Binding of nicotinamide–adenine dinucleotides to diphtheria toxin

1. Changes in protein fluorescence have been utilized in determining the stoicheiometry and dissociation constants of the complexes of diphtheria toxin with NADH(2), NAD, NADPH(2) and NADP. 2. The binding stoicheiometry is 2moles of NADH(2) and 1mole of NADPH(2)/mole of diphtheria toxin. The binding sites for NADH(2) appear to be equivalent and independent. 3. The toxin shows a higher affinity for the reduced than for the oxidized forms of the nucleotides. 4. Dissociation constants at 0.01I, pH7 and 25 degrees are 0.7x10(-6)m for NADH(2) and 0.45x10(-6)m for NADPH(2). Dissociation constants increase with increasing ionic strength, indicating that the binding is mainly electrostatic. 5. Bound NADH(2) and NADPH(2) may be activated to fluoresce by the transfer of energy from the excited aromatic amino acids of the toxin. Activation and emission spectra of bound and free nucleotides are compared. 6. Since NAD and NADH(2) are cofactors specifically required for the inhibition of protein synthesis by diphtheria toxin, the possible role of toxin-nucleotide complexes is discussed in this regard.     

Modeling of toxin–antibody interaction and toxin transport toward the endoplasmic reticulum

A model for toxin–antibody interaction and toxin trafficking towards the endoplasmic-reticulum is presented. Antibody and toxin (ricin) initially are delivered outside the cell. The model involves: the pinocytotic (cellular drinking) and receptor-mediated toxin internalization modes from the extracellular into the intracellular domain, its exocytotic excretion from the cytosol back to the extracellular medium, the intact toxin retrograde transport to the endoplasmic reticulum, the anterograde toxin movement outward from the cell across the plasma membrane, the lysosomal toxin degradation, and the toxin clearance (removal from the system) flux. The model consists of a set of coupled PDEs. Using an averaging procedure, the model is reduced to a system of coupled ODEs. Both PDEs and ODEs systems are solved numerically. Numerical results are illustrated by figures and discussed.     

The single transmembrane segment determines the modulatory function of the BK channel auxiliary γ subunit

The large-conductance, calcium-activated potassium (BK) channels consist of the pore-forming, voltage- and Ca²⁺-sensing α subunits (BKα) and the tissue-specific auxiliary β and γ subunits. The BK channel γ1 subunit is a leucine-rich repeat (LRR)–containing membrane protein that potently facilitates BK channel activation in many tissues and cell types through a vast shift in the voltage dependence of channel activation by ∼140 mV in the hyperpolarizing direction. In this study, we found that the single transmembrane (TM) segment together with its flanking charged residues is sufficient to fully modulate BK channels upon its transplantation into the structurally unrelated β1 subunit. We identified Phe273 and its neighboring residues in the middle of the TM segment and a minimum of three intracellular juxtamembrane Arg residues as important for the γ1 subunit’s modulatory function and observed functional coupling between residues of these two locations. We concluded that the TM segment is a key molecular determinant for channel association and modulation and that the intracellular positively charged cluster is involved mainly in channel association, likely through its TM-anchoring effect. Our findings provide insights into the structure–function relationship of the γ1 subunit in understanding its potent modulatory effects on BK channels.     

Is the K permeability of the resting membrane controlled by the excitable K channel?

To test whether or not the potassium permeability of the resting membrane is controlled by the excitable K channels (delayed rectifier), we examined changes in the Na and K permeability ratio, PNa/PK, of the squid axon before and after the excitable K channels were blocked. The blockage of the K channels was accomplished by three independent methods: internal application of tetraethylammonium, internal application of 4-aminopyridine plus Cs, and prolong internal perfusion of NaF solution. The permeability ratio was determined using two different methods: the conventional electrophysiological method and a new method based on the measurements of the hyperpolarizing effect of Na removal. We found that blocking the K channels did not cause a proportional decrease in the K permeability of the resting membrane, suggesting that the semipermeable property of the resting membrane is not determined by the excitable K channels.     

Mapping of the detergent-exposed surface of membrane proteins and peptides by 1H solution NMR in detergent: Application to the gramicidin A ion channel

The present work evaluates the use of intermolecular polypeptide–detergent 1H through-space connectivities to determine the bilayer exposed-surface and the bilayer topography of membrane polypeptides solubilized in non- deuterated detergents. For this purpose, the membrane peptide gramicidin A, solubilized in non-deuterated sodium dodecylsulfate as its dimeric 6,3 helix channel conformation was used. For this peptide, a high-resolution 3D structure, as well as reasonable assumptions concerning its membrane arrangement, exist. Band-selective 2D NOESY, ROESY and 3D NOESY-NOESY experiments were used to detect detergent–polypeptide through-space correlations in the presence of an excess of the non-deuterated detergent. The observed intermolecular NOEs appear to be strongly temperature- dependent. Based on the known 3D structure of the gramicidin channel, the detergent–polypeptide through-space correlations appear to be selective for 1H located on the hydrophobic surface of gramicidin A with very few contributions from interior 1H or water-exposed 1H. It is suggested that this method can be of general use to evaluate the bilayer-exposed surface and topography of membrane peptides and small proteins.     

Purification and membrane interactions of human KCNQ1100–370 potassium ion channel

KCNQ1 (Kv7.1 or KvLQT1) is a voltage-gated potassium ion channel that is involved in the ventricular repolarization following an action potential in the heart. It forms a complex with KCNE1 in the heart and is the pore forming subunit of slow delayed rectifier potassium current (I_ks). Mutations in KCNQ1, leading to a dysfunctional channel or loss of activity have been implicated in a cardiac disorder, long QT syndrome. In this study, we report the overexpression, purification, biochemical characterization of human KCNQ1_100–370, and lipid bilayer dynamics upon interaction with KCNQ1_100–370. The recombinant human KCNQ1 was expressed in Escherichia coli and purified into n-dodecylphosphocholine (DPC) micelles. The purified KCNQ1_100–370 was biochemically characterized by SDS-PAGE electrophoresis, western blot and nano-LC-MS/MS to confirm the identity of the protein. Circular dichroism (CD) spectroscopy was utilized to confirm the secondary structure of purified protein in vesicles. Furthermore, ³¹P and ²H solid-state NMR spectroscopy in DPPC/POPC/POPG vesicles (MLVs) indicated a direct interaction between KCNQ_100–370 and the phospholipid head groups. Finally, a visual inspection of KCNQ1_100–370 incorporated into MLVs was confirmed by transmission electron microscopy (TEM). The findings of this study provide avenues for future structural studies of the human KCNQ1 ion channel to have an in depth understanding of its structure-function relationship.     

Loop C and the mechanism of acetylcholine receptor–channel gating

Agonist molecules at the two neuromuscular acetylcholine (ACh) receptor (AChR) transmitter-binding sites increase the probability of channel opening. In one hypothesis for AChR activation (“priming”), the capping of loop C at each binding site transfers energy independently to the distant gate over a discrete structural pathway. We used single-channel analyses to examine the experimental support for this proposal with regard to brief unliganded openings, the effects of loop-C modifications, the effects of mutations to residues either on or off the putative pathway, and state models for describing currents at low [ACh]. The results show that (a) diliganded and brief unliganded openings are generated by the same essential, global transition; (b) the radical manipulation of loop C does not prevent channel opening but impairs agonist binding; (c) both on- and off-pathway mutations alter gating by changing the relative stability of the open-channel conformation by local interactions rather than by perturbing a specific site–gate communication link; and (d) it is possible to estimate directly the rate constants for agonist dissociation from and association to both the low and high affinity forms of the AChR-binding site by using a cyclic kinetic model. We conclude that the mechanism of energy transfer between the binding sites and the gate remains an open question.     

High-yield production of diphtheria toxin mutants by high-density culture of C7(β)tox+ strains grown in a non-deferrated medium

A high-density growth approach was utilized to produce mutated diphtheria toxin from two strains of Corynebacterium diphtheria: C7 ()^{(tox-201, tox-9)} and C7 ()^(tox-107). The cross-reacting mutants (CRM) of the diphtheria toxin are CRM9 and CRM107; both of them carry the mutation in their binding site and, as a result, have 1/300 of the systemic toxicity of the wild-type diphtheria toxin. Since iron inhibits diphtheria toxin production, the traditional approach has been to grow the bacteria in a very low iron concentration. The procedure described here involved the use of a modified, non-deferrated, growth medium that provided fast and high-density growth of the bacteria, and which, when associated with simultaneous depletion of glucose and iron, enhanced the toxin production. Oxygen-enriched air was supplied to enable the bacteria to grow to a cell density giving an absorbance of 70 at 600 nm (15–20 g/l dry weight). The maximum toxin concentration in the culture supernatant was 150 mg/l. The CRM products, which remained stable following microfiltration and ultrafiltration, could be easily purified using a two-step chromatography procedure.     

Pocket analysis of the full-length cholix toxin. An assessment of the structure–dynamics of the apo catalytic domain

Cholix toxin from Vibrio cholerae is the third member of the diphtheria toxin (DT) group of mono-ADP-ribosyltransferase (mART) bacterial toxins. It shares structural and functional properties with Pseudomonas aeruginosa exotoxin A and Corynebacterium diphtheriae DT. Cholix toxin is an important model for the development of antivirulence approaches and therapeutics against these toxins from pathogenic bacteria. Herein, we have used the high-resolution X-ray structure of full-length cholix complexed with NAD⁺ to describe the properties of the NAD⁺-binding pocket at the residue level, including the role of crystallographic water molecules in the NAD⁺ substrate interaction. The full-length apo cholix structure is used to describe the putative NAD⁺-binding site(s) and to correlate biochemical with crystallographic data to study the stoichiometry and orientation of bound NAD⁺ molecules. We quantitatively describe the NAD⁺ substrate interactions on a residue basis for the main 22 pocket residues in cholix_f, a glycerol and 5 contact water molecules as part of the recognition surface by the substrate according to the conditions of crystallization. In addition, the dynamic properties of an in silico version of the catalytic domain were investigated in order to understand the lack of electronic density for one of the main flexible loops (R-loop) in the pocket of X-ray complexes. Implications for a rational drug design approach for mART toxins are derived.     

Use of liposomes as membrane models to evaluate the contribution of drug–membrane interactions to antioxidant properties of etodolac

Abstract

This work stresses the need to combine antioxidant assays and drug–membrane interaction studies to describe more accurately the antioxidant profile of non-steroidal anti-inflammatory drugs (NSAIDs). Different experiments performed in liposomes and aqueous solution were compared and used to evaluate the protective effect of etodolac in lipid peroxidation. Lipid peroxidation was induced by the peroxyl radical (ROO?) derived from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and hydroxyl radical (HO?) generated by the Fenton reaction and was assessed by the fluorescence intensity decay of three fluorescence probes with distinct lipophilic properties – fluorescein; hexadecanoyl aminofluorescein (HDAF) and diphenylhexatriene propionic acid (DPHPA). Membrane fluidity changes due to lipid peroxidation were also evaluated by steady-state anisotropy measurements. Interactions of etodolac with lipid bilayers were evaluated by membrane zeta-potential measurements. Results indicate a drug location near the membrane surface and show that etodolac can scavenge the radicals studied but to a variable extent, depending on the assayed media and reactive species. The use of different probes and liposomes as membrane mimetic systems allowed us to conclude that membrane lipoperoxidation is not only related to the scavenging characteristics of the antioxidants, but also to their ability to interact with lipid bilayers.     

Contribution of the KCa3.1 channel–calmodulin interactions to the regulation of the KCa3.1 gating process

The Ca²⁺-activated potassium channel of intermediate conductance, KCa3.1, is now emerging as a therapeutic target for a large variety of health disorders. The Ca²⁺ sensitivity of KCa3.1 is conferred by the Ca²⁺-binding protein calmodulin (CaM), with the CaM C-lobe constitutively bound to an intracellular domain of the channel C terminus. It was proposed on the basis of the crystal structure obtained for the C-terminal region of the rat KCa2.2 channel (rSK2) with CaM that the binding of Ca²⁺ to the CaM N-lobe results in CaM interlocking the C-terminal regions of two adjacent KCa3.1 subunits, leading to the formation of a dimeric structure. A study was thus undertaken to identify residues of the CaM N-lobe–KCa3.1 complex that either contribute to the channel activation process or control the channel open probability at saturating Ca²⁺ (Pomax). A structural homology model of the KCa3.1–CaM complex was first generated using as template the crystal structure of the C-terminal region of the rat KCa2.2 channel with CaM. This model was confirmed by cross-bridging residues R362 of KCa3.1 and K75 of CaM. Patch-clamp experiments were next performed, demonstrating that the solvation energy of the residue at position 367 in KCa3.1 is a key determinant to the channel Pomax and deactivation time t_off. Mutations of residues M368 and Q364 predicted to form anchoring points for CaM binding to KCa3.1 had little impact on either t_off or Pomax. Finally, our results show that channel activation depends on electrostatic interactions involving the charged residues R362 and E363, added to a nonpolar energy contribution coming from M368. We conclude that electrostatic interactions involving residues R362 and E363 and hydrophobic effects at M368 play a prominent role in KCa3.1 activation, whereas hydrophobic interactions at S367 are determinant to the stability of the CaM–KCa3.1 complex throughout gating.     

Mapping the Distribution of Anthrax in Mainland China, 2005–2013

BackgroundAnthrax, a global re-emerging zoonotic disease in recent years is enzootic in mainland China. Despite its significance to the public health, spatiotemporal distributions of the disease in human and livestock and its potential driving factors remain poorly understood.Conclusions/SignificanceAnthrax in China was characterized by significant seasonality and spatial clustering. The spatial distribution of human anthrax was largely driven by livestock husbandry, human density, land cover, elevation, topsoil features and climate. Enhanced surveillance and intervention for livestock and human anthrax in the high-risk regions, particularly on the Qinghai-Tibetan Plateau, is the key to the prevention of human infections.     

Sticholysin I–membrane interaction: An interplay between the presence of sphingomyelin and membrane fluidity

Sticholysin I (St I) is a pore-forming toxin (PFT) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin protein family, a unique class of eukaryotic PFT exclusively found in sea anemones. As for actinoporins, it has been proposed that the presence of sphingomyelin (SM) and the coexistence of lipid phases increase binding to the target membrane. However, little is known about the role of membrane structure and dynamics (phase state, fluidity, presence of lipid domains) on actinoporins' activity or which regions of the membrane are the most favorable platforms for protein insertion. To gain insight into the role of SM on the interaction of St I to lipid membranes we studied their binding to monolayers of phosphatidylcholine (PC) and SM in different proportions. Additionally, the effect of acyl chain length and unsaturation, two features related to membrane fluidity, was evaluated on St I binding to monolayers. This study revealed that St I binds and penetrates preferentially and with a faster kinetic to liquid-expanded films with high lateral mobility and moderately enriched in SM. A high content of SM induces a lower lateral diffusion and/or liquid-condensed phases, which hinder St I binding and penetration to the lipid monolayer. Furthermore, the presence of lipid domain borders does not appear as an important factor for St I binding to the lipid monolayer.     

Differential N termini in epithelial Na+ channel δ-subunit isoforms modulate channel trafficking to the membrane

The epithelial Na(+) channel (ENaC) is a heteromultimeric ion channel that plays a key role in Na(+) reabsorption across tight epithelia. The canonical ENaC is formed by three analogous subunits, α, β, and γ. A fourth ENaC subunit, named δ, is expressed in the nervous system of primates, where its role is unknown. The human δ-ENaC gene generates at least two splice isoforms, δ(1) and δ(2) , differing in the N-terminal sequence. Neurons in diverse areas of the human and monkey brain differentially express either δ(1) or δ(2) , with few cells coexpressing both isoforms, which suggests that they may play specific physiological roles. Here we show that heterologous expression of δ(1) in Xenopus oocytes and HEK293 cells produces higher current levels than δ(2) . Patch-clamp experiments showed no differences in single channel current magnitude and open probability between isoforms. Steady-state plasma membrane abundance accounts for the dissimilarity in macroscopic current levels. Differential trafficking between isoforms is independent of β- and γ-subunits, PY-motif-mediated endocytosis, or the presence of additional lysine residues in δ(2)-N terminus. Analysis of δ(2)-N terminus identified two sequences that independently reduce channel abundance in the plasma membrane. The δ(1) higher abundance is consistent with an increased insertion rate into the membrane, since endocytosis rates of both isoforms are indistinguishable. Finally, we conclude that δ-ENaC undergoes dynamin-independent endocytosis as opposed to αβγ-channels.     

Characterization of variant diphtheria toxin–interleukin-3 fusion protein,DTIL3K116W,for phase I clinical trials

We have produced clinical grade of DTIL3K116W, a variant diphtheria toxin–interleukin-3 fusion protein, for treatment of acute myeloid leukemia. The product was filter sterilized, aseptically vialed, and stored at ?80 °C. It was characterized by Coomassie-stained SDS-PAGE, endotoxin assay, cytotoxicity assay, sterility, mass spectroscopy, receptor binding affinity, ADP-ribosylation, inhibition of normal human CFU-GM, disulfide bond analysis, immunoblots, stability, size exclusion chromatography–HPLC, sequencing, and immunohistochemistry. Vialed product was sterile in 0.25 M NaCl/5 mM Tris, pH 7.9, and had a protein concentration of 1.08 mg/ml. Purity by SDS-PAGE was >99%. Aggregates by HPLC were <1%. Endotoxin levels were 0.296 EU/mg. Peptide mapping and mass spectroscopy confirmed its composition and molecular weight. The vialed drug kept reactivity with anti-IL3 and DT antibodies. Potency study revealed a 48-h EC₅₀ of 0.5 pM on TF1/H-ras cell. Its binding properties were confirmed by competitive experiments showing IC₅₀ of 1.4 nM. ADP-ribosylation activity was equivalent to DTGM-CSF. Drug did not react with tested frozen human tissue sections by immunohistochemistry. There was no evidence of loss of solubility, proteolysis aggregation, or loss of potency over 6 months at ?80 °C. Further, the drug was stable at 4 and 25 °C in the plastic syringe and administration tubing for 48 h.     

Organization of the native ribosome–translocon complex at the mammalian endoplasmic reticulum membrane

BackgroundIn eukaryotic cells, many proteins have to be transported across or inserted into the endoplasmic reticulum membrane during their biogenesis on the ribosome. This process is facilitated by the protein translocon, a highly dynamic multi-subunit membrane protein complex.Scope of reviewThe aim of this review is to summarize the current structural knowledge about protein translocon components in mammals.Major conclusionsVarious structural biology approaches have been used in synergy to characterize the translocon in recent years. X-ray crystallography and cryoelectron microscopy single particle analysis have yielded highly detailed insights into the structure and functional mechanism of the protein-conducting channel Sec61, which constitutes the functional core of the translocon. Cryoelectron tomography and subtomogram analysis have advanced our understanding of the overall structure, molecular organization and compositional heterogeneity of the translocon in a native membrane environment. Tomography densities at subnanometer resolution revealed an intricate network of interactions between the ribosome, Sec61 and accessory translocon components that assist in protein transport, membrane insertion and maturation.General significanceThe protein translocon is a gateway for approximately one third of all synthesized proteins and numerous human diseases are associated with malfunctioning of its components. Thus, detailed insights into the structure and molecular organization of the translocon will not only advance our understanding of membrane protein biogenesis in general, but they can potentially pave the way for novel therapeutic approaches against human diseases.     

SAXS study of the snake toxin α-crotamine

-crotamine is a small toxic protein (42 amino acid residues with three disulphide bridges) present in the venom of Crotallus durissus terrificus. Molecular parameters (R _g=13.7 Å, S=3,000 Ų, V=9,200 ų and D _max=40 Å) were derived from SAXS curves obtained from a solution of this protein at pH=4.5. An excellent agreement between the experimental distance distribution curve and that calculated from a model consisting of two lobes linked by the Cys(18)-Cys(30) disulphide bridge.     

Deletion Mapping of the λ REX Gene

Deletion mapping has been used to order 12 λ rex^- mutants. Correlation of recombination data with physically-determined positions of deletion end-points (Szybalski 1971; Blattner et al. 1972) suggests that the left-most rex^- mutation, rex209, is located about 260-300 nucleotide pairs from the p_L mutation sex1 and about 475 nucleotide pairs from the left end-point of the region of nonhomology with λimm434.     

Modeling analysis of the lipid bilayer–cytoskeleton coupling in erythrocyte membrane

Studies of thermal fluctuations in discocytes, echinocytes, and spherocytes suggest that the coupling between lipid bilayer and cytoskeleton can affect viscoelastic behavior of single erythrocyte membranes. To test this hypothesis, we developed a 3D constitutive model describing viscoelastic behavior of erythrocyte membranes, at long relaxation times \(t \in [0.20\,\mathrm {s}, 1.05\,\mathrm {s}]\) and short relaxation times \(t \in [0.03\,\mathrm {s}, 0.20\,\mathrm {s}]\) . The model was constructed using combination of spring and spring pot rheological elements arranged in parallel. The rearrangement of cytoskeleton induced by changing the bending state of lipid bilayer was described by a modified Eyring model. The model predictions point to an anomalous nature of energy dissipation and an ordered harmonic nature of the coupling mechanism described by a series of fractional derivatives of the order n \(\alpha \) (where \( n \in [- 1, 2]\) ). As a result, the stress generated within the lipid bilayer is related to the rate of change of the irreversible stress within the cytoskeleton.