Enterotoxigenic Escherichia coli (ETEC) is a diarrhoeal pathogen associated with high morbidity and mortality especially among young children in developing countries. At present, there is no vaccine ...for ETEC. One candidate vaccine antigen, EtpA, is a conserved secreted adhesin that binds to the tips of flagellae to bridge ETEC to host intestinal glycans. EtpA is exported through a Gram-negative, two-partner secretion system (TPSS, type Vb) comprised of the secreted EtpA passenger (TpsA) protein and EtpB (TpsB) transporter that is integrated into the outer bacterial membrane. TpsA proteins share a conserved, N-terminal TPS domain followed by an extensive C-terminal domain with divergent sequence repeats. Two soluble, N-terminal constructs of EtpA were prepared and analysed respectively including residues 67 to 447 (EtpA67-447) and 1 to 606 (EtpA1-606). The crystal structure of EtpA67-447 solved at 1.76 Å resolution revealed a right-handed parallel β-helix with two extra-helical hairpins and an N-terminal β-strand cap. Analyses by circular dichroism spectroscopy confirmed the β-helical fold and indicated high resistance to chemical and thermal denaturation as well as rapid refolding. A theoretical AlphaFold model of full-length EtpA largely concurs with the crystal structure adding an extended β-helical C-terminal domain after an interdomain kink. We propose that robust folding of the TPS domain upon secretion provides a template to extend the N-terminal β-helix into the C-terminal domains of TpsA proteins.
The depletion of fossil fuels, associated pollution, and resulting health hazards are of concern worldwide. Woody biomass constitutes an alternative source of cleaner and renewable energy. The ...efficient use of woody biomass depends on xylan depolymerisation as the endo-β-1,4-xylopyranosyl homopolymer is the main component of hemicellulose, the second most abundant component of wood. Xylan depolymerisation is achieved by hemicellulolytic xylanases of glycoside hydrolase (GH) families 5, 8, 10, 11, 30 and 43 of the CAZY database. We analysed a multidomain xylanase (Xyl) from the hindgut metagenome of the snouted harvester termite Trinervitermes trinervoides that releases xylobiose and xylotriose from beech and birch xylan and wheat arabinoxylan. The four domains of Xyl include an N-terminal GH11 xylanase domain, two family 36-like carbohydrate-binding domains CBM36-1 and 2, and a C-terminal CE4 esterase domain. Previous analyses indicated that CBM36-1 deletion slightly increased GH11 catalysis at low pH whereas removal of both CBMs decreased xylanase activity at 60°C from 90 to 56%. Possible cooperativity between the domains suggested by these observations was explored. A crystal structure of the two-domain construct, GH11-CBM36-1, confirmed the structure of the GH11 domain whereas the CBM36-1 domain lacked electron density, possibly indicating a random orientation of the CBM36-1 domain around the GH11 domain. Isothermal titration calorimetry (ITC) experiments similarly did not indicate specific interactions between the individual domains of Xyl supporting a "beads-on-a-string" model for Xyl domains.
Nanoparticles represent promising carriers for controlled drug delivery. This work focuses on the size and molecular mass characterization of polyalkylcyanoacrylate nanoparticles formed by anionic ...emulsion polymerization of butylcyanoacrylate in the presence of poloxamer 188 as a stabilizer. Three different methods were used to determine the size and size distribution of the particle populations: scanning electron microscopy (SEM), dynamic light scattering (DLS), and analytical ultracentrifugation (ANUC). SEM on freeze-dried and Au-shadowed samples showed a relatively narrow distribution of virtually spherical particles with a mean diameter of 167 nm. DLS yielded a monomodal distribution with hydrodynamic diameters around 199 nm (in the absence of additional stabilizer) or 184 nm (in the presence of 1% poloxamer 188). The size distribution determined by ANUC using sedimentation velocity analysis was somewhat more complex, the size of the most abundant particles being around 184 nm. Molar particle mass distributions centered around 2.3×10
9 g/mol. The advantages and disadvantages of the three sizing techniques are discussed.
During (bacterio)chlorophyll biosynthesis of many photosynthetically active organisms, dark operative protochlorophyllide oxidoreductase (DPOR) catalyzes the two-electron reduction of ring D of ...protochlorophyllide to form chlorophyllide. DPOR is composed of the subunits ChlL, ChlN, and ChlB. Homodimeric ChlL2 bearing an intersubunit 4Fe-4S cluster is an ATP-dependent reductase transferring single electrons to the heterotetrameric (ChlN/ChlB)2 complex. The latter contains two intersubunit 4Fe-4S clusters and two protochlorophyllide binding sites, respectively. Here we present the crystal structure of the catalytic (ChlN/ChlB)2 complex of DPOR from the cyanobacterium Thermosynechococcus elongatus at a resolution of 2.4 Å. Subunits ChlN and ChlB exhibit a related architecture of three subdomains each built around a central, parallel β-sheet surrounded by α-helices. The (ChlN/ChlB)2 crystal structure reveals a 4Fe-4S cluster coordinated by an aspartate oxygen alongside three cysteine ligands. Two equivalent substrate binding sites enriched in aromatic residues for protochlorophyllide substrate binding are located at the interface of each ChlN/ChlB half-tetramer. The complete octameric (ChlN/ChlB)2(ChlL2)2 complex of DPOR was modeled based on the crystal structure and earlier functional studies. The electron transfer pathway via the various redox centers of DPOR to the substrate is proposed.
During a bacterial infection, each successive step is orchestrated by a dedicated set of virulence factors. In Gram-positive bacteria, the presentation or release of such factors is crucially ...dependent on the continual remodelling of the cell wall. We have investigated the autolysin or peptidoglycan hydrolase Auto (Lmo1076) from the human pathogen Listeria monocytogenes to structurally and biochemically underpin its role in host cell invasion. We demonstrate that Auto is an N-acetylglucosaminidase, that it is autoinhibited when newly secreted but activated by proteolytic cleavage, that it has an acidic pH optimum and that it preferentially cleaves acetylated over de-acetylated peptidoglycan. The crystal structure of Auto, the first for glycoside hydrolase family 73, and the first for a listerial autolysin, indicates that autoinhibition is due to an N-terminal α-helix unique to Auto that physically blocks the substrate-binding cleft. We identify Glu122 and Glu156 as the two catalytically essential carboxylate groups. The physical properties of Auto as well as its localization to lipoteichoic acid by its four C-terminal GW modules imply cell wall degradation by Auto to be highly co-ordinated. Its spatio-temporally controlled activation and localized activity in an acidified environment indicate that it facilitates remodelling of the cell wall and may be involved in co-ordinating the release of virulence factors at specific stages of an infection.
5‐Aminolevulinate synthase (ALAS) is the first and rate‐limiting enzyme of heme biosynthesis in humans, animals, other non‐plant eukaryotes, and α‐proteobacteria. It catalyzes the synthesis of ...5‐aminolevulinic acid, the first common precursor of all tetrapyrroles, from glycine and succinyl‐coenzyme A (sCoA) in a pyridoxal 5′‐phosphate (PLP)‐dependent manner. X‐linked sideroblastic anemias (XLSAs), a group of severe disorders in humans characterized by inadequate formation of heme in erythroblast mitochondria, are caused by mutations in the gene for erythroid eALAS, one of two human genes for ALAS. We present the first crystal structure of homodimeric ALAS from Rhodobacter capsulatus (ALASRc) binding its cofactor PLP. We, furthermore, present structures of ALASRc in complex with the substrates glycine or sCoA. The sequence identity of ALAS from R. capsulatus and human eALAS is 49%. XLSA‐causing mutations may thus be mapped, revealing the molecular basis of XLSA in humans. Mutations are found to obstruct substrate binding, disrupt the dimer interface, or hamper the correct folding. The structure of ALAS completes the structural analysis of enzymes in heme biosynthesis.
Photosynthesis uses chlorophylls for the conversion of light into chemical energy, the driving force of life on Earth. During chlorophyll biosynthesis in photosynthetic bacteria, cyanobacteria, green ...algae and gymnosperms, dark-operative protochlorophyllide oxidoreductase (DPOR), a nitrogenase-like metalloenzyme, catalyzes the chemically challenging two-electron reduction of the fully conjugated ring system of protochlorophyllide a . The reduction of the C-17=C-18 double bond results in the characteristic ring architecture of all chlorophylls, thereby altering the absorption properties of the molecule and providing the basis for light-capturing and energy-transduction processes of photosynthesis. We report the X-ray crystallographic structure of the substrate-bound, ADP-aluminium fluoride–stabilized (ADP·AlF ₃-stabilized) transition state complex between the DPOR components L ₂ and (NB) ₂ from the marine cyanobacterium Prochlorococcus marinus . Our analysis permits a thorough investigation of the dynamic interplay between L ₂ and (NB) ₂. Upon complex formation, substantial ATP-dependent conformational rearrangements of L ₂ trigger the protein–protein interactions with (NB) ₂ as well as the electron transduction via redox-active 4Fe–4S clusters. We also present the identification of artificial “small-molecule substrates” of DPOR in correlation with those of nitrogenase. The catalytic differences and similarities between DPOR and nitrogenase have broad implications for the energy transduction mechanism of related multiprotein complexes that are involved in the reduction of chemically stable double and/or triple bonds.
In causing disease, pathogens outmaneuver host defenses through a dedicated arsenal of virulence determinants that specifically bind or modify individual host molecules. This dedication limits the ...intruder to a defined range of hosts. Newly emerging diseases mostly involve existing pathogens whose arsenal has been altered to allow them to infect previously inaccessible hosts. We have emulated this chance occurrence by extending the host range accessible to the human pathogen Listeria monocytogenes by the intestinal route to include the mouse. Analyzing the recognition complex of the listerial invasion protein InlA and its human receptor E-cadherin, we postulated and verified amino acid substitutions in InlA to increase its affinity for E-cadherin. Two single substitutions increase binding affinity by four orders of magnitude and extend binding specificity to include formerly incompatible murine E-cadherin. By rationally adapting a single protein, we thus create a versatile murine model of human listeriosis.
gene encodes unconventional myosin VIIA, which, when mutated, causes a phenotypic spectrum ranging from recessive hearing loss DFNB2 to deaf-blindness, Usher Type 1B (USH1B).
mutations are reported ...in nine DFNB2 families to date, none from sub-Saharan Africa.In DNA, from a cohort of 94 individuals representing 92 families from the Limpopo province of South Africa, eight
variations were detected among 10 individuals. Family studies identified homozygous and compound heterozygous mutations in 17 individuals out of 32 available family members. Four mutations were novel, p.Gly329Asp, p.Arg373His, p.Tyr1780Ser, and p.Pro2126Leufs*5. Two variations, p.Ser617Pro and p.Thr381Met, previously listed as of uncertain significance (ClinVar), were confirmed to be pathogenic. The identified mutations are predicted to interfere with the conformational properties of myosin VIIA through interruption or abrogation of multiple interactions between the mutant and neighbouring residues. Specifically, p.Pro2126Leufs*5, is predicted to abolish the critical site for the interactions between the tail and the motor domain essential for the autoregulation, leaving a non-functional, unregulated protein that causes hearing loss. We have identified
as a possible key deafness gene among indigenous sub-Saharan Africans. The spectrum of
mutations in this South African population points to DFNB2 as a specific entity that may occur in a homozygous or in a compound heterozygous state.
Three's company: Rhizopodin is a cytostatic macrolide and a potent actin depolymerizer produced by the myxobacterium Myxococcus stipitatus. A crystal structure analysis of the rhizopodin/actin ...complex reveals that rhizopodin is a C2‐symmetric bislactone (see formula). The ternary complex supports the mode of rhizopodin‐induced actin dimerization and reveals the absolute configuration and biologically active conformation of this macrolide.
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