Directed epitope delivery across the Escherichia coli outer membrane through the porin OmpF.

The porins OmpF and OmpC are trimeric ß-barrel proteins with narrow channels running through each monomer that exclude molecules > 600 Da while mediating the passive diffusion of small nutrients and metabolites across the Gram-negative outer membrane (OM). Here, we elucidate the mechanism by which an entire soluble protein domain (> 6 kDa) is delivered through the lumen of such porins. Following high-affinity binding to the vitamin B(12) receptor in Escherichia coli, the bacteriocin ColE9 recruits OmpF or OmpC using an 83-residue intrinsically unstructured translocation domain (IUTD) to deliver a 16-residue TolB-binding epitope (TBE) in the center of the IUTD to the periplasm where it triggers toxin entry. We demonstrate that the IUTD houses two OmpF-binding sites, OBS1 (residues 2-18) and OBS2 (residues 54-63), which flank the TBE and bind with K(d)s of 2 and 24 µM, respectively, at pH 6.5 and 25 ºC. We show the two OBSs share the same binding site on OmpF and that the colicin must house at least one of them for antibiotic activity. Finally, we report the structure of the OmpF-OBS1 complex that shows the colicin bound within the porin lumen spanning the membrane bilayer. Our study explains how colicins exploit porins to deliver epitope signals to the bacterial periplasm and, more broadly, how the inherent flexibility and narrow cross-sectional area of an IUP domain can endow it with the ability to traverse a biological membrane via the constricted lumen of a ß-barrel membrane protein.

Non-equivalent role of inter- and intramolecular hydrogen bonds in the insulin dimer interface.

Apart from its role in insulin receptor (IR) activation, the C terminus of the B-chain of insulin is also responsible for the formation of insulin dimers. The dimerization of insulin plays an important role in the endogenous delivery of the hormone and in the administration of insulin to patients. Here, we investigated insulin analogues with selective N-methylations of peptide bond amides at positions B24, B25, or B26 to delineate their structural and functional contribution to the dimer interface. All N-methylated analogues showed impaired binding affinities to IR, which suggests a direct IR-interacting role for the respective amide hydrogens. The dimerization capabilities of analogues were investigated by isothermal microcalorimetry. Selective N-methylations of B24, B25, or B26 amides resulted in reduced dimerization abilities compared with native insulin (K(d) = 8.8 µM). Interestingly, although the N-methylation in [NMeTyrB26]-insulin or [NMePheB24]-insulin resulted in K(d) values of 142 and 587 µM, respectively, the [NMePheB25]-insulin did not form dimers even at high concentrations. This effect may be attributed to the loss of intramolecular hydrogen bonding between NHB25 and COA19, which connects the B-chain ß-strand to the core of the molecule. The release of the B-chain ß-strand from this hydrogen bond lock may result in its higher mobility, thereby shifting solution equilibrium toward the monomeric state of the hormone. The study was complemented by analyses of two novel analogue crystal structures. All examined analogues crystallized only in the most stable R(6) form of insulin oligomers (even if the dimer interface was totally disrupted), confirming the role of R(6)-specific intra/intermolecular interactions for hexamer stability.

Psychological correlates of affect in parents of children with cleft lip and/or palate.

OBJECTIVE: Parents of children with developmental malformations of different kinds are vulnerable to many consequences of the experienced stress and attempts to cope with it. The aim of the study was to determine the psychological correlates of affect for parents of such children. PATIENTS AND METHODS: The study included 78 respondents: 69 women and 9 men, aged between 20 and 45, all of them parents of children with craniofacial malformations who had their routine check-ups at an orthodontics clinic. The respondents were evaluated using pencil-and-paper questionnaires, the same survey set for all respondents. The following tools were used in the study: the Inventory for Measuring Coping with Stress (Mini-COPE), the Family Resilience Assessment Scale (FRAS), and the Positive and Negative Affect Schedule (PANAS). The guardians' demographic data and the details of the child's medical history were gathered using a questionnaire constructed for the purposes of the study. RESULTS: The present study confirmed significant correlations between affect and preferred stress coping strategies, as well as between affect and family resilience. Coping strategies and family resilience, treated as a resource, were also significantly correlated in the group of respondents. CONCLUSIONS: Mental resilience is an important resource contributing to effective stress coping in a situation where a child suffers from malformation.

Submandibular gland obstruction caused by an implant-retained lower denture: a report of three cases.

The aim of this study was to investigate the role of lower dentures in the development of distally located stenoses of Wharton's duct and to further identify contributing factors to this mechanism. In a database of 352 patients with submandibular gland obstruction, three patients with four obstructed glands with stenosis of the ostium of Wharton's duct suspected to be caused by a lower denture were identified and further retrospectively analysed by studying medical records, operation reports, and clinical photographs. In all three cases, the causative lower dental prosthesis was implant-retained. All affected sublingual caruncles were in close relationship with the implants or the implant bar. Initially, all patients were advised to leave out the lower denture for a period of several weeks. One patient was free of symptoms after this period and did not develop any complaints after adjustment and replacement of the prosthesis. Surgical treatment with posterior rerouting of the orificium of Wharton's duct was performed in the remaining two patients because of persistent signs of obstruction. All patients were free of symptoms after long-term follow-up. Although not frequently occurring, implant-retained dental prostheses seem to play a role in the development of some distally located stenoses of Wharton's duct.

A ternary complex of hydroxycinnamoyl-CoA hydratase-lyase (HCHL) with acetyl-CoA and vanillin gives insights into substrate specificity and mechanism.

HCHL (hydroxycinnamoyl-CoA hydratase-lyase) catalyses the biotransformation of feruloyl-CoA to acetyl-CoA and the important flavour-fragrance compound vanillin (4-hydroxy-3-methoxybenzaldehyde) and is exploited in whole-cell systems for the bioconversion of ferulic acid into natural equivalent vanillin. The reaction catalysed by HCHL has been thought to proceed by a two-step process involving first the hydration of the double bond of feruloyl-CoA and then the cleavage of the resultant beta-hydroxy thioester by retro-aldol reaction to yield the products. Kinetic analysis of active-site residues identified using the crystal structure of HCHL revealed that while Glu-143 was essential for activity, Ser-123 played no major role in catalysis. However, mutation of Tyr-239 to Phe greatly increased the K(M) for the substrate ferulic acid, fulfilling its anticipated role as a factor in substrate binding. Structures of WT (wild-type) HCHL and of the S123A mutant, each of which had been co-crystallized with feruloyl-CoA, reveal a subtle helix movement upon ligand binding, the consequence of which is to bring the phenolic hydroxyl of Tyr-239 into close proximity to Tyr-75 from a neighbouring subunit in order to bind the phenolic hydroxyl of the product vanillin, for which electron density was observed. The active-site residues of ligand-bound HCHL display a remarkable three-dimensional overlap with those of a structurally unrelated enzyme, vanillyl alcohol oxidase, that also recognizes p-hydroxylated aromatic substrates related to vanillin. The data both explain the observed substrate specificity of HCHL for p-hydroxylated cinnamate derivatives and illustrate a remarkable convergence of the molecular determinants of ligand recognition between the two otherwise unrelated enzymes.

Site-selective C-C modification of proteins at neutral pH using organocatalyst-mediated cross aldol ligations.

The bioconjugation of proteins with small molecules has proved an invaluable strategy for probing and perturbing biological mechanisms. The general use of chemical methods for protein functionalisation can be limited however by the requirement for complicated reaction partners to be present in large excess, and harsh conditions which are incompatible with many protein scaffolds. Herein we describe a site-selective organocatalyst-mediated protein aldol ligation (OPAL) that affords stable carbon-carbon linked bioconjugates at neutral pH. OPAL enables rapid modification of proteins using simple aldehyde probes in minimal excess, and is utilised here in the affinity tagging of proteins in cell lysate. Furthermore we demonstrate that the ß-hydroxy aldehyde OPAL product can be functionalised again at neutral pH in a tandem organocatalyst-mediated oxime ligation. This tandem strategy is showcased in the 'chemical mimicry' of a previously inaccessible natural dual post-translationally modified protein integral to the pathogenesis of the neglected tropical disease Leishmaniasis.

Structure of the ThDP-dependent enzyme benzaldehyde lyase refined to 1.65 A resolution.

Benzaldehyde lyase (BAL; EC 4.1.2.38) is a thiamine diphosphate (ThDP) dependent enzyme that catalyses the enantioselective carboligation of two molecules of benzaldehyde to form (R)-benzoin. BAL has hence aroused interest for its potential in the industrial synthesis of optically active benzoins and derivatives. The structure of BAL was previously solved to a resolution of 2.6 A using MAD experiments on a selenomethionine derivative [Mosbacher et al. (2005), FEBS J. 272, 6067-6076]. In this communication of parallel studies, BAL was crystallized in an alternative space group (P2(1)2(1)2(1)) and its structure refined to a resolution of 1.65 A, allowing detailed observation of the water structure, active-site interactions with ThDP and also the electron density for the co-solvent 2-methyl-2,4-pentanediol (MPD) at hydrophobic patches of the enzyme surface.

Structural insights into the mode of action of a pure antiestrogen.

BACKGROUND: Estrogens exert their effects on target tissues by binding to a nuclear transcription factor termed the estrogen receptor (ER). Previous structural studies have demonstrated that each class of ER ligand (agonist, partial agonist, and SERM antagonist) induces distinctive orientations in the receptor's carboxy-terminal transactivation helix. The conformation of this portion of the receptor determines whether ER can recruit and interact with the components of the transcriptional machinery, thereby facilitating target gene expression. RESULTS: We have determined the structure of rat ERbeta ligand binding domain (LBD) in complex with the pure antiestrogen ICI 164,384 at 2.3 A resolution. The binding of this compound to the receptor completely abolishes the association between the transactivation helix (H12) and the rest of the LBD. The structure reveals that the terminal portion of ICI's bulky side chain substituent protrudes from the hormone binding pocket, binds along the coactivator recruitment site, and physically prevents H12 from adopting either its characteristic agonist or AF2 antagonist orientation. CONCLUSIONS: The binding mode adopted by the pure antiestrogen is similar to that seen for other ER antagonists. However, the size and resultant positioning of the ligand's side chain substituent produces a receptor conformation that is distinct from that adopted in the presence of other classes of ER ligands. The novel observation that binding of ICI results in the complete destabilization of H12 provides some indications as to a possible mechanism for pure receptor antagonism.

11-fold symmetry of the trp RNA-binding attenuation protein (TRAP) from Bacillus subtilis determined by X-ray analysis.

The trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis has been crystallized and examined by crystallography using X-ray synchrotron radiation diffraction data. Crystals of TRAP complexed with L-tryptophan belong to space group C2 with a = 156.8 A, b = 114.05 A, c = 105.9 A, beta = 118.2 degrees. Crystals of a potential heavy-atom derivative of TRAP complexed with 5-bromo-L-tryptophan grow in the same space group with similar cell dimensions. X-ray data for the native crystals and for the derivative have been collected to 2.9 A and 2.2 A resolution, respectively. Peaks in the self-rotation function and in the Patterson synthesis could only be explained by two 11-subunit oligomers (each formed by an 11-fold axis of symmetry) in the asymmetric unit lying with the 11-fold rotation axes parallel to each other. The consequence is that the TRAP molecule has 11-fold symmetry and contains 11 subunits.

Anatomy of glycosynthesis: structure and kinetics of the Humicola insolens Cel7B E197A and E197S glycosynthase mutants.

The formation of glycoconjugates and oligosaccharides remains one of the most challenging chemical syntheses. Chemo-enzymatic routes using retaining glycosidases have been successfully harnessed but require tight kinetic or thermodynamic control. "Glycosynthases," specifically engineered glycosidases that catalyze the formation of glycosidic bonds from glycosyl donor and acceptor alcohol, are an emerging range of synthetic tools in which catalytic nucleophile mutants are harnessed together with glycosyl fluoride donors to generate powerful and versatile catalysts. Here we present the structural and kinetic dissection of the Humicola insolens Cel7B glycosynthases in which the nucleophile of the wild-type enzyme is mutated to alanine and serine (E197A and E197S). 3-D structures reveal the acceptor and donor subsites and the basis for substrate inhibition. Kinetic analysis shows that the E197S mutant is considerably more active than the corresponding alanine mutant due to a 40-fold increase in k(cat).

Structural insights into the mechanisms of agonism and antagonism in oestrogen receptor isoforms.

Here we summarise the results that have emerged from our structural studies on the oestrogen receptor (ER) ligand-binding domain. We have investigated the conformational effects of a variety of ligands on the structures of both ER isoforms. Each class of ligand (agonists, partial agonists and selective oestrogen receptor modulators) induces a unique conformation in the receptor's ligand-dependent transcriptional activation function. Together these studies have broadened our understanding of ER function by providing a unique insight into ER's ligand specificity and the structural changes that underlie receptor agonism and antagonism.

A structural biologist's view of the oestrogen receptor.

Here we review the results that have emerged from our structural studies on the oestrogen receptor ligand-binding domain (ER-LBD). The effects of agonists and antagonists on the structure of ERalpha- and ERbeta-LBDs are examined. In addition, the findings from structural studies of ER-LBD in complex with peptide fragments corresponding to the NR-box II and III modules of the p160 coactivator TIF2 are discussed in the context of the assembly of ER:coactivator complexes. Together these studies have broadened our understanding of ER function by providing a unique insight into ER's ligand specificity, it's ability to interact with coactivators and the structural changes that underlie receptor agonism and antagonism.

Transient expression and stable transformation of soybean using the jellyfish green fluorescent protein.

Embryogenic soybean [Glycine max (L.) Merrill.] suspension cultures were bombarded with five different gene constructions encoding the jellyfish (Aequorea victoria) green fluorescent protein (GFP). These constructions had altered codon usage compared to the native GFP gene and mutations that increased the solubility of the protein and/or altered the native chromophore. All of the constructions produced green fluorescence in soybean cultures upon blue light excitation, although a soluble modified red-shifted GFP (smRS-GFP) was the easiest to detect based on the brightness and number of foci produced. Expression of smRS-GFP was visible as early as 1.5 h after bombardment, with peak expression at approximately 6.5 h. Large numbers of smRS-GFP-expressing areas were visible for 48 h postbombardment and declined rapidly thereafter. Stably transformed cultures and plants exhibited variation in the intensity and location of GFP expression. PCR and Southern hybridization analyses confirmed the presence of introduced GFP genes in stably transformed cultures.

Synthesis, pharmacological investigations and X-ray studies on new 4,5-dihydro-1H-2,4-benzodiazepine derivatives.

The synthesis and biological activity of a series of 4,5-dihydro-1H-2,4-benzodiazepine is reported. The structure-activity relationship of these new compounds was studied based on X-ray investigations. Pharmacological investigations have shown that the compounds exert depressive action on the central nervous system and exhibit weak neuroleptic activity.

Design and synthesis of inhibitors of Plasmodium falciparum N-myristoyltransferase, a promising target for antimalarial drug discovery.

Design of inhibitors for N-myristoyltransferase (NMT), an enzyme responsible for protein trafficking in Plasmodium falciparum , the most lethal species of parasites that cause malaria, is described. Chemistry-driven optimization of compound 1 from a focused NMT inhibitor library led to the identification of two early lead compounds 4 and 25, which showed good enzyme and cellular potency and excellent selectivity over human NMT. These molecules provide a valuable starting point for further development.

The structure of monoamine oxidase from Aspergillus niger provides a molecular context for improvements in activity obtained by directed evolution.

Monoamine oxidase from Aspergillus niger (MAO-N) is a flavoenzyme that catalyses the oxidative deamination of primary amines. MAO-N has been used as the starting model for a series of directed evolution experiments, resulting in mutants of improved activity and broader substrate specificity, suitable for application in the preparative deracemisation of primary, secondary and tertiary amines when used as part of a chemoenzymatic oxidation-reduction cycle. The structures of a three-point mutant (Asn336Ser/Met348Lys/Ile246Met or MAO-N-D3) and a five-point mutant (Asn336Ser/Met348Lys/Ile246Met/Thr384Asn/Asp385Ser or MAO-N-D5) have been obtained using a multiple-wavelength anomalous diffraction experiment on a selenomethionine derivative of the truncated MAO-N-D5 enzyme. MAO-N exists as a homotetramer with a large channel at its centre and shares some structural features with human MAO B (MAO-B). A hydrophobic cavity extends from the protein surface to the active site, where a non-covalently bound flavin adenine dinucleotide (FAD) sits at the base of an 'aromatic cage,' the sides of which are formed by Trp430 and Phe466. A molecule of l-proline was observed near the FAD, and this ligand superimposed well with isatin, a reversible inhibitor of MAO-B, when the structures of MAO-N proline and MAO-B-isatin were overlaid. Of the mutations that confer the ability to catalyse the oxidation of secondary amines in MAO-N-D3, Asn336Ser reduces steric bulk behind Trp430 of the aromatic cage and Ile246Met confers greater flexibility within the substrate binding site. The two additional mutations, Thr384Asn and Asp385Ser, that occur in the MAO-N-D5 variant, which is able to oxidise tertiary amines, appear to influence the active-site environment remotely through changes in tertiary structure that perturb the side chain of Phe382, again altering the steric and electronic character of the active site near FAD. The possible implications of the change in steric and electronic environment caused by relevant mutations are discussed with respect to the improved catalytic efficiency of the MAO-N variants described in the literature.

Structural characterization of a beta-diketone hydrolase from the cyanobacterium Anabaena sp. PCC 7120 in native and product-bound forms, a coenzyme A-independent member of the crotonase suprafamily.

The gene alr4455 from the well-studied cyanobacterium Anabaena sp. PCC 7120 encodes a crotonase orthologue that displays beta-diketone hydrolase activity. Anabaena beta-diketone hydrolase (ABDH), in common with 6-oxocamphor hydrolase (OCH) from Rhodococcus sp. NCIMB 9784, catalyzes the desymmetrization of bicyclo[2.2.2]octane-2,6-dione to yield [(S)-3-oxocyclohexyl]acetic acid, a reaction unusual among the crotonase superfamily as the substrate is not an acyl-CoA thioester. The structure of ABDH has been determined to a resolution of 1.5 A in both native and ligand-bound forms. ABDH forms a hexamer similar to OCH and features one active site per enzyme monomer. The arrangement of side chains in the active site indicates that while the catalytic chemistry may be conserved in OCH orthologues, the structural determinants of substrate specificity are different. In the active site of ligand-bound forms that had been cocrystallized with the bicyclic diketone substrate bicyclo[2.2.2]octane-2,6-dione was found the product of the asymmetric enzymatic retro-Claisen reaction [(S)-3-oxocyclohexyl]acetic acid. The structures of ABDH in both native and ligand-bound forms reveal further details about structural variation and modes of coenzyme A-independent activity within the crotonases and provide further evidence of a wider suprafamily of enzymes that have recruited the crotonase fold for the catalysis of reactions other than those regularly attributed to canonical superfamily members.

The 1.8 A resolution structure of hydroxycinnamoyl-coenzyme A hydratase-lyase (HCHL) from Pseudomonas fluorescens, an enzyme that catalyses the transformation of feruloyl-coenzyme A to vanillin.

The crystal structure of hydroxycinnamoyl-CoA hydratase-lyase (HCHL) from Pseudomonas fluorescens AN103 has been solved to 1.8 A resolution. HCHL is a member of the crotonase superfamily and catalyses the hydration of the acyl-CoA thioester of ferulic acid [3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoic acid] and the subsequent retro-aldol cleavage of the hydrated intermediate to yield vanillin (4-hydroxy-3-methoxy-benzaldehyde). The structure contains 12 molecules in the asymmetric unit, in which HCHL assumes a hexameric structure of two stacked trimers. The substrate, feruloyl-CoA, was modelled into the active site based on the structure of enoyl-CoA hydratase bound to the feruloyl-CoA-like substrate 4-(N,N-dimethylamino)-cinnamoyl-CoA (PDB code 1ey3). Feruloyl-CoA was bound in this model between helix 3 of the A subunit and helix 9 of the B subunit. A highly ordered structural water in the HCHL structure coincided with the thioester carbonyl of feruloyl-CoA in the model, suggesting that the oxyanion hole for stabilization of a thioester-derived enolate, characteristic of coenzyme-A dependent members of the crotonase superfamily, is conserved. The model also suggested that a strong hydrogen bond between the phenolic hydroxyl groups of feruloyl-CoA and BTyr239 may be an important determinant of the enzyme's ability to discriminate between the natural substrate and cinnamoyl-CoA, which is not a substrate.

Structure and activity of two metal ion-dependent acetylxylan esterases involved in plant cell wall degradation reveals a close similarity to peptidoglycan deacetylases.

The enzymatic degradation of plant cell wall xylan requires the concerted action of a diverse enzymatic syndicate. Among these enzymes are xylan esterases, which hydrolyze the O-acetyl substituents, primarily at the O-2 position of the xylan backbone. All acetylxylan esterase structures described previously display a alpha/beta hydrolase fold with a "Ser-His-Asp" catalytic triad. Here we report the structures of two distinct acetylxylan esterases, those from Streptomyces lividans and Clostridium thermocellum, in native and complex forms, with x-ray data to between 1.6 and 1.0 A resolution. We show, using a novel linked assay system with PNP-2-O-acetylxyloside and a beta-xylosidase, that the enzymes are sugar-specific and metal ion-dependent and possess a single metal center with a chemical preference for Co2+. Asp and His side chains complete the catalytic machinery. Different metal ion preferences for the two enzymes may reflect the surprising diversity with which the metal ion coordinates residues and ligands in the active center environment of the S. lividans and C. thermocellum enzymes. These "CE4" esterases involved in plant cell wall degradation are shown to be closely related to the de-N-acetylases involved in chitin and peptidoglycan degradation (Blair, D. E., Schuettelkopf, A. W., MacRae, J. I., and Aalten, D. M. (2005) Proc. Natl. Acad. Sci. U. S. A., 102, 15429-15434), which form the NodB deacetylase "superfamily."