS.</p

Phosphomimetic/ phosphoablating mutants of NEIL1.

<p>Phosphomimetic/ phosphoablating mutants of NEIL1.</p

Phosphorylation of NEIL1 by JNK1 using an <i>in vitro</i> kinase assay.

<p>(A) <i>In vitro</i> kinase assays were performed with NEIL1 constructs expressed from E. coli cells and purified to homogeneity and active JNK1 kinase for 30 minutes at 32°C. γ-³²P incorporation was quantified using phosphor-autoradiography after SDS-PAGE analysis of the samples. Lane 1, no JNK1 control; lane 2, no NEIL1 control; lane 3–13 are WT, S207E, S207A, S306E, S306A, S61E, S61A, DE, DA, TE, TA, respectively. (B) Graphical representation of two experimental repeats of the <i>in vitro</i> kinase assay. Statistically significant values (at 95% confidence) were determined by a one-way Anova test where * denotes p-values <0.05, *** denotes p-values <0.0005, and **** denotes p values <0.0001. (C) Glycosylase activity assays were performed using Sp:C and AP:C substrates with increasing amounts of <i>in vitro</i> phosphorylated NEIL1 (pNEIL1), unphosphorylated NEIL1 (positive control), and JNK1 (negative control). S and P indicate substrate and product, respectively.</p

Identification of phosphorylation sites on NEIL1 by LC-MS/MS.

<p>(A) List of phosphorylated peptides identified from LC-MS/MS analysis of NEIL1-SBP with their corresponding monoisotopic mass m/z, charge-state (z), and Xcorr values. (B) Product-ion spectra (Scaffold software 4.3) for each phosphorylated peptide are displayed. The b- and y-ions are shown in red and blue, respectively, and the neutral loss peak resulting from the loss of a phosphogroup from the parent ion is indicated in green.</p

Glycosylase and lyase activity panel for human NEIL1-WT and the phosphomimetic/ablating mutants.

<p>Glycosylase assays were performed by incubating 20 nM of double-stranded DNA substrates (A) Sp:C and (B) AP:C and increasing amounts of enzyme with the following substrate to enzyme ratios: 1:0.5, 1:1, 1:4, and 1:16. “-” indicates a no enzyme negative control. Assays were performed at room temperature for 30 minutes. S and P indicate substrate and product, respectively. Data shown are representative of duplicate experiments.</p

Structural and Functional Analysis of the CspB Protease Required for <em>Clostridium</em> Spore Germination

<div><p>Spores are the major transmissive form of the nosocomial pathogen <em>Clostridium difficile</em>, a leading cause of healthcare-associated diarrhea worldwide. Successful transmission of <em>C. difficile</em> requires that its hardy, resistant spores germinate into vegetative cells in the gastrointestinal tract. A critical step during this process is the degradation of the spore cortex, a thick layer of peptidoglycan surrounding the spore core. In <em>Clostridium</em> sp., cortex degradation depends on the proteolytic activation of the cortex hydrolase, SleC. Previous studies have implicated Csps as being necessary for SleC cleavage during germination; however, their mechanism of action has remained poorly characterized. In this study, we demonstrate that CspB is a subtilisin-like serine protease whose activity is essential for efficient SleC cleavage and <em>C. difficile</em> spore germination. By solving the first crystal structure of a Csp family member, CspB, to 1.6 Å, we identify key structural domains within CspB. In contrast with all previously solved structures of prokaryotic subtilases, the CspB prodomain remains tightly bound to the wildtype subtilase domain and sterically occludes a catalytically competent active site. The structure, combined with biochemical and genetic analyses, reveals that Csp proteases contain a unique jellyroll domain insertion critical for stabilizing the protease <em>in vitro</em> and in <em>C. difficile</em>. Collectively, our study provides the first molecular insight into CspB activity and function. These studies may inform the development of inhibitors that can prevent clostridial spore germination and thus disease transmission.</p> </div

The jellyroll domain conformationally rigidifies CspB <i>perfringens</i>.

<p>(<b>a</b>) Overlay of jellyroll domain of CspB <i>perfringens</i> (green) and Tk-SP (grey). (<b>b</b>) Limited proteolysis profile of CspB and its variants. 15 µM of CspB and its variants were incubated with increasing concentrations of chymotrypsin for 60 min at 37°C. Reactions were resolved by SDS-PAGE and visualized by Coomassie staining. Schematic of CspB variants is shown below the Coomassie stained gel. “Pro” refers to the prodomain; black rectangle demarcates the jellyroll domain; thin white rectangle represents the jellyroll deletion; and white star denotes the S494A mutation. m-CspB refers to mature CspB, which is produced after autoprocessing.</p

CspB undergoes autoprocessing in a position-dependent manner.

<p>(<b>a</b>) Coomassie staining of recombinant <i>C. perfringens</i> and <i>C. difficile</i> CspB variants. 7.5 µg of each purified CspB variant was resolved by SDS-PAGE on a 4–12% Bis-Tris gel and visualized by Coomassie staining. The P3-P1 residues of the prodomain were mutated to Ala for the YTS/AAA and QTQ/AAA mutants, while the P3-P1 residues were deleted from CspB <i>perfringens</i> in the ΔYTS mutant. The products resulting from autoprocessing are indicated. (<b>b</b>) Sequence alignment of Csp prodomain cleavage sites mapped by Edman sequencing; the Csp <i>perfringens</i> cleavage sites were mapped in a previous study <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003165#ppat.1003165-Shimamoto1" target="_blank">[25]</a>. Completely conserved identical residues are blocked in black with white text, conserved identical residues in grey with white text, and conserved similar residues in light grey.</p