Solute channels of the outer membrane: from bacteria to chloroplasts

Chloroplasts, unique organelles of plants, originated from endosymbiosis of an ancestor of today's cyanobacteria with a mitochondria-containing host cell. It is assumed that the outer envelope membrane, which delimits the chloroplast from the surrounding cytosol, was thus inherited from its Gram-negative bacterial ancestor. This plastid-specific membrane is thus equipped with elements of prokaryotic and eukaryotic origin. In particular, the membrane-intrinsic outer envelope proteins (OEPs) form solute channels with properties reminiscent of porins and channels in the bacterial outer membrane. OEP channels are characterised by distinct specificities for metabolites and a quite peculiar expression pattern in specialised plant organs and plastids, thus disproving the assumption that the outer envelope is a non-specific molecular sieve. The same is true for the outer membrane of Gram-negative bacteria, which functions as a permeability barrier in addition to the cytoplasmic membrane, and embeds different classes of channel pores. The channels of these prokaryotic prototype proteins, ranging from unspecific porins to specific channels to ligand-gated receptors, are exclusively built of P-barrels. Although most of the OEP channels are formed by P-strands as well, phylogeny based on sequence homology alone is not feasible. Thus, the comparison of structural and functional properties of chloroplast outer envelope and bacterial outer membrane channels is required to pinpoint the ancestral OEP `portrait gallery'

Ferredoxin containing bacteriocins suggest a novel mechanism of iron uptake in <i>Pectobacterium spp</i>

In order to kill competing strains of the same or closely related bacterial species, many bacteria produce potent narrow-spectrum protein antibiotics known as bacteriocins. Two sequenced strains of the phytopathogenic bacterium &lt;i&gt;Pectobacterium carotovorum&lt;/i&gt; carry genes encoding putative bacteriocins which have seemingly evolved through a recombination event to encode proteins containing an N-terminal domain with extensive similarity to a [2Fe-2S] plant ferredoxin and a C-terminal colicin M-like catalytic domain. In this work, we show that these genes encode active bacteriocins, pectocin M1 and M2, which target strains of &lt;i&gt;Pectobacterium carotovorum&lt;/i&gt; and &lt;i&gt;Pectobacterium atrosepticum&lt;/i&gt; with increased potency under iron limiting conditions. The activity of pectocin M1 and M2 can be inhibited by the addition of spinach ferredoxin, indicating that the ferredoxin domain of these proteins acts as a receptor binding domain. This effect is not observed with the mammalian ferredoxin protein adrenodoxin, indicating that &lt;i&gt;Pectobacterium spp.&lt;/i&gt; carries a specific receptor for plant ferredoxins and that these plant pathogens may acquire iron from the host through the uptake of ferredoxin. In further support of this hypothesis we show that the growth of strains of &lt;i&gt;Pectobacterium carotovorum&lt;/i&gt; and &lt;i&gt;atrosepticum&lt;/i&gt; that are not sensitive to the cytotoxic effects of pectocin M1 is enhanced in the presence of pectocin M1 and M2 under iron limiting conditions. A similar growth enhancement under iron limiting conditions is observed with spinach ferrodoxin, but not with adrenodoxin. Our data indicate that pectocin M1 and M2 have evolved to parasitise an existing iron uptake pathway by using a ferredoxin-containing receptor binding domain as a Trojan horse to gain entry into susceptible cells

Molecular adaptation of a plant-bacterium outer membrane protease towards plague virulence factor Pla

<p>Abstract</p> <p>Background</p> <p>Omptins are a family of outer membrane proteases that have spread by horizontal gene transfer in Gram-negative bacteria that infect vertebrates or plants. Despite structural similarity, the molecular functions of omptins differ in a manner that reflects the life style of their host bacteria. To simulate the molecular adaptation of omptins, we applied site-specific mutagenesis to make Epo of the plant pathogenic <it>Erwinia pyrifoliae </it>exhibit virulence-associated functions of its close homolog, the plasminogen activator Pla of <it>Yersinia pestis</it>. We addressed three virulence-associated functions exhibited by Pla, i.e., proteolytic activation of plasminogen, proteolytic degradation of serine protease inhibitors, and invasion into human cells.</p> <p>Results</p> <p>Pla and Epo expressed in <it>Escherichia coli </it>are both functional endopeptidases and cleave human serine protease inhibitors, but Epo failed to activate plasminogen and to mediate invasion into a human endothelial-like cell line. Swapping of ten amino acid residues at two surface loops of Pla and Epo introduced plasminogen activation capacity in Epo and inactivated the function in Pla. We also compared the structure of Pla and the modeled structure of Epo to analyze the structural variations that could rationalize the different proteolytic activities. Epo-expressing bacteria managed to invade human cells only after all extramembranous residues that differ between Pla and Epo and the first transmembrane β-strand had been changed.</p> <p>Conclusions</p> <p>We describe molecular adaptation of a protease from an environmental setting towards a virulence factor detrimental for humans. Our results stress the evolvability of bacterial β-barrel surface structures and the environment as a source of progenitor virulence molecules of human pathogens.</p

Structure of human RNA polymerase III

In eukaryotes, RNA Polymerase (Pol) III is specialized for the transcription of tRNAs and other short, untranslated RNAs. Pol III is a determinant of cellular growth and lifespan across eukaryotes. Upregulation of Pol III transcription is observed in cancer and causative Pol III mutations have been described in neurodevelopmental disorders and hypersensitivity to viral infection. Here, we report a cryo-EM reconstruction at 4.0 Å of human Pol III, allowing mapping and rationalization of reported genetic mutations. Mutations causing neurodevelopmental defects cluster in hotspots affecting Pol III stability and/or biogenesis, whereas mutations affecting viral sensing are located in proximity to DNA binding regions, suggesting an impairment of Pol III cytosolic viral DNA-sensing. Integrating x-ray crystallography and SAXS, we also describe the structure of the higher eukaryote specific RPC5 C-terminal extension. Surprisingly, experiments in living cells highlight a role for this module in the assembly and stability of human Pol III

Enantioselective cyclopropanation of heterocycles and the use of high-pressure techniques for the conformational analysis of peptide foldamers

Cyclopropanated heterocycles have been shown to be invaluable building blocks for the synthesis of a broad variety of natural products and other biologically relevant compounds. The first part of the present thesis describes the enantioselective cyclopropanation of furan and pyrrole derivatives, as well as their further transformations of the derived compounds. Furan-3-carboxylic acid methyl ester 91 was used as starting material for the total synthesis of (-)-Paeonilide 27, however, only in 83% ee, as enantiopure material was not accessible at that time. In the course of this project an enantioselective cyclopropanation reaction of 91 was developed, which leads to cyclopropane 15 in > 99% ee and multi gram quantity. Key features for the success were the application of azabox ligand 8 as the chiral ligand and the modification of the ratio of catalyst to ligand from 1:1 to 1:2. On the other hand, cyclopropanation products of N-Boc-pyrrole 101, which already have been applied for various synthetic endeavors, could not be accessed by enantioselective cyclopropanation so far. In the present work, a strategy for the enantioselective cyclopropanation of 101 with the use of tert butyl diazoacetate as carbene source and again azabox ligand 8 was developed. Cyclopropane 103 can be obtained in > 99% ee and multi gram quantity, along with doubly cyclopropanated 104 (max. 41% ee). The application of tert butyl diazoacetate and the ligand 8 turned out to be crucial for achieving high enantiomeric excess of the product. In the course of this project tert butyl substituted azabox ligand 8 was identified to be superior to a great variety of other ligands for the asymmetric cylopropanation of both 91 and 101. Cyclopropane 103 was applied for a short and efficient synthesis of the biologically active GABA analogue (S)-(+)-homo-b-proline 109. Starting from N-Boc-pyrrole 101, (S)-(+)-109 could be prepared in three steps and 37% yield. However, the occurrence of epimerzation during the acid catalyzed cyclopropane ring opening step prohibited access to enantiomerically pure 109. In the second part of the present thesis the structural and conformational behavior of a/b-peptide foldamers were investigated by the application of high pressure NMR (up to 2 kbar) and FTIR (up to 6.5 kbar) spectroscopic methods. Short tripeptides 217a and 217b containing a central cyclopropane amino acid, were analyzed in terms of their catalytic performance related to the application of pressure. The fact that 217a acts as a highly efficient organocatalyst for intermolecular aldol reactions, while its enantiomer 217b gives poor results, led us to the investigation of this feature. A plausible model for explaining the discrepancy of catalytic performance of 217a and 217b was proposed. While 217b shows clear indications for a strong internal hydrogen bond that separates the two catalytic functional groups, 217b shows a significantly higher degree for flexibility that allows it for reaching an optimal conformation for catalysis. Experiments under high pressure conditions revealed a sixfold increase in reaction rate upon a pressure jump from 1 bar to 5 kbar, but this was rather assigned to the general effect of pressure than to a change of the equilibrium of conformations of 217a. In addition to short tripeptides 217a and 217b, the 13 residue a/b-peptide foldamer 226 was examined under variable pressure and temperature. Conformational studies by NMR, FTIR and CD spectroscopy revealed a partial unfolding of the helical structure of 226 at elevated pressures and temperatures, which was shown to be reversible

Enantioselective Three-Step Synthesis of Homo-β-proline: A Donor–Acceptor Cyclopropane as Key Intermediate

An enantioselective three-step synthesis of the GABA uptake inhibitor (S)-(+)-homo-beta-proline was developed. The basis for the synthesis was the enantioselective Cu-I-catalyzed cyclopropanation of N-Boc-pyrrole, a substrate that persistently has proved to be challenging in such transformations. The cyclopropanation can be performed on a 150 mmol scale, and the two subsequent steps (i.e., hydrogenation and in situ cyclopropane-opening/double-deprotection) toward the target molecule proceed smoothly in quantitative yield without loss of enantiopurity

Identification of Specific Residues in Colicin E1 Involved in Immunity Protein Recognition

The basis of specificity between pore-forming colicins and immunity proteins was explored by interchanging residues between colicins E1 (ColE1) and 10 (Col10) and testing for altered recognition by their respective immunity proteins, Imm and Cti. A total of 34 divergent residues in the pore-forming domain of ColE1 between residues 419 and 501, a region previously shown to contain the specificity determinants for Imm, were mutagenized to the corresponding Col10 sequences. The residue changes most effective in converting ColE1 to the Col10 phenotype are residue 448 at the N terminus of helix VI and residues 470, 472, and 474 at the C terminus of helix VII. Mutagenesis of helix VI residues 416 to 419 in Col10 to the corresponding ColE1 sequence resulted in increased recognition by Imm and loss of recognition by Cti

Sternal plate closure: indications, surgical procedure and follow-up

Objectives: Titanium plate osteosynthesis (Synthes) is an alternative option for sternal closure. The indications and time point of application are still debated. This study investigated the application and feasibility of this technique after median sternotomy. Methods: Forty-one patients (29 M/12F, mean age 63 ± 17 years) received the plate system for complicated sternal conditions. Indications, intraoperative course and postoperative follow-up were assessed. Results: Sternal deformity was present in 5 % (2/41), sternal fractures in 17 % (7/41), bone defect in 12 % (5/41), wire loosening in 39 % (16/41) and pseudoarthrosis in 27 % (11/41). 54 % (22/41) of patients showed concomitant sternal infection. Two intraoperative complications were noted: mammary artery injury (1 patient), pleural injury (1 patient). At discharge the patients reported no pain (90 %, 37/41) or only occasional discomfort (10 %, 4/41). Postoperative complications were subcutaneous hematoma in 12 % (5/41), seroma in 12 % (5/41) and sternal reinfection in 7 % (3/41). 12 % (5/41) showed occasional discomfort and 7 % (3/41) had persistent pain leading to plate removal. Conclusion: The Titanium Sternal Fixation System is comfortable and easy to use. It can be used to treat a wide spectrum of indications, especially for pseudoarthrosis, an entity which has not yet received sufficient attention