Progress in understanding the assembly process of bacterial O-antigen

The discovery that the surfaces of Gram-negative bacteria often carry unique polysaccharide signatures pre-dates most seminal discoveries of molecular biology and biochemistry of the 20th century. The O-antigen component of the lipopolysaccharide has been one of the most intensely studied bacterial polysaccharide surface structures for over 80 years. Yet, many questions about the mechanism of biosynthesis of the O-antigen and its transport to the cell surface remain unanswered. In this review we provide an overview of how the molecular basis of the O-antigen assembly and trafficking were unraveled in a historical context. We pay particular attention to the emergence of novel technological approaches and how they fueled the elucidation of the O-antigen maturation process. Moreover, we provide a brief perspective on the biosynthesis of enterobacterial common antigen and underline the similarities and differences between the pathways used to assemble these two surface polysaccharides. Finally, we highlight key discoveries that led to the understanding of the mechanistic basis of bacteriophage-induced O-antigen modifications. We place special emphasis on the regulation of the length of O-antigen polymers and provide a detailed overview of the models explaining the O-antigen length determination. Finally, we highlight outstanding questions that need to be addressed both structurally and functionally to advance our understanding of the O-antigen assembly, trafficking and export within cellular and molecular contexts.Sergei Kalynych, Renato Morona & Miroslaw Cygle

Structural Basis for the Inhibition of Host Protein Ubiquitination by Shigella Effector Kinase OspG

SummaryShigella invasion of its human host is assisted by T3SS-delivered effector proteins. The OspG effector kinase binds ubiquitin and ubiquitin-loaded E2-conjugating enzymes, including UbcH5b and UbcH7, and attenuates the host innate immune NF-kB signaling. We present the structure of OspG bound to the UbcH7∼Ub conjugate. OspG has a minimal kinase fold lacking the activation loop of regulatory kinases. UbcH7∼Ub binds OspG at sites remote from the kinase active site, yet increases its kinase activity. The ubiquitin is positioned in the “open” conformation with respect to UbcH7 using its I44 patch to interact with the C terminus of OspG. UbcH7 binds to OspG using two conserved loops essential for E3 ligase recruitment. The interaction of the UbcH7∼Ub with OspG is remarkably similar to the interaction of an E2∼Ub with a HECT E3 ligase. OspG interferes with the interaction of UbcH7 with the E3 parkin and inhibits the activity of the E3

5,6,7,8-Tetra­hydro­quinolin-8-one

In the quinoline fused-ring system of the title compound, C9H9NO, the pyridine ring is planar to within 0.011 (3) Å, while the partially saturated cyclo­hexene ring adopts a sofa conformation with an asymmetry parameter ΔC s(C6) = 1.5 (4)°. There are no classical hydrogen bonds in the crystal structure. Mol­ecules form mol­ecular layers parallel to (100) with a distance between the layers of a/2 = 3.468 Å

The α/β hydrolase fold

We have identified a new protein fold-the α/β hydrolase fold-that is common to several hydrolytic enzymes of widely differing phylogenetic origin and catalytic function. The core of each enzyme is similar: an α/β sheet, not barrel, of eight β-sheets connected by α-helices. These enzymes have diverged from a common ancestor so as to preserve the arrangement of the catalytic residues, not the binding site. They all have a catalytic triad, the elements of which are borne on loops which are the best-conserved structural features in the fold. Only the histidine in the nucleophile-histidine-acid catalytic triad is completely conserved, with the nucleophile and acid loops accommodating more than one type of amino acid. The unique topological and sequence arrangement of the triad residues produces a catalytic triad which is, in a sense, a mirror-image of the serine protease catalytic triad. There are now four groups of enzymes which contain catalytic triads and which are related by convergent evolution towards a stable, useful active site: the eukaryotic serine proteases, the cysteine proteases, subtilisins and the α/β hydrolase fold enzymes

Monte Carlo and analytic modeling of an Elekta Infinity linac with Agility MLC: Investigating the significance of accurate model parameters for small radiation fields

Purpose: To explain the deviation observed between measured and Monaco calculated dose profiles for a small field (i.e., alternating open-closed MLC pattern). A Monte Carlo (MC) model of an Elekta Infinity linac with Agility MLC was created and validated against measurements. In addition, an analytic model which predicts the fluence at the isocenter plane was used to study the impact of multiple beam parameters on the accuracy of dose calculations for small fields. Methods: A detailed MC model of a 6 MV Elekta Infinity linac with Agility MLC was created in EGSnrc/BEAMnrc and validated against measurements. An analytic model using primary and secondary virtual photon sources was created and benchmarked against the MC simulations and the impact of multiple beam parameters on the accuracy of the model for a small field was investigated. Both models were used to explain discrepancies observed between measured/EGSnrc simulated and Monaco calculated dose profiles for alternating open-closed MLC leaves. Results: MC-simulated dose profiles (PDDs, cross- and in-line profiles, etc.) were found to be in very good agreements with measurements. The bes

Revisiting fetal dose during radiation therapy: evaluating treatment techniques and a custom shield

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135233/1/acm20001i.pd

Regulatory Elements within the Prodomain of Falcipain-2, a Cysteine Protease of the Malaria Parasite Plasmodium falciparum

Falcipain-2, a papain family cysteine protease of the malaria parasite Plasmodium falciparum, plays a key role in parasite hydrolysis of hemoglobin and is a potential chemotherapeutic target. As with many proteases, falcipain-2 is synthesized as a zymogen, and the prodomain inhibits activity of the mature enzyme. To investigate the mechanism of regulation of falcipain-2 by its prodomain, we expressed constructs encoding different portions of the prodomain and tested their ability to inhibit recombinant mature falcipain-2. We identified a C-terminal segment (Leu155–Asp243) of the prodomain, including two motifs (ERFNIN and GNFD) that are conserved in cathepsin L sub-family papain family proteases, as the mediator of prodomain inhibitory activity. Circular dichroism analysis showed that the prodomain including the C-terminal segment, but not constructs lacking this segment, was rich in secondary structure, suggesting that the segment plays a crucial role in protein folding. The falcipain-2 prodomain also efficiently inhibited other papain family proteases, including cathepsin K, cathepsin L, cathepsin B, and cruzain, but it did not inhibit cathepsin C or tested proteases of other classes. A structural model of pro-falcipain-2 was constructed by homology modeling based on crystallographic structures of mature falcipain-2, procathepsin K, procathepsin L, and procaricain, offering insights into the nature of the interaction between the prodomain and mature domain of falcipain-2 as well as into the broad specificity of inhibitory activity of the falcipain-2 prodomain

Synthesis and in silico study of 2-furyl(4-{4-[(substituted)sulfonyl]benzyl}-1-piperazinyl)methanone derivatives as suitable therapeutic agents

Abstract In the study presented here, a new series of 2-furyl(4-{4-[(substituted)sulfonyl]benzyl}-1-piperazinyl)methanone derivatives was targeted. The synthesis was initiated by the treatment of different secondary amines (1a-h) with 4-bromomethylbenzenesulfonyl chloride (2) to obtain various 1-{[4-(bromomethyl)phenyl]sulfonyl}amines (3a-h). 2-Furyl(1-piperazinyl)methanone (2-furoyl-1-piperazine; 4) was then dissolved in acetonitrile, with the addition of K2CO3, and the mixture was refluxed for activation. This activated molecule was further treated with equi-molar amounts of 3a-h to form targeted 2-furyl(4-{4-[(substituted)sulfonyl]benzyl}-1-piperazinyl)methanone derivatives (5a-h) in the same reaction set up. The structure confirmation of all the synthesized compounds was carried out by EI-MS, IR and 1H-NMR spectral analysis. The compounds showed good enzyme inhibitory activity. Compound 5h showed excellent inhibitory effect against acetyl- and butyrylcholinesterase with respective IC50 values of 2.91±0.001 and 4.35±0.004 μM, compared to eserine, a reference standard with IC50 values of 0.04±0.0001 and 0.85±0.001 μM, respectively, against these enzymes. All synthesized molecules were active against almost all Gram-positive and Gram-negative bacterial strains tested. The cytotoxicity of the molecules was also checked to determine their utility as possible therapeutic agents

Structure-function analysis of Escherichia coli MnmG (GidA), a highly conserved tRNA-modifying enzyme

The MnmE-MnmG complex is involved in tRNA modification. We have determined the crystal structure of Escherichia coli MnmG at 2.4-A resolution, mutated highly conserved residues with putative roles in flavin adenine dinucleotide (FAD) or tRNA binding and MnmE interaction, and analyzed the effects of these mutations in vivo and in vitro. Limited trypsinolysis of MnmG suggests significant conformational changes upon FAD binding.This work was supported by grant GSP-48370 from the Canadian Institutes of Health Research (to M.C. and A.M.) and grant BFU2007-66509 from the Ministerio de Ciencia e Innovación (to M.-E.A.).Peer reviewe