Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour

SmSP2: A serine protease secreted by the blood fluke pathogen Schistosoma mansoni with anti-hemostatic properties.

BackgroundSerine proteases are important virulence factors for many pathogens. Recently, we discovered a group of trypsin-like serine proteases with domain organization unique to flatworm parasites and containing a thrombospondin type 1 repeat (TSR-1). These proteases are recognized as antigens during host infection and may prove useful as anthelminthic vaccines, however their molecular characteristics are under-studied. Here, we characterize the structural and proteolytic attributes of serine protease 2 (SmSP2) from Schistosoma mansoni, one of the major species responsible for the tropical infectious disease, schistosomiasis.Methodology/principal findingsSmSP2 comprises three domains: a histidine stretch, TSR-1 and a serine protease domain. The cleavage specificity of recombinant SmSP2 was determined using positional scanning and multiplex combinatorial libraries and the determinants of specificity were identified with 3D homology models, demonstrating a trypsin-like endopeptidase mode of action. SmSP2 displayed restricted proteolysis on protein substrates. It activated tissue plasminogen activator and plasminogen as key components of the fibrinolytic system, and released the vasoregulatory peptide, kinin, from kininogen. SmSP2 was detected in the surface tegument, esophageal glands and reproductive organs of the adult parasite by immunofluorescence microscopy, and in the excretory/secretory products by immunoblotting.Conclusions/significanceThe data suggest that SmSP2 is secreted, functions at the host-parasite interface and contributes to the survival of the parasite by manipulating host vasodilatation and fibrinolysis. SmSP2 may be, therefore, a potential target for anti-schistosomal therapy

Profiling of proteolytic enzymes in the gut of the tick Ixodes ricinus reveals an evolutionarily conserved network of aspartic and cysteine peptidases

<p>Abstract</p> <p>Background</p> <p>Ticks are vectors for a variety of viral, bacterial and parasitic diseases in human and domestic animals. To survive and reproduce ticks feed on host blood, yet our understanding of the intestinal proteolytic machinery used to derive absorbable nutrients from the blood meal is poor. Intestinal digestive processes are limiting factors for pathogen transmission since the tick gut presents the primary site of infection. Moreover, digestive enzymes may find practical application as anti-tick vaccine targets.</p> <p>Results</p> <p>Using the hard tick, <it>Ixodes ricinus</it>, we performed a functional activity scan of the peptidase complement in gut tissue extracts that demonstrated the presence of five types of peptidases of the cysteine and aspartic classes. We followed up with genetic screens of gut-derived cDNA to identify and clone genes encoding the cysteine peptidases cathepsins B, L and C, an asparaginyl endopeptidase (legumain), and the aspartic peptidase, cathepsin D. By RT-PCR, expression of asparaginyl endopeptidase and cathepsins B and D was restricted to gut tissue and to those developmental stages feeding on blood.</p> <p>Conclusion</p> <p>Overall, our results demonstrate the presence of a network of cysteine and aspartic peptidases that conceivably operates to digest host blood proteins in a concerted manner. Significantly, the peptidase components of this digestive network are orthologous to those described in other parasites, including nematodes and flatworms. Accordingly, the present data and those available for other tick species support the notion of an evolutionary conservation of a cysteine/aspartic peptidase system for digestion that includes ticks, but differs from that of insects relying on serine peptidases.</p

MemProtMD: Automated Insertion of Membrane Protein Structures into Explicit Lipid Membranes

SummaryThere has been exponential growth in the number of membrane protein structures determined. Nevertheless, these structures are usually resolved in the absence of their lipid environment. Coarse-grained molecular dynamics (CGMD) simulations enable insertion of membrane proteins into explicit models of lipid bilayers. We have automated the CGMD methodology, enabling membrane protein structures to be identified upon their release into the PDB and embedded into a membrane. The simulations are analyzed for protein-lipid interactions, identifying lipid binding sites, and revealing local bilayer deformations plus molecular access pathways within the membrane. The coarse-grained models of membrane protein/bilayer complexes are transformed to atomistic resolution for further analysis and simulation. Using this automated simulation pipeline, we have analyzed a number of recently determined membrane protein structures to predict their locations within a membrane, their lipid/protein interactions, and the functional implications of an enhanced understanding of the local membrane environment of each protein

Clinical and genetic risk factors for biofilm-forming Staphylococcus aureus

The molecular and clinical factors associated with biofilm-forming methicillin-resistant Staphylococcus aureus (MRSA) are incompletely understood. Biofilm production for 182 MRSA isolates obtained from clinical culture sites (2004 to 2013) was quantified. Microbiological toxins, pigmentation, and genotypes were evaluated, and patient demographics were collected. Logistic regression was used to quantify the effect of strong biofilm production (versus weak biofilm production) on clinical outcomes and independent predictors of a strong biofilm. Of the isolates evaluated, 25.8% (47/182) produced strong biofilms and 40.7% (74/182) produced weak biofilms. Strong biofilm-producing isolates were more likely to be from multilocus sequence typing (MLST) clonal complex 8 (CC8) (34.0% versus 14.9%; P = 0.01) but less likely to be from MLST CC5 (48.9% versus 73.0%; P = 0.007). Predictors for strong biofilms were spa type t008 (adjusted odds ratio [aOR], 4.54; 95% confidence interval [CI], 1.21 to 17.1) and receipt of chemotherapy or immunosuppressants in the previous 90 days (aOR, 33.6; 95% CI, 1.68 to 673). Conversely, patients with high serum creatinine concentrations (aOR, 0.33; 95% CI, 0.15 to 0.72) or who previously received vancomycin (aOR, 0.03; 95% CI, 0.002 to 0.39) were less likely to harbor strong biofilm-producing MRSA. Beta-toxin-producing isolates (aOR, 0.31; 95% CI, 0.11 to 0.89) and isolates with spa type t895 (aOR, 0.02 95% CI

Structural insights into the mechanism of the membrane integral N-acyltransferase step in bacterial lipoprotein synthesis

Lipoproteins serve essential roles in the bacterial cell envelope. The posttranslational modification pathway leading to lipoprotein synthesis involves three enzymes. All are potential targets for the development of new antibiotics. Here we report the crystal structure of the last enzyme in the pathway, apolipoprotein N-acyltransferase, Lnt, responsible for adding a third acyl chain to the lipoprotein’s invariant diacylated N-terminal cysteine. Structures of Lnt from Pseudomonas aeruginosa and Escherichia coli have been solved; they are remarkably similar. Both consist of a membrane domain on which sits a globular periplasmic domain. The active site resides above the membrane interface where the domains meet facing into the periplasm. The structures are consistent with the proposed ping-pong reaction mechanism and suggest plausible routes by which substrates and products enter and leave the active site. While Lnt may present challenges for antibiotic development, the structures described should facilitate design of therapeutics with reduced off-target effects

Crystal structure of undecaprenyl-pyrophosphate phosphatase and its role in peptidoglycan biosynthesis

As a protective envelope surrounding the bacterial cell, the peptidoglycan sacculus is a site of vulnerability and an antibiotic target. Peptidoglycan components, assembled in the cytoplasm, are shuttled across the membrane in a cycle that uses undecaprenyl-phosphate. A product of peptidoglycan synthesis, undecaprenyl-pyrophosphate, is converted to undecaprenyl-phosphate for reuse in the cycle by the membrane integral pyrophosphatase, BacA. To understand how BacA functions, we determine its crystal structure at 2.6 Å resolution. The enzyme is open to the periplasm and to the periplasmic leaflet via a pocket that extends into the membrane. Conserved residues map to the pocket where pyrophosphorolysis occurs. BacA incorporates an interdigitated inverted topology repeat, a topology type thus far only reported in transporters and channels. This unique topology raises issues regarding the ancestry of BacA, the possibility that BacA has alternate active sites on either side of the membrane and its possible function as a flippase

Hybridisation between two cyprinid fishes in a novel habitat: genetics, morphology and life-history traits

BACKGROUND: The potential role hybridisation in adaptive radiation and the evolution of new lineages has received much recent attention. Hybridisation between roach (Rutilus rutilus L.) and bream (Abramis brama L.) is well documented throughout Europe, however hybrids in Ireland occur at an unprecedented frequency, often exceeding that of both parental species. Utilising an integrated approach, which incorporates geometric morphometrics, life history and molecular genetic analyses we identify the levels and processes of hybridisation present, while also determining the direction of hybridisation, through the analysis of mitochondrial DNA. RESULTS: The presence of F2 hybrids was found to be unlikely from the studied populations, although significant levels of backcrossing, involving both parental taxa was observed in some lakes. Hybridisation represents a viable conduit for introgression of genes between roach and bream. The vast majority of hybrids in all populations studied exhibited bream mitochondrial DNA, indicating that bream are maternal in the majority of crosses. CONCLUSIONS: The success of roach × bream hybrids in Ireland is not due to a successful self reproducing lineage. The potential causes of widespread hybridisation between both species, along with the considerations regarding the role of hybridisation in evolution and conservation, are also discussed

Crystallizing membrane proteins using lipidic mesophases

peer-reviewedThis paper was obtained through PEER (Publishing and the Ecology of European Research) http://www.peerproject.euA detailed protocol for crystallizing membrane proteins that makes use of lipidic mesophases is described. This has variously been referred to as the lipid cubic phase or in meso method. The method has been shown to be quite general in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and α-helical and β-barrel proteins. Its most recent successes are the human engineered β2-adrenergic and adenosine A2A G protein-coupled receptors. Protocols are provided for preparing and characterizing the lipidic mesophase, for reconstituting the protein into the monoolein-based mesophase, for functional assay of the protein in the mesophase, and for setting up crystallizations in manual mode. Methods for harvesting micro-crystals are also described. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour