Structural insights into unique substrate selectivity of Thermoplasma acidophilum D-aldohexose dehydrogenase

The d-aldohexose dehydrogenase from the thermoacidophilic archaea Thermoplasma acidophilum (AldT) belongs to the short-chain dehydrogenase/reductase (SDR) superfamily and catalyzes the oxidation of several monosaccharides with a preference for NAD⁺ rather than NADP⁺ as a cofactor. It has been found that AldT is a unique enzyme that exhibits the highest dehydrogenase activity against d-mannose. Here, we describe the crystal structures of AldT in ligand-free form, in complex with NADH, and in complex with the substrate d-mannose, at 2.1 Å, 1.65 Å, and 1.6 Å resolution, respectively. The AldT subunit forms a typical SDR fold with an unexpectedly long C-terminal tail and assembles into an intertwined tetramer. The d-mannose complex structure reveals that Glu84 interacts with the axial C2 hydroxyl group of the bound d-mannose. Structural comparison with Bacillus megaterium glucose dehydrogenase (BmGlcDH) suggests that the conformation of the glutamate side-chain is crucial for discrimination between d-mannose and its C2 epimer d-glucose, and the conformation of the glutamate side-chain depends on the spatial arrangement of nearby hydrophobic residues that do not directly interact with the substrate. Elucidation of the d-mannose recognition mechanism of AldT further provides structural insights into the unique substrate selectivity of AldT. Finally, we show that the extended C-terminal tail completely shuts the substrate-binding pocket of the neighboring subunit both in the presence and absence of substrate. The elaborate inter-subunit interactions between the C-terminal tail and the entrance of the substrate-binding pocket imply that the tail may play a pivotal role in the enzyme activity.     

Identification of a B-cell epitope of hyaluronidase, a major bee venom allergen, from its crystal structure in complex with a specific Fab

The major allergens of honeybee venom, hyaluronidase (Hyal) and phospholipase A2, can induce life-threatening IgE-mediated allergic reactions in humans. Although conventional immunotherapy is effective, up to 40% of patients develop allergic side effects including anaphylaxis and thus, there is a need for an improved immunotherapy. A murine monoclonal anti-Hyal IgG1 antibody (mAb 21E11), that competed for Hyal binding with IgEs from sera of bee venom allergic patients, was raised. The fragment of these IgG antibodies which bind to antigen (Fab) was produced and complexed (1:1) with Hyal. The crystal structure determination of Hyal/Fab 21E11 complex (2.6 A) enabled the identification of the Hyal-IgG interface which provides indirect information on the Hyal-IgE interaction (B-cell epitope). The epitope is composed of a linear array of nine residues (Arg138, His141-Arg148) located at the tip of a helix-turn-helix motive which protrudes away from the globular core and fits tightly into the deep surface pocket formed by the residues from the six complementarity determining regions (CDRs) of the Fab. The epitope is continuous and yet its conformation appears to be essential for Ab recognition, since the synthetic 15-mer peptide comprising the entire epitope (Arg138-Glu152) is neither recognized by mAb 21E11 nor by human IgEs. The structure of the complex provides the basis for the rational design of Hyal derivatives with reduced allergenic activity, which could be used in the development of safer allergen-specific immunotherapy.     

In vitro stability and content release properties of phosphatidylglyceroglycerol containing thermosensitive liposomes

Recently, we reported that 1,2-dipalmitoyl-sn-glycero-3-phosphoglyceroglycerol (DPPGOG) prolongs the circulation time of thermosensitive liposomes (TSL). Since the only TSL formulation in clinical trials applies DSPE-PEG2000 and lysophosphatidylcholine (P-lyso-PC), the objective of this study was to compare the influence of these lipids with DPPGOG on in vitro stability and heat-induced drug release properties of TSL. The content release rate was significantly increased by incorporating DPPGOG or P-lyso-PC in TSL formulations. DPPC/DSPC/DPPGOG 50:20:30 (m/m) and DPPC/P-lyso-PC/DSPE-PEG2000 90:10:4 (m/m) did not differ significantly in their release rate of carboxyfluorescein with > 70% being released within the first 10s at their phase transition temperature. Furthermore, DPPC/DSPC/DPPGOG showed an improved stability at 37 °C in serum compared to the PEGylated TSL. The in vitro properties of DPPGOG-containing TSL remained unchanged when encapsulating doxorubicin instead of carboxyfluorescein. The TSL retained 89.1 ± 4.0% of doxorubicin over 3 h at 37  °C in the presence of serum. The drug was almost completely released within 120s at 42 °C. In conclusion, DPPGOG improves the in vitro properties in TSL formulations compared to DSPE-PEG2000, since it not only increases the in vivo half-life, it even increases the content release rate without negative effect on TSL stability at 37 °C which has been seen for DSPE-PEG2000/P-lyso-PC containing TSL.     

Probing the structure of the Ff bacteriophage major coat protein transmembrane helix dimer by solution NMR

The transmembrane (TM) segment of the major coat protein from Ff bacteriophage has been extensively studied as an example of dimerization in detergent and lipid bilayer systems. However, almost all the information regarding this interaction has been gained through mutagenesis studies, with little direct structural information being available. To this end solution NMR has the potential to provide new insights into structure of the dimer. In order to evaluate the utility of this approach we have studied a selectively ¹⁵N-labeled peptide containing the TM segment of MCP (MCP_TM) by solution NMR. This peptide was found to give rise to detergent concentration-dependent spectra that were assigned to monomeric and dimeric forms. The standard free energy of this interaction in SDS was estimated from these spectra and found to be consistent with weak but specific dimerization. In addition, similar spectra could be obtained in β-octyl glucoside with intermolecular paramagnetic relaxation experiments demonstrating a parallel arrangement of TM helices in the dimer. In both detergents backbone chemical shift differences between monomeric and dimeric forms of MCP_TM showed that the largest changes occur around its GXXXG motif. The resulting structural model is consistent with observations made for MCP mutants previously characterized in biological membranes, opening the door to detailed structural characterization of this form of MCP. These results also have general implications for the study of weakly interacting TM segments by solution NMR since the use of similar sample conditions should allow structural data to be accessed for oligomeric states from a wide range systems that undergo biologically relevant but weak associations in the membrane.     

Improved purification and PCR amplification of DNA from environmental samples

Purification and PCR amplification procedures for DNA extracted from environmental samples (soil, compost, and river sediment) were improved by introducing three modifications: precipitation of DNA with 5% polyethylene glycol 8000 (PEG) and 0.6 M NaCl; filtration with a Sepharose 4B-polyvinylpolypyrrolidone (PVPP) spin column; and addition of skim milk (0.3% w/v) to the PCR reaction solution. Humic substances' concentration after precipitation with 5% PEG was 2.57-, 5.3-, and 78.9-fold lower than precipitation with 7.5% PEG, 10% PEG, and isopropanol, respectively. After PEG precipitation, Sepharose, PVPP and the combined (Sepharose-PVPP) column removed 92.3%, 89.5%, and 98%, respectively, of the remaining humic materials. Each of the above-mentioned modifications improved PCR amplification of the 16S rRNA gene. DNA extracted by the proposed protocol is cleaner than DNA extracted by a commercial kit. Nevertheless, the improvement of DNA purification did not improve the detection limit of atrazine degradation gene atzA.     

P-selectin/ligand unbinding force measured with atomic force microscopy: comparison of two chemical protocols for the tethering of single molecules

Leukocytes, as an indispensable arm of the immune system, need to be recruited from the flowing blood and transferred to the sites of infection. Their extravasation is feasible due to their ability to tether and roll over the activated endothelium, which is much dependent on the association of their selectin molecules with ligands on the activated endothelial cells. In view of the importance of this interaction for the physiological immune functions as well as for autoimmune diseases, specifying the affinity of selectins to their ligands at the single molecule level appears a challenging task to gain insight into the mechanisms that control leukocyte–endothelial avidity. To this end we functionalized substrates with P‐selectin and cantilever probes with its major ligand, the P‐selectin glycoprotein ligand‐1, and used atomic force microscopy to measure their unbinding force. Two different chemical protocols were used for the tethering of the molecules on the substrates, one based on a homobifunctional poly(ethylene glycol) linker and the other on the use of antibody‐specific binding. The unbinding forces measured with the two methods were 312 ± 149 and 230 ± 57 pN, respectively. Measurements on activated endothelials, declaratory of single molecule interactions, gave comparable results. Copyright © 2011 John Wiley & Sons, Ltd.     

Comparison of covalent immobilization of amylase on polystyrene pellets with pentaethylenehexamine and pentaethylene glycol spacers

α-Amylase from Aspergillus oryzae was covalently immobilized onto polystyrene pellets with pentaethylenehexamine (PS-PEHA-Ald) and pentaethylene glycol (PS-PG-Ald) carrying a terminal aldehyde group. Optimum immobilization occured at pH 8.0 and 25 °C, and at pH 7.0 and 35 °C for PS-PEHA-Ald and PS-PG-Ald, respectively. PS-PEHA-Ald immobilized enzyme retained approximately 75% of the initial activity over 45 days of storage, 70% of the initial activity after nine runs of recycling and displayed the better resistance to detrimental metal ions. PS-PG-Ald immobilized enzyme retained approximately 50% of the initial activity in 8h at 70 °C. The catalytic efficiencies of PS-PEHA-Ald immobilized and PS-PG-Ald immobilized amylase were 1.42 and 1.29 times higher than that of native enzyme. The activation energy of the reaction mediated by the amylase was reduced by 58.1% and 57.3% when PS-PEHA-Ald and PS-PG-Ald used as support respectively.     

Enhancement of the adhesion of fibroblasts by peptide containing an Arg-Gly-Asp sequence with poly(ethylene glycol) into a thermo-reversible hydrogel as a synthetic extracellular matrix

In an effort to regulate the behavior of mammalian cell entrapped in a gel, the gels were functionalized with the putative cell-binding (-Arg-Gly-Asp-) (RGD) domain. The adhesion molecules composed of Gly-Arg-Gly-Asp-Ser (GRGDS) peptides and the cell recognition ligands were inculcated into the thermo-reversible hydrogel composed of N-isopropylacrylamide, with a small amount of succinyl poly(ethylene glycol) (PEG) acrylate (MW 2000) used as the biomimetic extracellular matrix (ECM). The GRGDS-containing p(NiPAAm-co-PEG) copolymer gel was examined in vitro for its ability to promote cell spreading and to increase the viability of the cells by introducing PEG spacers. ECM poorly adhered to hydrogel lacking adhesion molecules permitting only a 20% spread of the seeded cells after 10 days. When the PEG spacer arms, which were immobilized by a peptide linkage, had been integrated into the hydrogel, the conjugation of RGD improved cell spreading by 600% in a 10-day trial.