Anette Henriksen

Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein

To visualize the formation of disulfide bonds in living cells, a pair of redox‐active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2‐fold decrease in the intrinsic fluorescence. Inter conversion between the two redox states could thus be followed in vitro as well as in vivo by non‐invasive fluorimetric measurements. The 1.5 Å crystal structure of the oxidized protein revealed a disulfide bond‐induced distortion of the β‐barrel, as well as a structural reorganization of residues in the immediate chromophore environment. By combining this information with spectroscopic data, we propose a detailed mechanism accounting for the observed redox state‐dependent fluorescence. The redox potential of the cysteine couple was found to be within the physiological range for redox‐active cysteines. In the cytoplasm of Escherichia coli, the protein was a sensitive probe for the redox changes that occur upon disruption of the thioredoxin reductive pathway.

Dominant Epitopes and Allergic Cross-Reactivity: Complex Formation Between a Fab Fragment of a Monoclonal Murine IgG Antibody and the Major Allergen from Birch Pollen Bet v 1

The symptoms characteristic of allergic hypersensitivity are caused by the release of mediators, i.e., histamine, from effector cells such as basophils and mast cells. Allergens with more than one B cell epitope cross-link IgE Abs bound to high affinity FcεRI receptors on mast cell surfaces leading to aggregation and subsequent mediator release. Thus, allergen-Ab complexes play a crucial role in the cascade leading to the allergic response. We here report the structure of a 1:1 complex between the major birch pollen allergen Bet v 1 and the Fab fragment from a murine monoclonal IgG1 Ab, BV16, that has been solved to 2.9 Å resolution by x-ray diffraction. The mAb is shown to inhibit the binding of allergic patients’ IgE to Bet v 1, and the allergen-IgG complex may therefore serve as a model for the study of allergen-IgE interactions relevant in allergy. The size of the BV16 epitope is 931 Å2 as defined by the Bet v 1 Ab interaction surface. Molecular interactions predicted to occur in the interface are likewise in agreement with earlier observations on Ag-Ab complexes. The epitope is formed by amino acids that are conserved among major allergens from related species within the Fagales order. In combination with a surprisingly high inhibitory capacity of BV16 with respect to allergic patients’ serum IgE binding to Bet v 1, these observations provide experimental support for the proposal of dominant IgE epitopes located in the conserved surface areas. This model will facilitate the development of new and safer vaccines for allergen immunotherapy in the form of mutated allergens.

Major venom allergen of yellow jackets, Ves v 5: Structural characterization of a pathogenesis-related protein superfamily

Ves v 5 is one of three major allergens found in yellow‐jacket venom: phospholipase A1 (Ves v 1), hyaluronidase (Ves v 2), and antigen 5 (Ves v 5). Ves v 5 is related by high amino acid sequence identity to pathogenesis‐related proteins including proteins from mammals, reptiles, insects, fungi, and plants. The crystal structure of Ves v 5 has been solved and refined to a resolution of 1.9 Å. The majority of residues conserved between the pathogenesis‐related proteins can be rationalized in terms of hydrogen bonding patterns and hydrophobic interactions defining an α‐β‐α sandwich core structure. A small number of consensus residues are solvent exposed (including two adjacent histidines) and located in an elongated cavity that forms a putative active site. The site has no structural resemblance to previously characterized enzymes. Homologous antigen 5s from a large number of different yellow jackets, hornets, and paper wasps are known and patients show varying extents of cross‐reactivity to the related antigen 5s. The structure of Ves v 5 allows a detailed analysis of the epitopes that may participate in antigenic cross‐reactivity, findings that are useful for the development of a vaccine for treatment of insect allergy. Proteins 2001;45:438–448.

Concerted suppression of all starch branching enzyme genes in barley produces amylose-only starch granules

BackgroundStarch is stored in higher plants as granules composed of semi-crystalline amylopectin and amorphous amylose. Starch granules provide energy for the plant during dark periods and for germination of seeds and tubers. Dietary starch is also a highly glycemic carbohydrate being degraded to glucose and rapidly absorbed in the small intestine. But a portion of dietary starch, termed “resistant starch” (RS) escapes digestion and reaches the large intestine, where it is fermented by colonic bacteria producing short chain fatty acids (SCFA) which are linked to several health benefits. The RS is preferentially derived from amylose, which can be increased by suppressing amylopectin synthesis by silencing of starch branching enzymes (SBEs). However all the previous works attempting the production of high RS crops resulted in only partly increased amylose-content and/or significant yield loss.ResultsIn this study we invented a new method for silencing of multiple genes. Using a chimeric RNAi hairpin we simultaneously suppressed all genes coding for starch branching enzymes (SBE I, SBE IIa, SBE IIb) in barley (Hordeum vulgare L.), resulting in production of amylose-only starch granules in the endosperm. This trait was segregating 3:1. Amylose-only starch granules were irregularly shaped and showed peculiar thermal properties and crystallinity. Transgenic lines retained high-yield possibly due to a pleiotropic upregualtion of other starch biosynthetic genes compensating the SBEs loss. For gelatinized starch, a very high content of RS (65 %) was observed, which is 2.2-fold higher than control (29%). The amylose-only grains germinated with same frequency as control grains. However, initial growth was delayed in young plants.ConclusionsThis is the first time that pure amylose has been generated with high yield in a living organism. This was achieved by a new method of simultaneous suppression of the entire complement of genes encoding starch branching enzymes. We demonstrate that amylopectin is not essential for starch granule crystallinity and integrity. However the slower initial growth of shoots from amylose-only grains may be due to an important physiological role played by amylopectin ordered crystallinity for rapid starch remobilization explaining the broad conservation in the plant kingdom of the amylopectin structure.

Structure of soybean seed coat peroxidase: A plant peroxidase with unusual stability and haem-apoprotein interactions

Soybean seed coat peroxidase (SBP) is a peroxidase with extraordinary stability and catalytic properties. It belongs to the family of class III plant peroxidases that can oxidize a wide variety of organic and inorganic substrates using hydrogen peroxide. Because the plant enzyme is a heterogeneous glycoprotein, SBP was produced recombinant in Escherichia coli for the present crystallographic study. The three‐dimensional structure of SBP shows a bound tris(hydroxymethyl)aminomethane molecule (TRIS). This TRIS molecule has hydrogen bonds to active site residues corresponding to the residues that interact with the small phenolic substrate ferulic acid in the horseradish peroxidase C (HRPC):ferulic acid complex. TRIS is positioned in what has been described as a secondary substrate‐binding site in HRPC, and the structure of the SBP:TRIS complex indicates that this secondary substrate‐binding site could be of functional importance. SBP has one of the most solvent accessible δ‐meso haem edge (the site of electron transfer from reducing substrates to the enzymatic intermediates compound I and II) so far described for a plant peroxidase and structural alignment suggests that the volume of Ile74 is a factor that influences the solvent accessibility of this important site. A contact between haem C8 vinyl and the sulphur atom of Met37 is observed in the SBP structure. This interaction might affect the stability of the haem group by stabilisation/delocalisation of the porphyrin π‐cation of compound I.

Structure of barley grain peroxidase refined at 1.9-A resolution. A plant peroxidase reversibly inactivated at neutral pH.

The crystal structure of the major peroxidase of barley grain (BP 1) has been solved by molecular replacement and phase combination and refined to an R-factor of 19.2% for all data between 38 and 1.9 Å. The refined model includes amino acid residues 1–309, one calcium ion, one sodium ion, iron-protoporphyrin IX, and 146 solvent molecules. BP 1 has the apparently unique property of being unable to catalyze the reaction with the primary substrate hydrogen peroxide to form compound I at pH values > 5, a feature investigated by obtaining crystal structure data at pH 5.5, 7.5, and 8.5. Structural comparison shows that the overall fold of inactive barley grain peroxidase at these pH values resembles that of both horseradish peroxidase C and peanut peroxidase. The key differences between the structures of active horseradish peroxidase C and inactive BP 1 include the orientation of the catalytic distal histidine, disruption of a hydrogen bond between this histidine and a conserved asparagine, and apparent substitution of calcium at the distal cation binding site with sodium at pH 7.5. These profound changes are a result of a dramatic structural rearrangement to the loop region between helices B and C. This is the first time that structural rearrangements linked to active site chemistry have been observed by crystallography in the peroxidase domain distal to heme.

Allergy Vaccine Engineering: Epitope Modulation of Recombinant Bet v 1 Reduces IgE Binding but Retains Protein Folding Pattern for Induction of Protective Blocking-Antibody Responses

Human type 1 immediate allergic response symptoms are caused by mediator release from basophils and mast cells. This event is triggered by allergens aggregating preformed IgE Abs bound to the high-affinity receptor (FcεRI) on these cells. Thus, the allergen/IgE interaction is crucial for the cascade leading to the allergic and anaphylactic response. Two genetically engineered forms of the white birch pollen major allergen Bet v 1 with point mutations directed at molecular surfaces have been characterized. Four and nine point mutations led to a significant reduction of the binding to human serum IgE, suggesting a mutation-induced distortion of IgE-binding B cell epitopes. In addition, the mutated allergens showed a decrease in anaphylactic potential, because histamine release from human basophils was significantly reduced. Retained α-carbon backbone folding pattern of the mutated allergens was indicated by x-ray diffraction analysis and circular dichroism spectroscopy. The rBet v 1 mutants were able to induce proliferation of T cell lines derived from birch pollen allergic patients. The stimulation indices were similar to the indices of nonmutated rBet v 1 and natural Bet v 1 purified from birch pollen. The ability of anti-rBet v 1 mutant specific mouse IgG serum to block binding of human serum IgE to rBet v 1 demonstrates that the engineered rBet v 1 mutants are able to induce Abs reactive with nonmodified Bet v 1. rBet v 1 mutants may constitute vaccine candidates with improved efficacy/safety profiles for safer allergy vaccination.

Pancreatic spasmolytic polypeptide: first three-dimensional structure of a member of the mammalian trefoil family of peptides

BACKGROUND The trefoil peptides are a rapidly growing family of peptides, mainly found in the gastrointestinal tract. There is circumstantial evidence that they stabilize the mucus layer, and may affect the rate of healing of the mucosal epithelium. RESULTS We have determined the structure of porcine pancreatic spasmolytic polypeptide (PSP) to 2.5 A resolution. The polypeptide contains two trefoil domains. The domain structure is compact, and is composed of a central short antiparallel beta-sheet with one short helix above and one below it. This is a novel motif. The two domains are related by two-fold symmetry, and each domain contains a cleft. CONCLUSIONS The cleft within each domain could accommodate a polysaccharide chain, and may therefore be responsible for binding mucin glycoproteins. We suggest that PSP may cross-link glycoproteins, explaining its ability to stabilize the mucus layer.

Fatty acid synthesis. Role of active site histidines and lysine in Cys-His-His-type beta-ketoacyl-acyl carrier protein synthases.

β‐Ketoacyl‐acyl carrier protein (ACP) synthase enzymes join short carbon units to construct fatty acyl chains by a three‐step Claisen condensation reaction. The reaction starts with a trans thioesterification of the acyl primer substrate from ACP to the enzyme. Subsequently, the donor substrate malonyl‐ACP is decarboxylated to form a carbanion intermediate, which in the third step attacks C1 of the primer substrate giving rise to an elongated acyl chain. A subgroup of β‐ketoacyl‐ACP synthases, including mitochondrial β‐ketoacyl‐ACP synthase, bacterial plus plastid β‐ketoacyl‐ACP synthases I and II, and a domain of human fatty acid synthase, have a Cys‐His‐His triad and also a completely conserved Lys in the active site. To examine the role of these residues in catalysis, H298Q, H298E and six K328 mutants of Escherichia coliβ‐ketoacyl‐ACP synthase I were constructed and their ability to carry out the trans thioesterification, decarboxylation and/or condensation steps of the reaction was ascertained. The crystal structures of wild‐type and eight mutant enzymes with and/or without bound substrate were determined. The H298E enzyme shows residual decarboxylase activity in the pH range 6–8, whereas the H298Q enzyme appears to be completely decarboxylation deficient, showing that H298 serves as a catalytic base in the decarboxylation step. Lys328 has a dual role in catalysis: its charge influences acyl transfer to the active site Cys, and the steric restraint imposed on H333 is of critical importance for decarboxylation activity. This restraint makes H333 an obligate hydrogen bond donor at Nε, directed only towards the active site and malonyl‐ACP binding area in the fatty acid complex.

Structure of the human beta-ketoacyl [ACP] synthase from the mitochondrial type II fatty acid synthase.

Two distinct ways of organizing fatty acid biosynthesis exist: the multifunctional type I fatty acid synthase (FAS) of mammals, fungi, and lower eukaryotes with activities residing on one or two polypeptides; and the dissociated type II FAS of prokaryotes, plastids, and mitochondria with individual activities encoded by discrete genes. The β‐ketoacyl [ACP] synthase (KAS) moiety of the mitochondrial FAS (mtKAS) is targeted by the antibiotic cerulenin and possibly by the other antibiotics inhibiting prokaryotic KASes: thiolactomycin, platensimycin, and the α‐methylene butyrolactone, C75. The high degree of structural similarity between mitochondrial and prokaryotic KASes complicates development of novel antibiotics targeting prokaryotic KAS without affecting KAS domains of cytoplasmic FAS. KASes catalyze the C2 fatty acid elongation reaction using either a Cys‐His‐His or Cys‐His‐Asn catalytic triad. Three KASes with different substrate specificities participate in synthesis of the C16 and C18 products of prokaryotic FAS. By comparison, mtKAS carries out all elongation reactions in the mitochondria. We present the X‐ray crystal structures of the Cys‐His‐His‐containing human mtKAS and its hexanoyl complex plus the hexanoyl complex of the plant mtKAS from Arabidopsis thaliana. The structures explain (1) the bimodal (C6 and C10–C12) substrate preferences leading to the C8 lipoic acid precursor and long chains for the membranes, respectively, and (2) the low cerulenin sensitivity of the human enzyme; and (3) reveal two different potential acyl‐binding‐pocket extensions. Rearrangements taking place in the active site, including subtle changes in the water network, indicate a change in cooperativity of the active‐site histidines upon primer binding.