Following Natures Lead: On the Construction of Membrane-Inserted Toxins in Lipid Bilayer Nanodiscs

Bacterial toxin or viral entry into the cell often requires cell surface binding and endocytosis. The endosomal acidification induces a limited unfolding/refolding and membrane insertion reaction of the soluble toxins or viral proteins into their translocation competent or membrane inserted states. At the molecular level, the specific orientation and immobilization of the pre-transitioned toxin on the cell surface is often an important prerequisite prior to cell entry. We propose that structures of some toxin membrane insertion complexes may be observed through procedures where one rationally immobilizes the soluble toxin so that potential unfolding ↔ refolding transitions that occur prior to membrane insertion orientate away from the immobilization surface in the presence of lipid micelle pre-nanodisc structures. As a specific example, the immobilized prepore form of the anthrax toxin pore translocon or protective antigen can be transitioned, inserted into a model lipid membrane (nanodiscs), and released from the immobilized support in its membrane solubilized form. This particular strategy, although unconventional, is a useful procedure for generating pure membrane-inserted toxins in nanodiscs for electron microscopy structural analysis. In addition, generating a similar immobilized platform on label-free biosensor surfaces allows one to observe the kinetics of these acid-induced membrane insertion transitions. These platforms can facilitate the rational design of inhibitors that specifically target the toxin membrane insertion transitions that occur during endosomal acidification. This approach may lead to a new class of direct anti-toxin inhibitors

Monitoring the kinetics of the pH driven transition of the anthrax toxin prepore to the pore by biolayer interferometry and surface plasmon resonance

Domain 2 of the anthrax protective antigen (PA) prepore heptamer unfolds and refolds during endosome acidification to generate an extended 100 Å beta barrel pore that inserts into the endosomal membrane. The PA pore facilitates the pH dependent unfolding and translocation of bound toxin enzymic components, lethal factor (LF) and/or edema factor (EF), from the endosome into the cytoplasm. We constructed immobilized complexes of the prepore with the PA-binding domain of LF (LFN) to monitor the real-time prepore to pore kinetic transition using surface plasmon resonance (SPR) and bio-layer interferometry (BLI). The kinetics of this transition increased as the solution pH was decreased from pH 7.5 to pH 5.0, mirroring acidification of the endosome. Once transitioned, the LFN-PA pore complex was removed from the BLI biosensor tip and deposited onto EM grids, where the PA pore formation was confirmed by negative stain electron microscopy. When the soluble receptor domain (ANTRX2/CMG2) binds the immobilized PA prepore, the transition to the pore state was observed only after the pH was lowered to early or late endosomal pH conditions (5.5 to 5.0 respectively). Once the pore formed, the soluble receptor readily dissociated from the PA pore. Separate binding experiments with immobilized PA pores and soluble receptor indicate that the receptor has a weakened propensity to bind to the transitioned pore. This immobilized anthrax toxin platform can be used to identify or validate potential antimicrobial lead compounds capable of regulating and/or inhibiting anthrax toxin complex formation or pore transitions

Targeting Medium-Chain Acyl-CoA Dehydrogenase for Glioblastoma

View full abstracthttps://openworks.mdanderson.org/leading-edge/1041/thumbnail.jp

Tryptophan exposure and accessibility in the chitooligosaccharide-specific phloem exudate lectin from pumpkin (Cucurbita maxima). A fluorescence study

The exposure and accessibility of the tryptophan residues in the chitooligosaccharide-specific pumpkin (Cucurbita maxima) phloem exudate lectin (PPL) have been investigated by fluorescence spectroscopy. The emission λmax of native PPL, seen at 338 nm was red-shifted to 348 nm upon denaturation by 6 M Gdn.HCl in the presence of 10 mM β-mercaptoethanol, indicating near complete exposure of the tryptophan residues to the aqueous medium, whereas a blue-shift to 335 nm was observed in the presence of saturating concentrations of chitotriose, suggesting that ligand binding leads to a decrease in the solvent exposure of the tryptophan residues. The extent of quenching was maximum with the neutral molecule, acrylamide whereas the ionic species, iodide and Cs+ led to significantly lower quenching, which could be attributed to the presence of charged amino acid residues in close proximity to some of the tryptophan residues. The Stern-Volmer plot for acrylamide was linear for native PPL and upon ligand binding, but became upward curving upon denaturation, indicating that the quenching occurs via a combination of static and dynamic mechanisms. In time-resolved fluorescence experiments, the decay curves could be best fit to biexponential patterns, for native protein, in the presence of ligand and upon denaturation. In each case both lifetimes systematically decreased with increasing acrylamide concentrations, indicating that quenching occurs predominantly via a dynamic process

Rapid affinity-purification and physicochemical characterization of pumpkin (Cucurbita maxima) phloem exudate lectin

The chito-oligosaccharide-specific lectin from pumpkin (Cucurbita maxima) phloem exudate has been purified to homogeneity by affinity chromatography on chitin. After SDS/PAGE in the presence of 2-mercaptoethanol, the pumpkin phloem lectin yielded a single band corresponding to a molecular mass of 23.7 kDa, whereas ESI-MS (electrospray ionization MS) gave the molecular masses of the subunit as 24645 Da. Analysis of the CD spectrum of the protein indicated that the secondary structure of the lectin consists of 9.7% α-helix, 35.8% β-sheet, 22.5% β-turn and 32.3% unordered structure. Saccharide binding did not significantly affect the secondary and tertiary structures of the protein. The haemagglutinating activity of pumpkin phloem lectin was mostly unaffected in the temperature range 4-70 °C, but a sharp decrease was seen between 75 and 85 °C. Differential scanning calorimetric and CD spectroscopic studies suggest that the lectin undergoes a co-operative thermal unfolding process centred at approx. 81.5 °C, indicating that it is a relatively stable protein

Spatial Restrictions in Chemotaxis Signaling Arrays: A Role for Chemoreceptor Flexible Hinges across Bacterial Diversity

The chemotactic sensory system enables motile bacteria to move toward favorable environments. Throughout bacterial diversity, the chemoreceptors that mediate chemotaxis are clustered into densely packed arrays of signaling complexes. In these arrays, rod-shaped receptors are in close proximity, resulting in limited options for orientations. A recent geometric analysis of these limitations in Escherichia coli, using published dimensions and angles, revealed that in this species, straight chemoreceptors would not fit into the available space, but receptors bent at one or both of the recently-documented flexible hinges would fit, albeit over a narrow window of shallow bend angles. We have now expanded our geometric analysis to consider variations in receptor length, orientation and placement, and thus to species in which those parameters are known to be, or might be, different, as well as to the possibility of dynamic variation in those parameters. The results identified significant limitations on the allowed combinations of chemoreceptor dimensions, orientations and placement. For most combinations, these limitations excluded straight chemoreceptors, but allowed receptors bent at a flexible hinge. Thus, our analysis identifies across bacterial diversity a crucial role for chemoreceptor flexible hinges, in accommodating the limitations of molecular crowding in chemotaxis core signaling complexes and their arrays

A new chitooligosaccharide specific lectin from snake gourd (Trichosanthes anguina) phloem exudate. Purification, physico-chemical characterization and thermodynamics of saccharide binding

A new lectin has been purified to homogeneity from the phloem exudate of snake gourd (Trichosanthes anguina) by affinity chromatography on chitin. The snake gourd phloem lectin (SGPL) specifically binds chitooligosaccharides and their inhibitory potency increased with increase in size. PAGE and SDS-PAGE studies indicate that SGPL is a heterodimer, in which the two subunits (48 and 53 kDa) are joined by disulfide bonds. Consistent with this, electrospray-ionization mass spectrum yielded the exact mass of the protein as 104,621.8 Daltons. CD studies showed that SGPL contains about 9% α-helix, 39% β-sheet, 20% β-turns and 32% unordered structures and that saccharide binding does not significantly affect its secondary and tertiary structures. Titration calorimetric studies indicate that the dimeric lectin binds two ligand molecules [(GlcNAc)<SUB>3-6</SUB>] with association constants determined at 25 °C being 1.7 × 10<SUP>5</SUP> and 3.6 × 10<SUP>5</SUP> M<SUP>−1</SUP>, for chitotriose and chitohexaose, respectively. Binding of all the chitooligosaccharides is governed by enthalpic forces, whereas the contribution from binding entropies was unfavorable. These results suggest that the SGPL-saccharide interaction is stabilized by hydrogen bonding and van der Waals' interactions. Enthalpy-entropy compensation was observed for the SGPL-chitooligosaccharide interaction, suggesting that water molecules play a key role in the binding process

Spatial restrictions in chemotaxis signaling arrays: a role for chemoreceptor flexible hinges across bacterial diversity [supporting material]

Modeling tool available as a Mathematica Computable Document Format (cdf) file that can be downloaded here. It requires installation of the free Wolfram Player [https://www.wolfram.com/player/] or the full Mathematica suite. The free CDF player restricts file import/export, so models and plots are saved as screenshots. The utility generates interactive models and plots based on user-defined parameters starting with default values for E. coli (Akkaladevi et al., 2018). Interested scientists can change other variables such as receptor length, trimer separation, dimensions of the periplasmic domain and the position or number of flexible hinges by using sliders (figure below, left panel) and the utility displays the resulting geometry of a single core complex (figure below, center panel). Optional graphics controls adjust the angle from which the model is viewed. Two-dimensional plots showing forbidden and allowed combinations of two, operator-chosen variable parameters are generated from operator-chosen values for fixed parameters and the range of variable parameters. A ‘plot marker’ option places a point on the plot corresponding to the current values, and will adjust as those values are altered. This allows users to correlate the plot to the physical geometry in real space. Related publication (pending): Spatial restrictions in chemotaxis signaling arrays: a role for chemoreceptor flexible hinges across bacterial diversity.Authors: David Stalla (Electron Microscopy Core Facility, University of Missouri), Narahari Akkaladevi (Department of Biochemistry, University of Missouri), Tommi A. White (Department of Biochemistry and Electron Microscopy Core Facility, University of Missouri), and Gerald L. Hazelbauer (Department of Biochemistry, University of Missouri)

Disulphide bond reduction and S-carboxamidomethylation of PSP94 affects its conformation but not the ability to bind immunoglobulin

Prostate secretory protein of 94 amino acids (PSP94) is a small non-glycosylated, cysteine rich protein with a molecular mass of 10 kDa. It has also been referred to as β-microseminoprotein (β-MSP) and proteins homologous to it have been reported in a number of species. Comparison of the amino acid sequence of these proteins suggests that, it is a rapidly evolving protein. However, all the ten cysteine residues are well conserved in these homologues, indicating their possible role in maintaining the structure and function of these proteins. In the present study, PSP94 was purified from human seminal plasma and characterized further and it showed the presence of five disulfide bonds. Reduction of disulphide bonds of PSP94 led to significant changes in the secondary and tertiary structure of PSP94. CD of disulphide bond reduced PSP94 indicates an overall decrease in the beta sheet content from 79.8% to 46.4%. Tertiary structural changes as monitored by fluorescence quenching reveal that reduction of disulphide bonds of PSP94 followed by the modification of the free thiol groups leads to complete exposure of Trp32 and Trp92 and that one or more side chain carboxyl groups move closer to their indole side chains. Antibodies against native and modified PSP94 demonstrated that the changes following reduction of disulphide linkages are within the immunodominant region of the protein. Changes induced in the functional properties of PSP94, if any, by modification were investigated with respect to IgG binding as PSP94 has been reported to be similar to immunoglobulin binding factor purified from seminal plasma. A novel finding from this study is that both native PSP94 as well as modified protein have the ability to bind human IgG, suggesting the involvement of sequential epitopes of PSP94 in IgG binding