A Novel C-Terminal Homologue of Aha1 Co-Chaperone Binds to Heat Shock Protein 90 and Stimulates Its ATPase Activity in Entamoeba histolytica

Cytosolic heat shock protein 90 (Hsp90) has been shown to be essential for many infectious pathogens and is considered a potential target for drug development. In this study, we have carried out biochemical characterization of Hsp90 from a poorly studied protozoan parasite of clinical importance, Entamoeba histolytica. We have shown that Entamoeba Hsp90 can bind to both ATP and its pharmacological inhibitor, 17-AAG (17-allylamino-17-demethoxygeldanamycin), with K_d values of 365.2 and 10.77 μM, respectively, and it has a weak ATPase activity with a catalytic efficiency of 4.12 × 10^− 4 min^− 1 μM^− 1. Using inhibitor 17-AAG, we have shown dependence of Entamoeba on Hsp90 for its growth and survival. Hsp90 function is regulated by various co-chaperones. Previous studies suggest a lack of several important co-chaperones in E. histolytica. In this study, we describe the presence of a novel homologue of co-chaperone Aha1 (activator of Hsp90 ATPase), EhAha1c, lacking a canonical Aha1 N-terminal domain. We also show that EhAha1c is capable of binding and stimulating ATPase activity of EhHsp90. In addition to highlighting the potential of Hsp90 inhibitors as drugs against amoebiasis, our study highlights the importance of E. histolytica in understanding the evolution of Hsp90 and its co-chaperone repertoire.     

Thermodynamic Profiling of HIV RREIIB RNA-Zinc Finger Interactions

The interactions between the HIV Rev-responsive element (RRE) RNA and the HIV regulatory protein Rev, are crucial for the HIV life-cycle. Earlier, we showed that single C₂H₂ zinc fingers (znfs) have the same binding site as the Rev peptide and exhibit nanomolar affinity. In this study, the specific role of amino acid side chains and molecular processes involved with complex formation were investigated by perturbation of the binding energetics via changes in temperature, pH, buffers, and salt concentrations, as well as znf and RNA mutations, by isothermal titration calorimetry. Interestingly, despite the large cationic charge on the znfs, the number of interactions with the RNA phosphate backbone was lower than intuitively expected. The presence of binding induced protonation was established by ITC and localized by NMR to a histidine on the znf β-sheet. The ΔC_p of znf-RNA binding was observed to be substantially negative and could not be accounted for by conventional solvent-accessible surface area models. An alternative model, based on the extent of hydrogen bond changes as a result of differences in ligand-induced water displacement at the binding site, provided reasonable explanation of the trends in ΔC_p, as well as ΔH and ΔS. Our studies show that incorporation of favorable interactions at the solvent-excluded binding interface can be used to alleviate the unfavorable enthalpic penalties of displacing water molecules from the hydrated RNA surface.     

Decomposition analysis of free energy profile for Hsp90-ADP association

The functional cycle of heat shock protein 90 (Hsp90) is driven and inhibited by the association/dissociation of ligand molecules. In order to understand the molecular mechanism of the association of N-terminal domain of Hsp90 (N-Hsp90) and its ligand molecule, it is necessary to investigate which part in the target system promotes or inhibits the association of N-Hsp90 and its ligand molecule. We apply the decomposition analysis for the association free energy of N-Hsp90 and ADP. The mean force calculated by thermodynamic integration method combined with molecular dynamic simulations is divided into the contributions from molecules in the target system. Van der Waals interaction of the solvent water molecules strongly stabilises the association. Three lysine residues on the surface of the N-Hsp90 pull ADP toward the binding pocket of N-Hsp90. This study elucidates the association process of ADP from the bulk region to the binding pocket of the N-terminal domain Hsp90. This approach is applicable to elucidate the association process of biomolecules.     

Crystal structure of the polyextremophilic alpha-amylase AmyB from Halothermothrix orenii: details of a productive enzyme-substrate complex and an N domain with a role in binding raw starch

The gene for a membrane-bound, halophilic, and thermostable α-amylase, AmyB, from Halothermothrix orenii was cloned and sequenced. The crystal structure shows that, in addition to the typical domain organization of family 13 glycoside hydrolases, AmyB carries an additional N-terminal domain (N domain) that forms a large groove—the N-C groove—some 30 Å away from the active site. The structure of AmyB with the inhibitor acarbose at 1.35 Å resolution shows that a nonasaccharide has been synthesized through successive transglycosylation reactions of acarbose. Unexpectedly, in a complex of wild-type AmyB with α-cyclodextrin and maltoheptaose at 2.2 Å resolution, a maltotetraose molecule is bound in subsites − 1 to + 3, spanning the cleavage point at − 1/+ 1, with the − 1 glucosyl residue present as a ²S_o skew boat. This wild-type AmyB complex was obtained in the presence of a large excess of substrate, a condition under which it is possible to capture Michaelis complexes, which may explain the observed binding across − 1/+ 1 and ring distortion. We observe three methionine side chains that serve as “binding platforms” for glucosyl rings in AmyB, a seemingly rare occurrence in carbohydrate-binding proteins. The structures and results from the biochemical characterization of AmyB and AmyB lacking the N domain show that the N domain increases binding of the enzyme to raw starch. Furthermore, theoretical modeling suggests that the N-C groove can accommodate, spatially and chemically, large substrates such as A-starch.     

A quaternary SNARE-synaptotagmin-Ca2+-phospholipid complex in neurotransmitter release

The function of synaptotagmin as a Ca²⁺ sensor in neurotransmitter release involves Ca²⁺-dependent phospholipid binding to its two C₂ domains, but this activity alone does not explain why Ca²⁺ binding to the C₂B domain is more critical for release than Ca²⁺ binding to the C₂A domain. Synaptotagmin also binds to SNARE complexes, which are central components of the membrane fusion machinery, and displaces complexins from the SNAREs. However, it is unclear how phospholipid binding to synaptotagmin is coupled to SNARE binding and complexin displacement. Using supported lipid bilayers deposited within microfluidic channels, we now show that Ca²⁺ induces simultaneous binding of synaptotagmin to phospholipid membranes and SNARE complexes, resulting in an intimate quaternary complex that we name SSCAP complex. Mutagenesis experiments show that Ca²⁺ binding to the C₂B domain is critical for SSCAP complex formation and displacement of complexin, providing a clear rationale for the preponderant role of the C₂B domain in release. This and other correlations between the effects of mutations on SSCAP complex formation and their functional effects in vivo suggest a key role for this complex in release. We propose a model whereby the highly positive electrostatic potential at the tip of the SSCAP complex helps to induce membrane fusion during release.     

Aromatic N-oxide bridged copper(II) coordination polymers: Synthesis, characterization and magnetic properties

The complexes [Cu₂(o-NO₂-C₆H₄COO)₄(PNO)₂] (1), [Cu₂(C₆H₅COO)₄(2,2′-BPNO)]_n (2), [Cu₂(C₆H₅COO)₄(4,4′-BPNO)]_n (3), [Cu(p-OH-C₆H₄COO)₂(4,4′-BPNO)₂·H₂O]_n (4), (where PNO = pyridine N-oxide, 2,2′-BPNO = 2,2′-bipyridyl-N,N′-dioxide, 4,4′-BPNO = 4,4′-bipyridyl-N,N′-dioxide) are prepared and characterized and their magnetic properties are studied as a function of temperature. Complex 1 is a discrete dinuclear complex while complexes 2-4 are polymeric of which 2 and 3 have paddle wheel repeating units. Magnetic susceptibility measurements from polycrystalline samples of 1-4 revealed strong antiferromagnetic interactions within the {Cu₂}⁴⁺ paddle wheel units and no discernible interactions between the units. The complex 5, [Cu(NicoNO)₂·2H₂O]_n·4nH₂O, in which the bridging ligand to the adjacent copper(II) ions is nicotinate N-oxide (NicoNO) the transmitted interaction is very weakly antiferromagnetic.     

Binding thermodynamics of phosphorylated inhibitors to triosephosphate isomerase and the contribution of electrostatic interactions

Electrostatic interactions have a central role in some biological processes, such as recognition of charged ligands by proteins. We characterized the binding energetics of yeast triosephosphate isomerase (TIM) with phosphorylated inhibitors 2-phosphoglycollate (2PG) and phosphoglycolohydroxamate (PGH). We determined the thermodynamic parameters of the binding process (K_b, ΔG_b, ΔH_b, ΔS_b and ΔC_p) with different concentrations of NaCl, using fluorimetric and calorimetric titrations in the conventional mode of ITC and a novel method, multithermal titration calorimetry (MTC), which enabled us to measure ΔC_p in a single experiment. We ruled out specific interactions of Na⁺ and Cl^- with the native enzyme and did not detect significant linked protonation effects upon the binding of inhibitors. Increasing ionic strength (I) caused K_b, ΔG_b and ΔH_b to become less favorable, while ΔS_b became less unfavorable. From the variation of K_b with I, we determined the electrostatic contribution of TIM−2PG and TIM−PGH to ΔG_b at I = 0.06 M and 25 °C to be 36% and 26%, respectively. The greater affinity of PGH for TIM is due to a more favorable ΔH_b compared to 2PG (by 19-24 kJ mol^-1 at 25 °C). This difference is compatible with PGH establishing up to five more hydrogen bonds with TIM. Both binding ΔC_ps were negative, and less negative with increasing ionic strength. ΔC_ps at I = 0.06 M were much more negative than predicted by surface area models. Water molecules trapped in the interface when ligands bind to protein could explain the highly negative ΔCps. Thermodynamic binding functions for TIM−2PG changed more with ionic strength than those for TIM−PGH. This greater dependence is consistent with linked, but compensated, protonation equilibriums yielding the dianionic species of 2PG that binds to TIM, process that is not required for PGH.     

Role of the Transmembrane Potential in the Membrane Proton Leak

The molecular mechanism responsible for the regulation of the mitochondrial membrane proton conductance (G) is not clearly understood. This study investigates the role of the transmembrane potential (ΔΨ_m) using planar membranes, reconstituted with purified uncoupling proteins (UCP1 and UCP2) and/or unsaturated FA. We show that high ΔΨ_m (similar to ΔΨ_m in mitochondrial State IV) significantly activates the protonophoric function of UCPs in the presence of FA. The proton conductance increases nonlinearly with ΔΨ_m. The application of ΔΨ_m up to 220 mV leads to the overriding of the protein inhibition at a constant ATP concentration. Both, the exposure of FA-containing bilayers to high ΔΨ_m and the increase of FA membrane concentration bring about the significant exponential G_m increase, implying the contribution of FA in proton leak. Quantitative analysis of the energy barrier for the transport of FA anions in the presence and absence of protein suggests that FA⁻ remain exposed to membrane lipids while crossing the UCP-containing membrane. We believe this study shows that UCPs and FA decrease ΔΨ_m more effectively if it is sufficiently high. Thus, the tight regulation of proton conductance and/or FA concentration by ΔΨ_m may be key in mitochondrial respiration and metabolism.     

Heat Shock Protein 90 as a Drug Target against Protozoan Infections: BIOCHEMICAL CHARACTERIZATION OF HSP90 FROM PLASMODIUM FALCIPARUM AND TRYPANOSOMA EVANSI AND EVALUATION OF ITS INHIBITOR AS A CANDIDATE DRUG*

Using a pharmacological inhibitor of Hsp90 in cultured malarial parasite, we have previously implicated Plasmodium falciparum Hsp90 (PfHsp90) as a drug target against malaria. In this study, we have biochemically characterized PfHsp90 in terms of its ATPase activity and interaction with its inhibitor geldanamycin (GA) and evaluated its potential as a drug target in a preclinical mouse model of malaria. In addition, we have explored the potential of Hsp90 inhibitors as drugs for the treatment of Trypanosoma infection in animals. Our studies with full-length PfHsp90 showed it to have the highest ATPase activity of all known Hsp90s; its ATPase activity was 6 times higher than that of human Hsp90. Also, GA brought about more robust inhibition of PfHsp90 ATPase activity as compared with human Hsp90. Mass spectrometric analysis of PfHsp90 expressed in P. falciparum identified a site of acetylation that overlapped with Aha1 and p23 binding domain, suggesting its role in modulating Hsp90 multichaperone complex assembly. Indeed, treatment of P. falciparum cultures with a histone deacetylase inhibitor resulted in a partial dissociation of PfHsp90 complex. Furthermore, we found a well known, semisynthetic Hsp90 inhibitor, namely 17-(allylamino)-17-demethoxygeldanamycin, to be effective in attenuating parasite growth and prolonging survival in a mouse model of malaria. We also characterized GA binding to Hsp90 from another protozoan parasite, namely Trypanosoma evansi. We found 17-(allylamino)-17-demethoxygeldanamycin to potently inhibit T. evansi growth in a mouse model of trypanosomiasis. In all, our biochemical characterization, drug interaction, and animal studies supported Hsp90 as a drug target and its inhibitor as a potential drug against protozoan diseases.     

Enthalpic switch-points and temperature dependencies of DNA binding and nucleotide incorporation by Pol I DNA polymerases

This study examines the relationship between the DNA binding thermodynamics and the enzymatic activity of the Klenow and Klentaq Pol I DNA polymerases from Escherichia coli and Thermus aquaticus. Both polymerases bind DNA with nanomolar affinity at temperatures down to at least 5 °C, but have lower than 1% enzymatic activity at these lower temperatures. For both polymerases it is found that the temperature of onset of significant enzymatic activity corresponds with the temperature where the enthalpy of binding (ΔH_binding) crosses zero (T_H) and becomes favorable (negative). This T_H/activity upshift temperature is 15 °C for Klenow and 30 °C for Klentaq. The results indicate that a negative free energy of DNA binding alone is not sufficient to proceed to catalysis, but that the enthalpic versus entropic balance of binding may be a modulator of the temperature dependence of enzymatic function. Analysis of the temperature dependence of the catalytic activity of Klentaq polymerase using expanded Eyring theory yields thermodynamic patterns for ΔG^‡, ΔH^‡, and TΔS^‡ that are highly analogous to those commonly observed for direct DNA binding. Eyring analysis also finds a significant ΔCp^‡ of formation of the activated complex, which in turn indicates that the temperature of maximal activity, after which incorporation rate slows with increasing temperature, will correspond with the temperature where the activation enthalpy (ΔH^‡) switches from positive to negative.     

Thermodynamic analysis reveals that GTP binding affects the interaction between the alpha- and gamma-subunits of translation initiation factor 2

Eukaryotic and archaeal translation initiation factors 2, heterotrimers that consist of α-, β-, and γ-subunits, deliver methionylated initiator tRNA to a small ribosomal subunit in a manner that depends on GTP. To evaluate correlation of the function and association of the subunits, we used isothermal titration calorimetry to analyze the thermodynamics of the interactions between the α- and γ-subunits in the presence or absence of a nonhydrolyzable GTP analog or GDP. The α-subunits bound to the γ-subunit with large heat capacity change (ΔC_p) values. The ΔH and ΔC_p values for the interaction between the α- and γ-subunits varied in the presence of the GTP analog but not in the presence of GDP. These results suggest that the binding of both the α-subunit and GTP changes the conformation of the switch region of the γ-subunit and increases the affinity of the γ-subunit for tRNA.     

Galectin-3 Interactions with Glycosphingolipids

Galectins have essential roles in pathological states including cancer, inflammation, angiogenesis and microbial infections. Endogenous receptors include members of the lacto- and neolacto-series glycosphingolipids present on mammalian cells and contain the tetrasaccharides lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) that form their core structural components and also ganglio-series glycosphingolipids. We present crystallographic structures of the carbohydrate recognition domain of human galectin-3, both wild type and a mutant (K176L) that influenced ligand affinity, in complex with LNT, LNnT and acetamido ganglioside a-G_M3 (α2,3-sialyllactose). Key structural features revealed include galectin-3's demonstration of a binding mode towards gangliosides distinct from that to the lacto/neolacto-glycosphingolipids, with its capacity for recognising the core β-galactoside region being challenged when the core oligosaccharide epitope of ganglio-series glycosphingolipids (G_M3) is embedded within particular higher-molecular-weight glycans. The lacto- and neolacto- glycosphingolipids revealed different orientations of their terminal galactose in the galectin-3-bound LNT and LNnT structures that has significant ramifications for the capacity of galectin-3 to interact with higher-order lacto/neolacto-series glycosphingolipids such as ABH blood group antigens and the HNK-1 antigen that is common on leukocytes. LNnT also presents an important model for poly-N-acetyllactosamine-containing glycans and provides insight into galectin-3's accommodation of extended oligosaccharides such as the poly-N-acetyllactosamine-modified N- and O-glycans that, via galectin-3 interaction, facilitate progression of lung and bladder cancers, respectively. These findings provide the first atomic detail of galectin-3's interactions with the core structures of mammalian glycosphingolipids, providing information important in understanding the capacity of galectin-3 to engage with receptors identified as facilitators of major disease.     

Extreme temperature tolerance of a hyperthermophilic protein coupled to residual structure in the unfolded state

Understanding the mechanisms that dictate protein stability is of large relevance, for instance, to enable design of temperature-tolerant enzymes with high enzymatic activity over a broad temperature interval. In an effort to identify such mechanisms, we have performed a detailed comparative study of the folding thermodynamics and kinetics of the ribosomal protein S16 isolated from a mesophilic (S16_meso) and hyperthermophilic (S16_thermo) bacterium by using a variety of biophysical methods. As basis for the study, the 2.0 Å X-ray structure of S16_thermo was solved using single wavelength anomalous dispersion phasing. Thermal unfolding experiments yielded midpoints of 59 and 111 °C with associated changes in heat capacity upon unfolding (ΔC_p⁰) of 6.4 and 3.3 kJ mol^− 1 K^− 1, respectively. A strong linear correlation between ΔC_p⁰ and melting temperature (T_m) was observed for the wild-type proteins and mutated variants, suggesting that these variables are intimately connected. Stopped-flow fluorescence spectroscopy shows that S16_meso folds through an apparent two-state model, whereas S16_thermo folds through a more complex mechanism with a marked curvature in the refolding limb indicating the presence of a folding intermediate. Time-resolved energy transfer between Trp and N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide of proteins mutated at selected positions shows that the denatured state ensemble of S16_thermo is more compact relative to S16_meso. Taken together, our results suggest the presence of residual structure in the denatured state ensemble of S16_thermo that appears to account for the large difference in quantified ΔC_p⁰ values and, in turn, parts of the observed extreme thermal stability of S16_thermo. These observations may be of general importance in the design of robust enzymes that are highly active over a wide temperature span.     

Synthesis of regioisomeric 17β-N-phenylpyrazolyl steroid derivatives and their inhibitory effect on 17α-hydroxylase/C17,20-lyase

The reaction of 3β-hydroxy-21-hydroxymethylidenepregn-5-en-3β-ol-20-one (1) with phenylhydrazine (2a) affords two regioisomers, 17β-(1-phenyl-3-pyrazolyl)androst-3-en-3β-ol (5a) and 17β-(1-phenyl-5-pyrazolyl)androst-5-en-3β-ol (6a). The direction of the ring-closure reactions of 1 with p-substituted phenylhydrazines (2b-e) depends strongly on the electronic features of the substituents. Oppenauer oxidation of 3β-hydroxy-17β-exo-heterocyclic steroids 5a-e and 6a-e yielded the corresponding Δ⁴-3-ketosteroids 9a-e and 10a-e. The inhibitory effects (IC₅₀) of these compounds on rat testicular C_17,20-lyase were investigated by means of an in vitro radioligand incubation technique.     

Enzymatic biosynthesis and biological evaluation of novel 17-AAG glucoside as potential anti-cancer agents

A novel 17-allylamino-17-demethoxygeldanamycin (17-AAG) glucoside (1) was obtained from in vitro enzymatic glycosylation using a UDP-glycosyltransferase (YjiC). The water-solubility of compound 1 was approximately 10.5 times higher than that of the substrate, 17-AAG. Compound 1 showed potential anti-proliferative activities against five human cancer cell lines, with IC₅₀ values ranging from 5.26 to 28.52 μM. Further studies also indicated that compound 1 could inhibit the growth of CNE-2Z cells by inducing the degradation of Hsp90 client proteins (Akt, c-Raf, Bcl-2, and HIF-1α). In addition, compound 1 showed greater potential anti-tumor efficacy than 17-AAG in nude mice xenografted with CNE-2Z cells. Therefore, we suggest that in vitro enzymatic glycosylation is a powerful approach for the structural optimization of 17-AAG.     

Detergents as intrinsic P-glycoprotein substrates and inhibitors

We assessed the interaction of three electrically neutral detergents (Triton X-100, C₁₂EO₈, and Tween 80) with P-glycoprotein (ABCB1, MDR1) and identified the molecular elements responsible for this interaction. To this purpose we titrated P-glycoprotein in inside-out plasma membrane vesicles of MDR1-transfected mouse embryo fibroblasts (NIH-MDR1-G185) with the detergents below their critical micelle concentration, CMC. The P-glycoprotein ATPase measured as a function of the detergent concentration yielded bell-shaped activity curves which were evaluated with a two-site binding model. The lipid-water partition coefficient and the transporter-water binding constant of the detergents were measured independently. Knowledge of these two parameters allowed assessment of the free energy of detergent binding to P-glycoprotein in the lipid membrane, ΔG_tl⁰, that reflects the direct detergent-transporter affinity. It increased as the number of ethoxyl groups increased, suggesting that these hydrogen bond acceptor groups are the key elements for the detergent-transporter interaction in the lipid membrane. The free energy of binding to P-glycoprotein per ethoxyl group (EO) was determined as approximately ΔG_EO⁰ = − 1.6 kJ/mol. The present findings moreover document that, depending on the concentration applied, detergents are intrinsic substrates for, or inhibitors of P-glycoprotein.     

Conserved Hydrophobic Clusters on the Surface of the Caf1A Usher C-Terminal Domain Are Important for F1 Antigen Assembly

The outer membrane usher protein Caf1A of the plague pathogen Yersinia pestis is responsible for the assembly of a major surface antigen, the F1 capsule. The F1 capsule is mainly formed by thin linear polymers of Caf1 (capsular antigen fraction 1) protein subunits. The Caf1A usher promotes polymerization of subunits and secretion of growing polymers to the cell surface. The usher monomer (811 aa, 90.5 kDa) consists of a large transmembrane β-barrel that forms a secretion channel and three soluble domains. The periplasmic N-terminal domain binds chaperone-subunit complexes supplying new subunits for the growing fiber. The middle domain, which is structurally similar to Caf1 and other fimbrial subunits, serves as a plug that regulates the permeability of the usher. Here we describe the identification, characterization, and crystal structure of the Caf1A usher C-terminal domain (Caf1A_C). Caf1A_C is shown to be a periplasmic domain with a seven-stranded β-barrel fold. Analysis of C-terminal truncation mutants of Caf1A demonstrated that the presence of Caf1A_C is crucial for the function of the usher in vivo, but that it is not required for the initial binding of chaperone-subunit complexes to the usher. Two clusters of conserved hydrophobic residues on the surface of Caf1A_C were found to be essential for the efficient assembly of surface polymers. These clusters are conserved between the FGL family and the FGS family of chaperone-usher systems.     

Synthesis of LeLe oligosaccharide fragments and efficient one-step deprotection

We describe here the synthesis of two oligosaccharide fragments of the tumor associated carbohydrate antigen Le^aLe^x. While the linear lacto-N-triose I: β-d-Galp-(1→4)-β-d-GlcNAcp-(1→3)-β-d-Galp-OMe is a known compound, this is the first reported preparation of the branched tetrasaccharide β-d-GlcNAcp-(1→3)-β-d-Galp-(1→4)-[α-l-Fucp-(1→3)]-β-d-GlcNAcp-OMe. Our synthetic schemes involved using an N-trichloroacetylated trichloroacetimidate glucosaminyl donor activated with excess TMSOTf at 0 °C for glycosylation at O-3 of galactosyl residues and that of trichloroacetimidate galactosyl donors activated with excess BF₃·OEt₂ to glycosylate either O-3 or O-4 of glucosamine residues. The fucosylation at O-3 of the glucosamine acceptor was accomplished using a thiofucoside donor activated with copper(II) bromide and tetrabutylammonium bromide. Thus, syntheses of the protected tri- and tetrasaccharides were achieved easily and efficiently using known building blocks. Of particular interest, we also report that these protected oligosaccharides were submitted to dissolving metal conditions (Na-NH₃) to provide in one single step the corresponding deprotected compounds. Under these conditions all protecting groups (O-acyl, benzylidene, benzyl, and N-trichloroacetyl) were efficiently cleaved. The work-up procedure for such reactions usually involves quenching with excess methanol and then neutralization with acetic acid. In our work the neutralization was carried out using acetic anhydride rather than acetic acid to ensure N-acetylation of the glucosamine residue. Both fully deprotected compounds were then simply purified and desalted by gel permeation chromatography on a Biogel P2 column eluted with water.     

Effect of “helper lipid” on lipoplex electrostatics

Lipoplexes, which are complexes between cationic liposomes (L⁺) and nucleic acids, are commonly used as a nucleic acid delivery system in vitro and in vivo. This study aimed to better characterize cationic liposome and lipoplex electrostatics, which seems to play a major role in the formation and the performance of lipoplexes in vitro and in vivo. We characterized lipoplexes based on two commonly used monocationic lipids, DOTAP and DMRIE, and one polycationic lipid, DOSPA—each with and without helper lipid (cholesterol or DOPE). Electrical surface potential (Ψ₀) and surface pH were determined using several surface pH-sensitive fluorophores attached either to a one-chain lipid (4-heptadecyl hydroxycoumarin (C17HC)) or to the primary amino group of the two-chain lipids (1,2-dioleyl-sn-glycero-3-phosphoethanolamine-N-carboxyfluorescein (CFPE) and 1,2-dioleyl-sn-glycero-3-phosphoethanolamine-N-7-hydroxycoumarin) (HC-DOPE). Zeta potentials of the DOTAP-based cationic liposomes and lipoplexes were compared with Ψ₀ determined using C17HC. The location and relatively low sensitivity of fluorescein to pH changes explains why CFPE is the least efficient in quantifying the differences between the various cationic liposomes and lipoplexes used in this study. The fact that, for all cationic liposomes studied, those containing DOPE as helper lipid have the least positive Ψ₀ indicates neutralization of the cationic charge by the negatively-charged phosphodiester of the DOPE. Zeta potential is much less positively charged than Ψ₀ determined by C17HC. The electrostatics affects size changes that occurred to the cationic liposomes upon lipoplex formation. The largest size increase (based on static light scattering measurements) for all formulations occurred at DNA⁻/L⁺ charge ratios 0.5-1. Comparing the use of the one-chain C17HC and the two-chain HC-DOPE for monitoring lipoplex electrostatics reveals that both are suitable, as long as there is no serum (or other lipidic assemblies) present in the medium; in the latter case, only the two-chain HC-DOPE gives reliable results. Increasing NaCl concentrations decrease surface potential. Neutralization by DNA is reduced in a NaCl-concentration-dependent manner.     

The X-ray Crystal Structure of an Arthrobacter protophormiae Endo-β-N-Acetylglucosaminidase Reveals a (β/α)8 Catalytic Domain, Two Ancillary Domains and Active Site Residues Key for Transglycosylation Activity

Glycoside hydrolase family GH85 is a family of endo-β-N-acetylglucosaminidases that is responsible for the hydrolysis of β-1,4 linkage in the N,N-diacetylchitobiose core of N-linked glycans. The endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) is of particular interest, given its increasing use for the chemoenzymatic synthesis of bespoke N-glycans using N-glycan oxazolines as glycosyl donors. The E173Q variant of Endo-A is especially attractive for synthesis, as it is hydrolytically impaired but still able to catalyze N-glycan synthesis by transglycosylation using activated oxazoline donors. Here we present the three-dimensional structure of the A. protophormiae Endo-A E173Q variant, solved by multiple-wavelength anomalous scattering methods and refined at 1.8 Å resolution. The structure reveals that GH85 enzymes display a trimodular architecture in which a (β/α)₈ catalytic domain occurs with two ancillary β-sheet modules. The active centre is fully consistent with the known neighboring-group catalytic mechanism in which E173 acts as the catalytic acid/base for reaction via an oxazoline intermediate. Of note is the presence of an asparagine in the active centre, in a position likely to interact with the acetyl NH group that, in all other known families of glycosidase using this mechanism, is an aspartate or glutamate residue. The substrate-binding surface reveals an open topography, consistent with the ability to accept a large range of glycoprotein substrates and the ability to transglycosylate other acceptors. The three-dimensional structure of this important biocatalyst reveals that residues implicated in the enhancement of transglycosylation and synthetic capacity are proximal to the active centre, where they may act to favor binding of acceptor substrates.