Phosphatidylethanolamine mediates insertion of the catalytic domain of leader peptidase in membranes

AbstractLeader peptidase is an integral membrane protein of E. coli and it catalyses the removal of most signal peptides from translocated precursor proteins. In this study it is shown that when the transmembrane anchors are removed in vivo, the remaining catalytic domain can bind to inner and outer membranes of E. coli. Furthermore, the purified catalytic domain binds to inner membrane vesicles and vesicles composed of purified inner membrane lipids with comparable efficiency. It is shown that the interaction is caused by penetration of a part of the catalytic domain between the lipids. Penetration is mediated by phosphatidylethanolamine, the most abundant lipid in E. coli, and does not seem to depend on electrostatic interactions. A hydrophobic segment around the catalytically important residue serine 90 is required for the interaction with membranes

Novel Avian Influenza H7N3 Strain Outbreak, British Columbia

Genome sequences of chicken (low pathogenic avian influenza [LPAI] and highly pathogenic avian influenza [HPAI]) and human isolates from a 2004 outbreak of H7N3 avian influenza in Canada showed a novel insertion in the HA0 cleavage site of the human and HPAI isolate. This insertion likely occurred by recombination between the hemagglutination and matrix genes in the LPAI virus

MR fluoroscopy in vascular and cardiac interventions (review)

Vascular and cardiac disease remains a leading cause of morbidity and mortality in developed and emerging countries. Vascular and cardiac interventions require extensive fluoroscopic guidance to navigate endovascular catheters. X-ray fluoroscopy is considered the current modality for real time imaging. It provides excellent spatial and temporal resolution, but is limited by exposure of patients and staff to ionizing radiation, poor soft tissue characterization and lack of quantitative physiologic information. MR fluoroscopy has been introduced with substantial progress during the last decade. Clinical and experimental studies performed under MR fluoroscopy have indicated the suitability of this modality for: delivery of ASD closure, aortic valves, and endovascular stents (aortic, carotid, iliac, renal arteries, inferior vena cava). It aids in performing ablation, creation of hepatic shunts and local delivery of therapies. Development of more MR compatible equipment and devices will widen the applications of MR-guided procedures. At post-intervention, MR imaging aids in assessing the efficacy of therapies, success of interventions. It also provides information on vascular flow and cardiac morphology, function, perfusion and viability. MR fluoroscopy has the potential to form the basis for minimally invasive image–guided surgeries that offer improved patient management and cost effectiveness

Purification of a Tat leader peptide by co-expression with its chaperone

a b s t r a c t We present a method for the purification of the 45 residue long leader peptide of Escherichia coli dimethyl sulfoxide reductase subunit A (DmsA L ), a substrate of the twin arginine translocase, by co-expressing the leader peptide with its specific chaperone protein, DmsD. The peptide can be isolated from the soluble DmsA L /DmsD complex or conveniently from the lysate pellet fraction. The recombinant leader peptide is functionally intact as the peptide/chaperone complex can be reconstituted from purified DmsA L and DmsD. A construct with DmsA L fused to the N-terminus of DmsD (DmsA L -DmsD fusion) was created to further explore the properties of the leader peptide-chaperone interactions. Analytical size-exclusion chromatography in-line with multi-angle light scattering reveals that the DmsA L -DmsD fusion construct forms a dimer wherein each protomer binds the neighboring leader peptide. A model of this homodimeric interaction is presented

Structure of Signal Peptide Peptidase A with C‑Termini Bound in the Active Sites: Insights into Specificity, Self-Processing, and Regulation

Bacterial signal peptide peptidase A (SppA) is a membrane-bound enzyme that utilizes a serine/lysine catalytic dyad mechanism to cleave remnant signal peptides within the cellular membrane. <i>Bacillus subtilis</i> SppA (SppA_BS) oligomerizes into a homo-octameric dome-shaped complex with eight active sites, located at the interface between each protomer. In this study, we show that SppA_BS self-processes its own C-termini. We have determined the crystal structure of a proteolytically stable fragment of SppA_BSK199A that has its C-terminal peptide bound in each of the eight active sites, creating a perfect circle of peptides. Substrate specificity pockets S1, S3, and S2′ are identified and accommodate C-terminal residues Tyr331, Met329, and Tyr333, respectively. Tyr331 at the P1 position is conserved among most <i>Bacillus</i> species. The structure reveals that the C-terminus binds within the substrate-binding grooves in an antiparallel β-sheet fashion. We show, by C-terminal truncations, that the C-terminus is not essential for oligomeric assembly. Kinetic analysis shows that a synthetic peptide corresponding to the C-terminus of SppA_BS competes with a fluorometric peptide substrate for the SppA_BS active site. A model is proposed for how the C-termini of SppA may function in the regulation of this membrane-bound self-compartmentalized protease

Crystal structure of the VP4 protease from infectious pancreatic necrosis virus reveals the acyl-enzyme complex for an intermolecular self-cleavage reaction

Infectious pancreatic necrosis virus (IPNV), an aquatic birnavirus that infects salmonid fish, encodes a large polyprotein (NH2-pVP2-VP4-VP3-COOH) that is processed through the proteolytic activity of its own protease, VP4, to release the proteins pVP2 and VP3. pVP2 is further processed to give rise to the capsid protein VP2 and three peptides that are incorporated into the virion. Reported here are two crystal structures of the IPNV VP4 protease solved from two different crystal symmetries. The electron density at the active site in the triclinic crystal form, refined to 2.2-A˚ resolution, reveals the acyl-enzyme complex formed with an internalVP4cleavage site. The complex was generated using a truncated enzyme in which the general base lysine was substituted. Inside the complex, the nucleophilic Ser633O_ forms an ester bond with the main-chain carbonyl of the C-terminal residue, Ala716, of a neighboring VP4. The structure of this substrate-VP4 complex allows us to identify the S1, S3, S5, and S6 substrate binding pockets as well as other substrate- VP4 interactions and therefore provides structural insights into the substrate specificity of this enzyme. The structure from the hexagonal crystal form, refined to 2.3-A˚ resolution, reveals the free-binding site of the protease. Three-dimensional alignment with the VP4 of blotched snakehead virus, another birnavirus, shows that the overall structure of VP4 is conserved despite a low level of sequence identity (_19%). The structure determinations of IPNV VP4, the first of an acyl-enzyme complex for a Ser/Lys dyad protease, provide insights into the catalytic mechanism and substrate recognition of this type of protease