Feeding Anthrax: The Crystal Structure of Bacillus anthracis InhA Protease

Pathogenic bacteria secrete proteases to evade host defense and to acquire nutrients. In this issue of Structure, Arolas et al. (2016) describe the structural basis of activation and latency of InhA, a major secreted protease of Bacillus anthracis

Structural characterization of the ribonuclease H-like type ASKHA superfamily kinase MK0840 from Methanopyrus kandleri

Murein recycling is a process in which microorganisms recover peptidoglycan-degradation products in order to utilize them in cell wall biosynthesis or basic metabolic pathways. Methanogens such as Methanopyrus kandleri contain pseudomurein, which differs from bacterial murein in its composition and branching. Here, four crystal structures of the putative sugar kinase MK0840 from M. kandleri in apo and nucleotide-bound states are reported. MK0840 shows high similarity to bacterial anhydro-N-acetylmuramic acid kinase, which is involved in murein recycling. The structure shares a common fold with panthothenate kinase and the 2-hydroxyglutaryl-CoA dehydratase component A, both of which are members of the ASKHA (acetate and sugar kinases/Hsc70/actin) superfamily of phosphotransferases. Local conformational changes in the nucleotide-binding site between the apo and holo forms are observed upon nucleotide binding. Further insight is given into domain movements and putative active-site residues are identified

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a target for future development of inhibitors to decrease the virulence of the pathogen. To perform biophysical and structural characterization as well as inhibitor screening, large amounts of pure and active protein will be needed. We have developed a protocol for efficient production and purification of PPEP-1 by the use of E. coli as the expression host yielding sufficient amounts and purity of protein for crystallization and structure determination. Additionally, using microseeding, highly intergrown crystals of PPEP-1 can be grown to well-ordered crystals suitable for X-ray diffraction analysis. The methods could also be used to produce other recombinant proteins and to study the structures of other proteins producing intergrown crystals

Structural Basis of Proline-Proline Peptide Bond Specificity of the Metalloprotease Zmp1 Implicated in Motility of Clostridium difficile

Clostridium difficile is a pathogenic bacterium causing gastrointestinal diseases from mild diarrhea to toxic megacolon. In common with other pathogenic bacteria, C. difficile secretes proteins involved in adhesion, colonization, and dissemination. The recently identified Zmp1 is an extracellular metalloprotease showing a unique specificity for Pro-Pro peptide bonds. The endogenous substrates of Zmp1 are two surface proteins implicated in adhesion of C. difficile to surface proteins of human cells. Thus, Zmp1 is believed to be involved in the regulation of the adhesion-motility balance of C. difficile. Here, we report crystal structures of Zmp1 from C. difficile in its unbound and peptide-bound forms. The structure analysis revealed a fold similar to Bacillus anthracis lethal factor. Crystal structures in the open and closed conformation of the S-loop shed light on the mode of binding of the substrate, and reveal important residues for substrate recognition and the strict specificity of Zmp1 for Pro-Pro peptide bonds

The first crystal structure of the peptidase domain of the U32 peptidase family

The U32 family is a collection of over 2500 annotated peptidases in the MEROPS database with unknown catalytic mechanism. They mainly occur in bacteria and archaea, but a few representatives have also been identified in eukarya. Many of the U32 members have been linked to pathogenicity, such as proteins from Helicobacter and Salmonella. The first crystal structure analysis of a U32 catalytic domain from Methanopyrus kandleri (gene mk0906) reveals a modified (beta alpha) 8 TIM-barrel fold with some unique features. The connecting segment between strands beta 7 and beta 8 is extended and helix alpha 7 is located on top of the C-terminal end of the barrel body. The protein exhibits a dimeric quaternary structure in which a zinc ion is symmetrically bound by histidine and cysteine side chains from both monomers. These residues reside in conserved sequence motifs. No typical proteolytic motifs are discernible in the three-dimensional structure, and biochemical assays failed to demonstrate proteolytic activity. A tunnel in which an acetate ion is bound is located in the C-terminal part of the beta-barrel. Two hydrophobic grooves lead to a tunnel at the C-terminal end of the barrel in which an acetate ion is bound. One of the grooves binds to a Strep-Tag II of another dimer in the crystal lattice. Thus, these grooves may be binding sites for hydrophobic peptides or other ligands

Molecular determinants of the mechanism and substrate specificity of Clostridium difficile proline-proline endopeptidase-1

Pro-Pro endopeptidase-1 (PPEP-1) is a secreted metalloprotease from the bacterial pathogen Clostridium difficile that cleaves two endogenous adhesion proteins. PPEP-1 is therefore important for bacterial motility and hence for efficient gut colonization during infection. PPEP-1 exhibits a unique specificity for Pro-Pro peptide bonds within the consensus sequence VNP down arrow PVP. In this study, we combined information from crystal and NMR structures with mutagenesis and enzyme kinetics to investigate the mechanism and substrate specificity of PPEP-1. Our analyses revealed that the substrate-binding cleft of PPEP-1 is shaped complementarily to the major conformation of the substrate in solution. We found that it possesses features that accept a tertiary amide and help discriminate P1 ' residues by their amide hydrogen bond-donating potential. We also noted that residues Lys-101, Trp-103, and Glu-184 are crucial for proteolytic activity. Upon substrate binding, these residues position a flexible loop over the substrate-binding cleft and modulate the second coordination sphere of the catalytic zinc ion. On the basis of these findings, we propose an induced-fit model in which prestructured substrates are recognized followed by substrate positioning within the active-site cleft and a concomitant increase in the Lewis acidity of the catalytic Zn2+ ion. In conclusion, our findings provide detailed structural and mechanistic insights into the substrate recognition and specificity of PPEP-1 from the common gut pathogen C. difficile

The two conformations of <i>Af</i>AmzAs substrate-binding site.

<p>Superposition of NHis-<i>Af</i>AmzA (yellow) and nat-<i>Af</i>AmzA (green) substrate binding site. Changes in the position of side chains in the bulge edge segment (Met78, Phe80,82), side chains and the main chain of the S1′-wall forming segment (Phe136, Asn138) and in the position of the catalytic water molecule (H₂O_cat) are indicated by the arrows.</p

Multiple sequence alignment of the amino acid sequences of <i>Af</i>AmzA with other archaemetzincins and metzincins.

<p>Archaemetzincins from <i>M. kandleri</i> (AMZA_METKA), <i>M. labreanum</i> (AMZA_METLZ), <i>H. sapiens</i> (AMZ2_HUMAN), non-catalytic archaemetzincin from <i>M. xanthus</i> (Q50888_MYXXA) and metzincins <i>H. sapiens</i> ADAM33 (ADA33_HUMAN), <i>P. flavoviridis</i> H2-proteinase (VMHR2_PROFL), <i>B. asper</i> Bap1 (VMBP1_BOTAS) and <i>A. acutus</i> acutolysin A (VMACA_DEIAC). Sequences were aligned using Chimera <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043863#pone.0043863-Meng1" target="_blank">[38]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043863#pone.0043863-Pettersen1" target="_blank">[39]</a> and visualized with ESPript <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043863#pone.0043863-Gouet1" target="_blank">[40]</a>. 3₁₀-Helices are indicated by η, β-turns by TT. Conserved residues in all sequences are highlighted in red. Similar sequences are in red letters, orange color indicates residues similar in each group (1, 2 or 3) but significantly different from the other groups.</p

Structural differences between NHis-<i>Af</i>AmzA and nat-<i>Af</i>AmzA.

<p>Simulated annealing F_o–F_c omit map of (A) the N-terminus of a symmetry-related molecule (Gly-3*-Ser-2*) in NHis-<i>Af</i>AmzA, (B) a malonate molecule (MLI) in nat-<i>Af</i>AmzA::malonate and (C) a citrate molecule (FLC) in nat-<i>Af</i>AmzA::citrate, contoured at 2σ level. The maps were generated using phenix.omit_map <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043863#pone.0043863-Terwilliger1" target="_blank">[37]</a> and converted to the ccp4 format with FFT (V6.1) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043863#pone.0043863-Collaborative1" target="_blank">[32]</a>. Important residues are shown as sticks. The zinc ions and water molecules are shown as grey and red spheres, respectively. (D) Surface representation of NHis-<i>Af</i>AmzA active site cleft with the N-terminus of a symmetry related molecule bound to the catalytic zinc ion (primed site in closed conformation). (E) Surface representation of nat-<i>Af</i>AmzA active site cleft with the zinc-bound malonate molecule (MLI; primed site in open conformation). Important residues and the catalytic water molecule (H₂O_cat) are labeled. The surface is transparent to allow a view on the residues involved in zinc ion and ligand binding.</p