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

Dual salt bridges are required for prodomain intramolecular chaperone activity.

<p>(<b>a</b>) Overlay of prodomains from CspB <i>perfringens</i> (teal), Tk-SP (grey), and PCSK9 (pink). (<b>b</b>) PDBe PISA analyses of free energy of prodomain dissociation from mature subtilase, with CspB in teal, PCSK9 in pink, and others in grey. (<b>c</b>) Close-up view of dual salt-bridge interaction at prodomain-subtilase interface. The C-terminus of the prodomain (C, teal) extends toward the substrate-binding pocket. Prodomain Glu35, Glu59 and Arg91 residues are shown in teal; subtilase domain Arg231 and D257 residues are shown in magenta. (<b>d</b>) Analysis of CspB prodomain mutant solubility using Western blotting and Coomassie staining. Cultures expressing <i>cspB</i> variants were induced with IPTG, and aliquots were removed 30 minutes later (“induced-IPTG” sample). Cells were lysed by sonication and centrifuged at high speed; the “cleared lysate” sample represents the soluble fraction. CspB variants were purified by affinity chromatography. Equivalent amounts of samples were resolved by SDS-PAGE and analyzed either by Western blotting using anti-CspB <i>perfringens</i> antisera or by Coomassie staining (bottom gel, affinity-purified CspB).</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

GTP molecule is bound in both activation and nucleotidyl transfer sites.

<p>(a) Two GTP molecules were observed in the BtTLP structure. The interactions with the guanine base in the activation site come mostly from protein main-chain atoms. Several conserved, catalytically important residues interact with the triphosphate tail in the nucleotidyl transfer site. Some of the interactions to the triphosphate and base originate from the adjacent monomer (Residues shown in pink). (b) Activation and nucleotidyl transfer sites of hTHG1. The two enzymes employ similar residues to bind GTP in both sites. Residues originating from the adjacent monomer are shown in teal.</p

The jellyroll domain and catalytic serine of CspBA are required for efficient germination.

<p>(<b>a</b>) Schematic of CspBA variants produced by <i>cspBAC</i> complementation constructs. “Pro” denotes the prodomain; black rectangle demarcates the jellyroll domain; a thin white rectangle represents the jellyroll deletion; and white star indicates S461A mutation. (<b>b</b>) Western blot analyses of sporulating cells expressing <i>cspBAC</i> complementation constructs and (<b>c</b>) germinating spores expressing <i>cspBAC</i> complementation constructs. Purified spores of the indicated strain were either untreated (−) or exposed to 0.2% w/v sodium taurocholate (+, germinant) for 15 min at 37°C and analyzed by Western blotting with the indicted antibodies. Germination efficiency was determined via colony forming unit (cfu) determination. Representative clones of each construct are shown, but more than two clones of each complementation construct were tested. m-CspBA reflects the mature form of CspBA following autoprocessing, and m-CspB reflects the mature form of CspB following autoprocessing. The different mutant CspB variants are indicated.</p

Structural Studies of a Bacterial tRNA^HIS Guanylyltransferase (Thg1)-Like Protein, with Nucleotide in the Activation and Nucleotidyl Transfer Sites

<div><p>All nucleotide polymerases and transferases catalyze nucleotide addition in a 5′ to 3′ direction. In contrast, tRNA^His guanylyltransferase (Thg1) enzymes catalyze the unusual reverse addition (3′ to 5′) of nucleotides to polynucleotide substrates. In eukaryotes, Thg1 enzymes use the 3′–5′ addition activity to add G₋₁ to the 5′-end of tRNA^His, a modification required for efficient aminoacylation of the tRNA by the histidyl-tRNA synthetase. Thg1-like proteins (TLPs) are found in Archaea, Bacteria, and mitochondria and are biochemically distinct from their eukaryotic Thg1 counterparts TLPs catalyze 5′-end repair of truncated tRNAs and act on a broad range of tRNA substrates instead of exhibiting strict specificity for tRNA^His. Taken together, these data suggest that TLPs function in distinct biological pathways from the tRNA^His maturation pathway, perhaps in tRNA quality control. Here we present the first crystal structure of a TLP, from the gram-positive soil bacterium <i>Bacillus thuringiensis</i> (BtTLP). The enzyme is a tetramer like human THG1, with which it shares substantial structural similarity. Catalysis of the 3′–5′ reaction with 5′-monophosphorylated tRNA necessitates first an activation step, generating a 5′-adenylylated intermediate prior to a second nucleotidyl transfer step, in which a nucleotide is transferred to the tRNA 5′-end. Consistent with earlier characterization of human THG1, we observed distinct binding sites for the nucleotides involved in these two steps of activation and nucleotidyl transfer. A BtTLP complex with GTP reveals new interactions with the GTP nucleotide in the activation site that were not evident from the previously solved structure. Moreover, the BtTLP-ATP structure allows direct observation of ATP in the activation site for the first time. The BtTLP structural data, combined with kinetic analysis of selected variants, provide new insight into the role of key residues in the activation step.</p></div

The CspBA fusion protein undergoes processing during sporulation.

<p>(<b>a</b>) Schematic of Csps and SleC in <i>C. perfringens</i> and <i>C. difficile</i>. Intact catalytic residues are black, while catalytic mutations are grey. The prodomain of <i>C. perfringens</i> Csps are shown in light grey, with their lengths indicated. The predicted prodomain of CspBA is also indicated. SleC is outlined in black, with the prepeptide (Pre), propeptide (Pro), and Csp cleavage site indicated for <i>C. perfringens</i> SleC <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003165#ppat.1003165-Miyata1" target="_blank">[21]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003165#ppat.1003165-Okamura1" target="_blank">[23]</a> (<b>b</b>) Western blot analysis of sporulating <i>C. difficile</i> and purified spores. Purified spores of the indicated strain were either untreated (−) or exposed to 0.2% w/v sodium taurocholate <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003165#ppat.1003165-Sorg1" target="_blank">[16]</a> (+, germinant) for 15 min at 37°C and analyzed by Western blotting and for germination efficiency via colony forming unit (cfu) determination. The processing products of CspB and SleC are indicated. CD1433 was previously shown to be a component of <i>C. difficile</i> spores and is used as a loading control <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003165#ppat.1003165-Permpoonpattana1" target="_blank">[61]</a>; the anti-CD1433 antiserum primarily recognizes the chitinase domain of CD1433. CspB levels were 3.5-fold lower in <i>sleC⁻</i> spores relative to wildtype spores, despite containing similar amounts of CD1433. (<b>c</b>) Phase-contrast microscopy of sporulating <i>C. difficile</i> strains used in (b) showing equivalent levels of sporulation as measured by particle counting. The white triangles indicate mature phase-bright spores that have been released from the mother cell; the black triangles highlight immature forespores in the mother cell.</p

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

CspBA activity downstream of autoprocessing is required for efficient SleC cleavage.

<p>(<b>a</b>) Schematic of CspBA variants produced by <i>cspBAC</i> transcomplementation constructs. “Pro” denotes the prodomain; black rectangle demarcates the jellyroll domain; a thin white rectangle represents the jellyroll deletion; and white star indicates S461A mutation. (<b>b</b>) Western blot analyses of sporulating cells expressing <i>cspBAC</i> transcomplementation constructs and (<b>c</b>) germinating spores expressing transcomplementation constructs. Purified spores of the indicated strain were either untreated (−) or exposed to 0.2% w/v sodium taurocholate (+, germinant) for 15 min at 37°C and analyzed by Western blotting with the indicated antibody. Germination efficiency was determined via colony forming unit (cfu) determination. Representative clones of each construct are shown, but more than two clones of each complementation construct were tested. m-CspBA reflects the mature form of CspBA following autoprocessing, and m-CspB reflects the mature form of CspB following autoprocessing.</p

C-terminal prodomain residues sterically occlude a catalytically competent active site.

<p>(<b>a</b>) Close-up of interaction between prodomain C-terminus and substrate binding pocket. Subtilase, jellyroll and prodomains are shown in semi-transparent surface representation (purple, green, and teal, respectively). Residues 89–96 of prodomain are shown in yellow. (<b>b</b>) Structure of fluorophosphonate-rhodamine (FP-Rh) activity-based probe. Rhodamine dye is shown in red. (<b>c</b>) Schematic of CspB variants. “Pro” refers to the prodomain; “+” reflects co-expression of the prodomain in trans, with the number reflecting the prodomain length. (<b>d</b>) Labeling of CspB variants by FP-Rh. CspB variants (10 µM) were incubated with 1 µM FP-Rh probe for 20 min at RT in triplicate. The labeling reactions were resolved by SDS-PAGE on a 15% gel and visualized by fluorescent scanning followed by Coomassie staining. A single representative replicate is shown. m-CspB refers to mature CspB lacking its prodomain.</p