Inositol Hexakisphosphate-Induced Autoprocessing of Large Bacterial Protein Toxins

Large bacterial protein toxins autotranslocate functional effector domains to the eukaryotic cell cytosol, resulting in alterations to cellular functions that ultimately benefit the infecting pathogen. Among these toxins, the clostridial glucosylating toxins (CGTs) produced by Gram-positive bacteria and the multifunctional-autoprocessing RTX (MARTX) toxins of Gram-negative bacteria have distinct mechanisms for effector translocation, but a shared mechanism of post-translocation autoprocessing that releases these functional domains from the large holotoxins. These toxins carry an embedded cysteine protease domain (CPD) that is activated for autoprocessing by binding inositol hexakisphosphate (InsP6), a molecule found exclusively in eukaryotic cells. Thus, InsP6-induced autoprocessing represents a unique mechanism for toxin effector delivery specifically within the target cell. This review summarizes recent studies of the structural and molecular events for activation of autoprocessing for both CGT and MARTX toxins, demonstrating both similar and potentially distinct aspects of autoprocessing among the toxins that utilize this method of activation and effector delivery

Side chain residues from CPD that contact InsP₆ in the structural models derived from crystal structures of MARTX_Vc and TcdA CPD.

<p>All key residues that contact InsP₆ in the CPD of (A) MARTX_Vc and (B) TcdA are shown labeled with a single letter code, with the three Lys residues determined to be most critical for InsP₆ binding shown in bold text. Interestingly, despite strong conservation of the critical Lys residues in the primary amino acid sequence, contacts with InsP₆ and the orientation of InsP₆ differ in the two structures. Diagram is colored to represent residues originating from the N-terminal strand (yellow), the core structure (green), and β-strands G₁-G₅ (blue), a structure also known as β8-β12 or the β-flap. Structural models were based on PDB (A) 3FZY <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000942#ppat.1000942-Prochazkova1" target="_blank">[48]</a> and (B) 3HO6 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000942#ppat.1000942-Pruitt1" target="_blank">[57]</a>, and figures were prepared with MacPyMol software (DeLano Scientific).</p

Schematic diagrams representing CPD-dependent autoprocessing sites within CGTs and MARTX toxins.

<p>Diagrams are shown for (A) CGTs represented by TcdA and TcdB or (B) MARTX toxins represented by MARTX_Vc. In (A), the CGT holotoxins contain C-terminal repeats required for receptor interactions and a hydrophobic region (HR) postulated to function in translocation of the GT across the membrane of the endosome. Upon autoprocessing, the catalytically active glucosyltransferase effector (GT) is delivered to cells where it targets RhoGTPases. In (B), the MARTX holotoxin contains both N- and C-terminal repeats that likely function in translocation. Upon autoprocessing, MARTX_Vc delivers three effectors that have distinct cellular targets as indicated. For both diagrams, the CPD catalytic Cys and His are marked, as are processing site Leu residues (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000942#ppat-1000942-t001" target="_blank">Table 1</a>) found in unstructured segments between effectors (indicated by arrows). For CGTs, sequence numbering above the diagram represents TcdA while numbering below the diagram represents TcdB. For MARTX_Vc, sequence numbering is based on the original annotation of the <i>rtxA</i> gene by Lin et al. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000942#ppat.1000942-Lin1" target="_blank">[37]</a> and may be different than that found in cited references.</p

Crystal structures of MARTX_Vc and TcdA CPDs.

<p>Crystal structures of the (A–D) CPD catalytic sites with distances between residues designated in angstroms and (E–G) the CPD proteins are shown at various stages of processing. (A, E) Pre-processing and (B, F) post-processing structures of MARTX_Vc CPD bound to InsP₆ (PDB 3FZY <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000942#ppat.1000942-Prochazkova1" target="_blank">[48]</a> and PDB 3EEB <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000942#ppat.1000942-Lupardus1" target="_blank">[56]</a>, respectively). (C, G) Post-processing structures of TcdA CPD bound to InsP₆ (PDB 3HO6 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000942#ppat.1000942-Pruitt1" target="_blank">[57]</a>). (D, H) Post-processing structure of MARTX_Vc bound to <i>z</i>-Leu-Leu-azaLeu-epoxide inhibitor JCP598 as a surrogate substrate representing the structure of CPD after reactivation (PDB 3GCD <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000942#ppat.1000942-Shen1" target="_blank">[49]</a>). Structures are identically oriented at a conserved Trp (purple) in the G₁/G₂ β-hairpin that is critical to InsP₆ induction of autoprocessing <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000942#ppat.1000942-Lupardus1" target="_blank">[56]</a>. The catalytic Cys and His residues are shown in pink with InsP₆ present at the backside of each structure in red. The P1 Leu (turquoise) is found only in the unprocessed structure (A) with the scissile bond oriented between the catalytic residues. Figures were prepared with MacPyMol software (DeLano Scientific).</p

Proposed model for cooperative activation and reactivation of MARTX_Vc CPD by InsP₆.

<p>I. Apo-CPD without InsP₆ is an unstable protein susceptible to thermal denaturation at physiological temperature. The core structure (green) is folded but the β-flap (blue) is susceptible to proteolysis, indicating it may be only partially structured. II. Upon binding InsP₆, the structure rearranges such that the N-terminus (yellow) becomes locked within the active site between the catalytic Cys (C) and His (H) in a rigid alignment amenable to substrate-activated autoprocessing. III. After autoprocessing, the MARTX_Vc CPD enters a transitional state that has distinct biochemical properties, including a 500-fold reduced affinity for InsP₆. IV. After first binding a new substrate (grey) and then a new molecule of InsP₆, the enzyme–substrate complex structure of the MARTX_Vc CPD is restored for additional processing events. Figure is based on multisite processing model for MARTX_Vc proposed by Prochazkova et al. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000942#ppat.1000942-Prochazkova1" target="_blank">[48]</a>. Current evidence from NMR studies supports the idea that stage I and II also occur for TcdA <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000942#ppat.1000942-Pruitt1" target="_blank">[57]</a>. However, binding studies with TcdB suggest CGTs likely do not undergo stage III deactivation or stage IV reactivation <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000942#ppat.1000942-Egerer2" target="_blank">[55]</a>.</p

InsP₆-induced autoprocessing CGTs and MARTX toxins.

a<p>Based on analysis of effector domains as reviewed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000942#ppat.1000942-Belyi1" target="_blank">[15]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000942#ppat.1000942-Satchell1" target="_blank">[22]</a>.</p>b<p>Processing site is indicated by inverted arrow. m, processing site as mapped experimentally by N-terminal sequencing or mass spectrometry; p, processing site predicted by homology to mapped processing site from closely related toxin; a, alternative processing site identified by mass spectrometry. Numbering of MARTX_Vc processing sites is based on amino acid sequence as originally annotated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000942#ppat.1000942-Lin1" target="_blank">[37]</a> and may be different than that found in cited references. For other MARTX toxins, not all processing sites are known and only those previously reported in the literature are listed.</p