Alexey A. Schulga

Unique dimeric structure of BNip3 transmembrane domain suggests membrane permeabilization as a cell death trigger.

BNip3 is a prominent representative of apoptotic Bcl-2 proteins with rather unique properties initiating an atypical programmed cell death pathway resembling both necrosis and apoptosis. Many Bcl-2 family proteins modulate the permeability state of the outer mitochondrial membrane by forming homo- and hetero-oligomers. The structure and dynamics of the homodimeric transmembrane domain of BNip3 were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics energy relaxation in an explicit lipid bilayer. The right-handed parallel helix-helix structure of the domain with a hydrogen bond-rich His-Ser node in the middle of the membrane, accessibility of the node for water, and continuous hydrophilic track across the membrane suggest that the domain can provide an ion-conducting pathway through the membrane. Incorporation of the BNip3 transmembrane domain into an artificial lipid bilayer resulted in pH-dependent conductivity increase. A possible biological implication of the findings in relation to triggering necrosis-like cell death by BNip3 is discussed.

Spatial Structure and pH-dependent Conformational Diversity of Dimeric Transmembrane Domain of the Receptor Tyrosine Kinase EphA1

Eph receptors are found in a wide variety of cells in developing and mature tissues and represent the largest family of receptor tyrosine kinases, regulating cell shape, movements, and attachment. The receptor tyrosine kinases conduct biochemical signals across plasma membrane via lateral dimerization in which their transmembrane domains play an important role. Structural-dynamic properties of the homodimeric transmembrane domain of the EphA1 receptor were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics in explicit lipid bilayer. EphA1 transmembrane segments associate in a right-handed parallel α-helical bundle, region (544-569)2, through the N-terminal glycine zipper motif A550X3G554X3G558. Under acidic conditions, the N terminus of the transmembrane helix is stabilized by an N-capping box formed by the uncharged carboxyl group of Glu547, whereas its deprotonation results in a rearrangement of hydrogen bonds, fractional unfolding of the helix, and a realignment of the helix-helix packing with appearance of additional minor dimer conformation utilizing seemingly the C-terminal GG4-like dimerization motif A560X3G564. This can be interpreted as the ability of the EphA1 receptor to adjust its response to ligand binding according to extracellular pH. The dependence of the pKa value of Glu547 and the dimer conformational equilibrium on the lipid head charge suggests that both local environment and membrane surface potential can modulate dimerization and activation of the receptor. This makes the EphA1 receptor unique among the Eph family, implying its possible physiological role as an “extracellular pH sensor,” and can have relevant physiological implications.

Left-Handed Dimer of EphA2 Transmembrane Domain: Helix Packing Diversity among Receptor Tyrosine Kinases

The Eph receptor tyrosine kinases and their membrane-bound ephrin ligands control a diverse array of cell-cell interactions in the developing and adult organisms. During signal transduction across plasma membrane, Eph receptors, like other receptor tyrosine kinases, are involved in lateral dimerization and subsequent oligomerization presumably with proper assembly of their single-span transmembrane domains. Spatial structure of dimeric transmembrane domain of EphA2 receptor embedded into lipid bicelle was obtained by solution NMR, showing a left-handed parallel packing of the transmembrane helices (535-559)(2). The helices interact through the extended heptad repeat motif L(535)X(3)G(539)X(2)A(542)X(3)V(546)X(2)L(549) assisted by intermolecular stacking interactions of aromatic rings of (FF(557))(2), whereas the characteristic tandem GG4-like motif A(536)X(3)G(540)X(3)G(544) is not used, enabling another mode of helix-helix association. Importantly, a similar motif AX(3)GX(3)G as was found is responsible for right-handed dimerization of transmembrane domain of the EphA1 receptor. These findings serve as an instructive example of the diversity of transmembrane domain formation within the same family of protein kinases and seem to favor the assumption that the so-called rotation-coupled activation mechanism may take place during the Eph receptor signaling. A possible role of membrane lipid rafts in relation to Eph transmembrane domain oligomerization and Eph signal transduction was also discussed.

Expression, purification, and characterization of human enteropeptidase catalytic subunit in Escherichia coli

Enteropeptidase (synonym:enterokinase, EC 3.4.21.9) is a heterodimeric serine protease of the intestinal brush border that activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)(4)-Lys. The DNA sequence encoding the light chain (catalytic subunit) of human enteropeptidase (GenBank Accession No. U09860) was synthesized from 26 oligonucleotides by polymerase chain reaction and cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin immediately after the DNA sequence encoding enteropeptidase recognition site. The fusion protein thioredoxin/human enteropeptidase light chain was expressed in Escherichia coli BL21(DE3) strain in both soluble and insoluble forms. The soluble recombinant fusion protein failed to undergo autocatalytic cleavage and activation; however, autocatalytic cleavage and activation of recombinant human enteropeptidase light chain (L-HEP) were achieved by solubilization and renaturation of the fusion protein from inclusion bodies and the active L-HEP was purified on agarose-linked soybean trypsin inhibitor. The purified L-HEP cleaved the synthetic peptide substrate Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide with kinetic parameters K(m)=0.16 mM and k(cat)=115 s(-1) and small ester Z-Lys-SBzl with K(m)=140 microM, k(cat)=133 s(-1). L-HEP associated with soybean trypsin inhibitor slowly and small ester Z-Lys-SBzl cleavage was inhibited with K(i)(*)=2.3 nM. L-HEP digested thioredoxin/human epidermal growth factor fusion protein five times faster than equal activity units of bovine recombinant light chain (EKMax, Invitrogen) at the same conditions.

De novo design, synthesis and study of albebetin, a polypeptide with a predetermined three-dimensional structure. Probing the structure at the nanogram level.

The de novo polypeptide named albebetin was designed to form the tertiary fold that has not yet been observed in natural proteins. The design was based on the molecular theory of protein structures. The gene coding for this polypeptide was chemically synthesized. For the initial characterization of a protein structure, a new approach has been developed that uses only nanogram amounts of a polypeptide without its previous purification. This approach includes the biosynthesis of radiolabeled protein in a cell-free translation system with subsequent analysis of its compactness and structure by size-exclusion chromatography, urea-gradient electrophoresis and limited proteolysis. According to all tests used, albebetin has a compact stable structure.

Towards structure determination of neurotoxin II bound to nicotinic acetylcholine receptor: a solid-state NMR approach.

Solid‐state magic‐angle spinning nuclear magnetic resonance (NMR) has sufficient resolving power for full assignment of resonances and structure determination of immobilised biological samples as was recently shown for a small microcrystalline protein. In this work, we show that highly resolved spectra may be obtained from a system composed of a receptor–toxin complex. The NMR sample used for our studies consists of a membrane preparation of the nicotinic acetylcholine receptor from the electric organ of Torpedo californica which was incubated with uniformly 13C‐,15N‐labelled neurotoxin II. Despite the large size of the ligand–receptor complex (>290 kDa) and the high lipid content of the sample, we were able to detect and identify residues from the ligand. The comparison with solution NMR data of the free toxin indicates that its overall structure is very similar when bound to the receptor, but significant changes were observed for one isoleucine.

Bacterial Expression, NMR, and Electrophysiology Analysis of Chimeric Short/Long-chain α-Neurotoxins Acting on Neuronal Nicotinic Receptors

Different snake venom neurotoxins block distinct subtypes of nicotinic acetylcholine receptors (nAChR). Short-chain α-neurotoxins preferentially inhibit muscle-type nAChRs, whereas long-chain α-neurotoxins block both muscle-type and α7 homooligomeric neuronal nAChRs. An additional disulfide in the central loop of α- and κ-neurotoxins is essential for their action on the α7 and α3β2 nAChRs, respectively. Design of novel toxins may help to better understand their subtype specificity. To address this problem, two chimeric toxins were produced by bacterial expression, a short-chain neurotoxin II Naja oxiana with the grafted disulfide-containing loop from long-chain neurotoxin I from N. oxiana, while a second chimera contained an additional A29K mutation, the most pronounced difference in the central loop tip between long-chain α-neurotoxins and κ-neurotoxins. The correct folding and structural stability for both chimeras were shown by 1H and 1H-15N NMR spectroscopy. Electrophysiology experiments on the nAChRs expressed in Xenopus oocytes revealed that the first chimera and neurotoxin I blockα7 nAChRs with similar potency (IC50 6.1 and 34 nm, respectively). Therefore, the disulfide-confined loop endows neurotoxin II with full activity of long-chain α-neurotoxin and the C-terminal tail in neurotoxin I is not essential for binding. The A29K mutation of the chimera considerably diminished the affinity for α7 nAChR (IC50 126 nm) but did not convey activity at α3β2 nAChRs. Docking of both chimeras toα7 andα3β2 nAChRs was possible, but complexes with the latter were not stable at molecular dynamics simulations. Apparently, some other residues and dimeric organization of κ-neurotoxins underlie their selectivity for α3β2 nAChRs.

Loop 3 of Short Neurotoxin II is an Additional Interaction Site with Membrane-bound Nicotinic Acetylcholine Receptor as Detected by Solid-state NMR Spectroscopy

The contact area of neurotoxin II from Naja naja oxiana when interacting with the membrane-bound nicotinic acetylcholine receptor from Torpedo californica was determined by solid-state, magic-angle spinning NMR spectroscopy. For this purpose, the carbon signals for more than 90% of the residues of the bound neurotoxin were assigned. Differences between the solution and solid-state chemical shifts of the free and bound form of the toxin are confined to distinct surface regions. Loop II of the short toxin was identified as the main interaction site. In addition, loop III of neurotoxin II shows several strong responses defining an additional interaction site. A comparison with the structures of alpha-cobratoxin bound to the acetylcholine-binding protein from snail species Lymnaea stagnalis and Aplysia californica, and of alpha-bungarotoxin bound to an extracellular domain of an alpha-subunit of the receptor reveals different contact areas for long and short alpha-neurotoxins.

Thermodynamics of denaturation of complexes of barnase and binase with barstar.

Differential scanning calorimetry was used to study the thermodynamics of denaturation of protein complexes for which the free energy stabilizing the complexes varied between -8 and -16 kcal/mol. The proteins studied were the ribonucleases barnase and binase, their inhibitor barstar and mutants thereof, and complexes between the two. The results are in good agreement with the model developed by Brandts and Lin for studying the thermodynamics of denaturation for tight complexes between two proteins which undergo two-state thermal unfolding transitions.

Recombinant targeted toxin based on HER2-specific DARPin possesses a strong selective cytotoxic effect in vitro and a potent antitumor activity in vivo.

DARPins fused with other proteins are promising non-immunoglobulin scaffolds for specific binding to target cells. In this study HER2-specific DARPin (DARPin_9-29) was used as a tumor-targeting moiety for the delivery of a cytotoxic agent - the fragment of Pseudomonas aeruginosa exotoxin A. It was determined that DARPin-PE40 possesses a considerable cytotoxic activity and induces apoptosis in HER2-positive cells. Cytotoxic effect of DARPin-PE40 strongly correlates with the HER2 expression level. The effect of intravenous administration of DARPin-PE40 was tested in the xenograft model of breast cancer. It was shown that treatment of animals with DARPin-PE40 caused strong and prolonged suppression of xenograft tumor growth.