Alexander Arseniev | Moscow Institute of Physics and Technology (original) (raw)

Papers by Alexander Arseniev

Journal of Biological Chemistry, 2015

Background: Cobra's "three-finger" nonconventional toxin WTX allosterically modulates muscarinic ... more Background: Cobra's "three-finger" nonconventional toxin WTX allosterically modulates muscarinic receptors (mAChRs). Results: Activity of several WTX mutants was analyzed; toxin spatial structure and dynamics were determined; and complexes of toxin with M1 and M3 mAChRs were modeled. Conclusion: Flexible loop II is the major determinant for toxin binding to different mAChRs. Significance: Structural framework for rationalization of target-specific positive/negative allosteric regulation of mAChRs is provided. Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional "three-finger" snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1-and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by "three-finger" snake neurotoxins.

Journal of Biological Chemistry, 2015

Background: Cobra's "three-finger" nonconventional toxin WTX allosterically modulates muscarinic ... more Background: Cobra's "three-finger" nonconventional toxin WTX allosterically modulates muscarinic receptors (mAChRs). Results: Activity of several WTX mutants was analyzed; toxin spatial structure and dynamics were determined; and complexes of toxin with M1 and M3 mAChRs were modeled. Conclusion: Flexible loop II is the major determinant for toxin binding to different mAChRs. Significance: Structural framework for rationalization of target-specific positive/negative allosteric regulation of mAChRs is provided. Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional "three-finger" snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1-and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by "three-finger" snake neurotoxins.

Biochemistry, 2008

Latarcins, linear peptides from the Lachesana tarabaevi spider venom, exhibit a broad-spectrum an... more Latarcins, linear peptides from the Lachesana tarabaevi spider venom, exhibit a broad-spectrum antimicrobial activity, likely acting on the bacterial cytoplasmic membrane. We study their spatial structures and interaction with model membranes by a combination of experimental and theoretical methods to reveal the structure-activity relationship. In this work, a 26 amino acid peptide, Ltc1, was investigated. Its spatial structure in detergent micelles was determined by (1)H nuclear magnetic resonance (NMR) and refined by Monte Carlo simulations in an implicit water-octanol slab. The Ltc1 molecule was found to form a straight uninterrupted amphiphilic helix comprising 8-23 residues. A dye-leakage fluorescent assay and (31)P NMR spectroscopy established that the peptide does not induce the release of fluorescent marker nor deteriorate the bilayer structure of the membranes. The voltage-clamp technique showed that Ltc1 induces the current fluctuations through planar membranes when the sign of the applied potential coincides with the one across the bacterial inner membrane. This implies that Ltc1 acts on the membranes via a specific mechanism, which is different from the carpet mode demonstrated by another latarcin, Ltc2a, featuring a helix-hinge-helix structure with a hydrophobicity gradient along the peptide chain. In contrast, the hydrophobic surface of the Ltc1 helix is narrow-shaped and extends with no gradient along the axis. We have also disclosed a number of peptides, structurally homologous to Ltc1 and exhibiting similar membrane activity. This indicates that the hydrophobic pattern of the Ltc1 helix and related antimicrobial peptides specifies their activity mechanism. The latter assumes the formation of variable-sized lesions, which depend upon the potential across the membrane.

Biochemical Journal, 2005

The CTs (cytotoxins) I and II are positively charged three-finger folded proteins from venom of N... more The CTs (cytotoxins) I and II are positively charged three-finger folded proteins from venom of Naja oxiana (the Central Asian cobra). They belong to S- and P-type respectively based on Ser-28 and Pro-30 residues within a putative phospholipid bilayer binding site. Previously, we investigated the interaction of CTII with multilamellar liposomes of dipalmitoylphosphatidylglycerol by wide-line 31P-NMR spectroscopy. To compare interactions of these proteins with phospholipids, we investigated the interaction of CTI with the multilamellar liposomes of dipalmitoylphosphatidylglycerol analogously. The effect of CTI on the chemical shielding anisotropy and deformation of the liposomes in the magnetic field was determined at different temperatures and lipid/protein ratios. It was found that both the proteins do not affect lipid organization in the gel state. In the liquid crystalline state of the bilayer they disturb lipid packing. To get insight into the interactions of the toxins with mem...

Theoretical Chemistry Accounts: Theory, Computation, and Modeling (Theoretica Chimica Acta), 2001

A heterogeneous implicit membrane-mimetic model is applied to simulations of membrane proteins. T... more A heterogeneous implicit membrane-mimetic model is applied to simulations of membrane proteins. The model employs atomic solvation parameters for gas± water and gas±cyclohexane transfer. It is used to analyze structure, energetics, and orientation with respect to the bilayer of two polypeptides with dierent modes of membrane binding ± hydrophobic segment of human glycophorin A (GpA) and cytotoxin II from Naja naja oxiana snake venom (CTX). The native state of GpA represents a transmembrane (TM) a helix, while CTX is a water-soluble protein, which is able to interact with the cell membrane. The conformational space of the polypeptides was explored in Monte Carlo simulations. The results show that the most stable conformers of GpA represent a TM a helix. They are additionally stabilized by an applied TM voltage. The results also show that CTX inserts with its three loops, does not cross the hydrophobic layer, and stays partially immersed in the membrane. This agrees well with the experimental data, thus con®rming the validity of the solvation model.

Biophysical Journal, 2002

Incorporation of ␤-sheet proteins into membrane is studied theoretically for the first time, and ... more Incorporation of ␤-sheet proteins into membrane is studied theoretically for the first time, and the results are validated by the direct experimental data. Using Monte Carlo simulations with implicit membrane, we explore spatial structure, energetics, polarity, and mode of insertion of two cardiotoxins with different membrane-destabilizing activity. Both proteins, classified as P-and S-type cardiotoxins, are found to retain the overall "three-finger" fold interacting with membrane core and lipid/water interface by the tips of the "fingers" (loops). The insertion critically depends upon the structure, hydrophobicity, and electrostatics of certain regions. The simulations reveal apparently distinct binding modes for S-and P-type cardiotoxins via the first loop or through all three loops, respectively. This rationalizes an earlier empirical classification of cardiotoxins into S-and P-type, and provides a basis for the analysis of experimental data on their membrane affinities. Accomplished with our previous simulations of membrane ␣-helices, the computational method may be used to study partitioning of proteins with diverse folds into lipid bilayers.

FEBS letters, Jan 3, 2014

Toll-like receptors (TLRs) take part in both the innate and adaptive immune systems. The role of ... more Toll-like receptors (TLRs) take part in both the innate and adaptive immune systems. The role of the transmembrane domain in TLR signaling is still elusive, while its importance for the TLR activation was clearly demonstrated. In the present study the ability of the TLR3 transmembrane domain to form dimers and trimers in detergent micelles was shown by solution NMR spectroscopy. Spatial structures and free energy magnitudes were determined for the TLR3 transmembrane domain in dimeric and trimeric states, and two possible surfaces that may be used for the helix-helix interaction by the full-length TLR3 were revealed.

Biochemical and biophysical research communications, Jan 22, 2014

A new defensin Lc-def, isolated from germinated seeds of the lentil Lens culinaris, has molecular... more A new defensin Lc-def, isolated from germinated seeds of the lentil Lens culinaris, has molecular mass 5440.4Da and consists of 47 amino acid residues. Lc-def and its (15)N-labeled analog were overexpressed in Escherichia coli. Antimicrobial activity of the recombinant protein was examined, and its spatial structure, dynamics, and interaction with lipid vesicles were studied by NMR spectroscopy. It was shown that Lc-def is active against fungi, but does not inhibit the growth of Gram-positive and Gram-negative bacteria. The peptide is monomeric in aqueous solution and contains one α-helix and triple-stranded β-sheet, which form cysteine-stabilized αβ motif (CSαβ) previously found in other plant defensins. The sterically neighboring loop1 and loop3 protrude from the defensin core and demonstrate significant mobility on the μs-ms timescale. Lc-def does not bind to the zwitterionic lipid (POPC) vesicles but interacts with the partially anionic (POPC/DOPG, 7:3) membranes under low-salt ...

Structure (London, England : 1993), Jan 5, 2014

Transmembrane signaling by receptor tyrosine kinases (RTKs) entails ligand-mediated dimerization ... more Transmembrane signaling by receptor tyrosine kinases (RTKs) entails ligand-mediated dimerization and structural rearrangement of the extracellular domains. RTK activation also depends on the specific orientation of the transmembrane domain (TMD) helices, as suggested by pathogenic, constitutively active RTK mutants. Such mutant TMDs carry polar amino acids promoting stable transmembrane helix dimerization, which is essential for kinase activation. We investigated the effect of polar amino acids introduced into the TMD of vascular endothelial growth factor receptor 2, regulating blood vessel homeostasis. Two mutants showed constitutive kinase activity, suggesting that precise TMD orientation is mandatory for kinase activation. Nuclear magnetic resonance spectroscopy revealed that TMD helices in activated constructs were rotated by 180° relative to the interface of the wild-type conformation, confirming that ligand-mediated receptor activation indeed results from transmembrane helix r...

Journal of Biological Chemistry, 2014

We present a structural and functional study of a sodium channel activation inhibitor from crab s... more We present a structural and functional study of a sodium channel activation inhibitor from crab spider venom. Hm-3 is an insecticidal peptide toxin consisting of 35 amino acid residues from the spider Heriaeus melloteei (Thomisidae). We produced Hm-3 recombinantly in Escherichia coli and determined its structure by NMR spectroscopy. Typical for spider toxins, Hm-3 was found to adopt the so-called "inhibitor cystine knot" or "knottin" fold stabilized by three disulfide bonds. Its molecule is amphiphilic with a hydrophobic ridge on the surface enriched in aromatic residues and surrounded by positive charges. Correspondingly, Hm-3 binds to both neutral and negatively charged lipid vesicles. Electrophysiological studies showed that at a concentration of 1 μm Hm-3 effectively inhibited a number of mammalian and insect sodium channels. Importantly, Hm-3 shifted the dependence of channel activation to more positive voltages. Moreover, the inhibition was voltage-dependent, and strong depolarizing prepulses attenuated Hm-3 activity. The toxin is therefore concluded to represent the first sodium channel gating modifier from an araneomorph spider and features a "membrane access" mechanism of action. Its amino acid sequence and position of the hydrophobic cluster are notably different from other known gating modifiers from spider venom, all of which are described from mygalomorph species. We hypothesize parallel evolution of inhibitor cystine knot toxins from Araneomorphae and Mygalomorphae suborders.

Theoretical Chemistry Accounts: Theory, Computation, and Modeling (Theoretica Chimica Acta), 2001

A heterogeneous implicit membrane-mimetic model is applied to simulations of membrane proteins. T... more A heterogeneous implicit membrane-mimetic model is applied to simulations of membrane proteins. The model employs atomic solvation parameters for gas± water and gas±cyclohexane transfer. It is used to analyze structure, energetics, and orientation with respect to the bilayer of two polypeptides with dierent modes of membrane binding ± hydrophobic segment of human glycophorin A (GpA) and cytotoxin II from Naja naja oxiana snake venom (CTX). The native state of GpA represents a transmembrane (TM) a helix, while CTX is a water-soluble protein, which is able to interact with the cell membrane. The conformational space of the polypeptides was explored in Monte Carlo simulations. The results show that the most stable conformers of GpA represent a TM a helix. They are additionally stabilized by an applied TM voltage. The results also show that CTX inserts with its three loops, does not cross the hydrophobic layer, and stays partially immersed in the membrane. This agrees well with the experimental data, thus con®rming the validity of the solvation model.

FEBS Journal, 2006

Antimicrobial peptides (AMPs) are ubiquitous agents that play a crucial role in the host defense ... more Antimicrobial peptides (AMPs) are ubiquitous agents that play a crucial role in the host defense systems of bacteria, fungi, plants and animals . In higher vertebrates, these peptides work in synergy with the adaptive immune system and form the basis of 'so-called' innate immunity . At the same time, AMPs of plants provide one of the major barriers for invading pathogens and significantly enhance the Keywords cystine knot; divalent cation-binding site; natural abundance 13 C-NMR spectroscopy; knottin; quantitative J-correlation Correspondence A. S. Arseniev, Shemyakin-Ovchinnikov

FEBS Journal, 2006

alpha-Conotoxins are small peptides from cone snail venoms that function as nicotinic acetylcholi... more alpha-Conotoxins are small peptides from cone snail venoms that function as nicotinic acetylcholine receptor (nAChR)-competitive antagonists differentiating between nAChR subtypes. Current understanding about the mechanism of these selective interactions is based largely on mutational analyses, which identify amino acids in the toxin and nAChR that determine the energetics of ligand binding. To identify regions of the nAChR involved in alpha-conotoxin binding by use of photoactivated cross-linking, two benzoylphenylalanine (Bpa) analogs of alpha-conotoxin GI, GI(Bpa12) and GI(Bpa4), were synthesized by replacing the respective residues with Bpa, and their (1)H-NMR structures were determined. Both analogs preserved the GI conformation, but only GI(Bpa12) displaced (125)I-labeled GI from the Torpedo californica nAChR. (125)I-labeled GI(Bpa12) bound to two sites on the receptor (K(d) 13 and 1800 nM), and on UV irradiation specifically photolabeled the alpha, gamma and delta subunits. Photolabeling sites were mapped by selective proteolysis and enzymatic deglycosylation, combined with SDS/PAGE, HPLC and Edman degradation. In the alpha subunit, cobratoxin-inhibited incorporation was limited to the 22-kDa fragment beginning at alphaSer173 and containing the agonist-binding site segment C. In the gamma subunit, radioactivity was localized to two distinct peptides containing agonist-binding site segments F and D: nonglycosylated 24-kDa and glycosylated 13-kDa fragments starting at gammaAla167 and gammaAla49, respectively. The labeling of these fragments is discussed in terms of a model of GI(Bpa12) bound to the extracellular domain of the Torpedo nAChR homology model derived from the cryo-electron microscopy structure of Torpedo marmorata nAChR and X-ray crystal structures of snail acetylcholine-binding protein complexes with agonists and antagonists.

Journal of Biological Chemistry, 2014

Journal of Biological Chemistry, 2014

Background: Protein folds differ in size and complexity and hence in their utility for engineerin... more Background: Protein folds differ in size and complexity and hence in their utility for engineering purposes. Results: The three-dimensional structure of wheat antifungal peptide Tk-AMP-X2 was investigated, and a new functionality was engineered based on its ␣-hairpin scaffold. Conclusion: ␣-Hairpinins are an attractive simple structural template for functional engineering and drug design. Significance: The repertoire of available scaffolds for protein engineering is broadened.

Current Medicinal Chemistry, 2007

Hydrophobic interactions play a key role in the folding and maintenance of the 3-dimensional stru... more Hydrophobic interactions play a key role in the folding and maintenance of the 3-dimensional structure of proteins, as well as in the binding of ligands (e.g. drugs) to protein targets. Therefore, quantitative assessment of spatial hydrophobic (lipophilic) properties of these molecules is indispensable for the development of efficient computational methods in drug design. One possible solution to the problem lies in application of a concept of the 3-dimensional molecular hydrophobicity potential (MHP). The formalism of MHP utilizes a set of atomic physicochemical parameters evaluated from octanol-water partition coefficients (log P) of numerous chemical compounds. It permits detailed assessment of the hydrophobic and/or hydrophilic properties of various parts of molecules and may be useful in analysis of protein-protein and protein-ligand interactions. This review surveys recent applications of MHP-based techniques to a number of biologically relevant tasks. Among them are: (i) Detailed assessment of hydrophobic/hydrophilic organization of proteins; (ii) Application of this data to the modeling of structure, dynamics, and function of globular and membrane proteins, membrane-active peptides, etc. (iii) Employment of the MHP-based criteria in docking simulations for ligands binding to receptors. It is demonstrated that the application of the MHP-based techniques in combination with other molecular modeling tools (e.g. Monte Carlo and molecular dynamics simulations, docking, etc.) permits significant improvement to the standard computational approaches, provides additional important insights into the intimate molecular mechanisms driving protein assembling in water and in biological membranes, and helps in the computer-aided drug discovery process.

Chemistry & Biodiversity, 2007

Antiamoebin I (Aam-I) is a membrane-active peptaibol antibiotic isolated from fungal species belo... more Antiamoebin I (Aam-I) is a membrane-active peptaibol antibiotic isolated from fungal species belonging to the genera Cephalosporium, Emericellopsis, Gliocladium, and Stilbella. Antiamoebin I has the amino acid sequence: Ac-Phe 1 -Aib-Aib-Aib-Iva-Gly-Leu-Aib 8 -Aib-Hyp-Gln-Iva-Hyp-Aib-Pro-Phl 16 . By using the uniformly 13 C, 15 N-labeled sample of Aam-I, the set of conformationally dependent J couplings and 3h J NC couplings through H-bonds were measured. Analysis of these data along with the data on magnetic nonequivalence of the 13 C b nuclei (Dd( 13 C b )) in Aib and Iva residues allowed us to draw the univocal conclusion that the N-terminal part (Phe 1 -Gly 6 ) of Aam-I in MeOH solution is in fast exchange between the right-handed and left-handed 3 10 -helical conformations, with an approximately equal population of both states. An additional conformational exchange process was found at the Aib 8 residue. The 15 N-NMR-relaxation and CD-spectroscopy measurements confirmed these findings. Molecular modeling and Monte Carlo simulations revealed that both exchange processes are correlated and coupled with significant hinge-bending motions around the Aib 8 residue. Our results explain relatively low activity of Aam-I with respect to other 15-amino acid residue peptaibols (for example, zervamicin) in functional and biological tests. The high dynamic propensity possibly prevents both initial binding of the antiamoebin to the membrane and subsequent formation of stable ionic channels according to the barrel-stave mechanism. ¼ 139.4 ms). Peaks are labeled by residue numbers and subdivided in four classes: I denotes intraresidual connectivity through 2 J NC coupling constant; S denotes sequential connectivity through 1 J NC coupling constant, H denotes long-range connectivity through 3h J NC (across H-bond) coupling constant, B denotes possible bifurcated H-bond connectivity. The low intensity (invisible under drawing threshold) cross-peaks are encircled. b)

Biophysical Journal, 2011

proteins, even alpha-helical ones, because the contribution of the backbone conformation is subtr... more proteins, even alpha-helical ones, because the contribution of the backbone conformation is subtracted from our final measurements. We were also able to use OMPLA to measure how the energetics of side-chain insertion vary with depth in the bilayer. Both Arginine and Leucine have their most extreme insertion energies when they are closest to the middle of the hydrophobic region of OMPLA. Further, we carried out a double mutant cycle with Arginines and discovered that the insertion of a second Arginine is aided by the insertion of the first. This result is particularly relevant for understanding the function of the voltage sensing domains of some ion channels, which may involve multiple Arginines penetrating the lipid bilayer.

Journal of Biological Chemistry, 2015

Background: Cobra's "three-finger" nonconventional toxin WTX allosterically modulates muscarinic ... more Background: Cobra's "three-finger" nonconventional toxin WTX allosterically modulates muscarinic receptors (mAChRs). Results: Activity of several WTX mutants was analyzed; toxin spatial structure and dynamics were determined; and complexes of toxin with M1 and M3 mAChRs were modeled. Conclusion: Flexible loop II is the major determinant for toxin binding to different mAChRs. Significance: Structural framework for rationalization of target-specific positive/negative allosteric regulation of mAChRs is provided. Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional "three-finger" snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1-and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by "three-finger" snake neurotoxins.

Journal of Biological Chemistry, 2015

Background: Cobra's "three-finger" nonconventional toxin WTX allosterically modulates muscarinic ... more Background: Cobra's "three-finger" nonconventional toxin WTX allosterically modulates muscarinic receptors (mAChRs). Results: Activity of several WTX mutants was analyzed; toxin spatial structure and dynamics were determined; and complexes of toxin with M1 and M3 mAChRs were modeled. Conclusion: Flexible loop II is the major determinant for toxin binding to different mAChRs. Significance: Structural framework for rationalization of target-specific positive/negative allosteric regulation of mAChRs is provided. Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional "three-finger" snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1-and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by "three-finger" snake neurotoxins.

Biochemistry, 2008

Latarcins, linear peptides from the Lachesana tarabaevi spider venom, exhibit a broad-spectrum an... more Latarcins, linear peptides from the Lachesana tarabaevi spider venom, exhibit a broad-spectrum antimicrobial activity, likely acting on the bacterial cytoplasmic membrane. We study their spatial structures and interaction with model membranes by a combination of experimental and theoretical methods to reveal the structure-activity relationship. In this work, a 26 amino acid peptide, Ltc1, was investigated. Its spatial structure in detergent micelles was determined by (1)H nuclear magnetic resonance (NMR) and refined by Monte Carlo simulations in an implicit water-octanol slab. The Ltc1 molecule was found to form a straight uninterrupted amphiphilic helix comprising 8-23 residues. A dye-leakage fluorescent assay and (31)P NMR spectroscopy established that the peptide does not induce the release of fluorescent marker nor deteriorate the bilayer structure of the membranes. The voltage-clamp technique showed that Ltc1 induces the current fluctuations through planar membranes when the sign of the applied potential coincides with the one across the bacterial inner membrane. This implies that Ltc1 acts on the membranes via a specific mechanism, which is different from the carpet mode demonstrated by another latarcin, Ltc2a, featuring a helix-hinge-helix structure with a hydrophobicity gradient along the peptide chain. In contrast, the hydrophobic surface of the Ltc1 helix is narrow-shaped and extends with no gradient along the axis. We have also disclosed a number of peptides, structurally homologous to Ltc1 and exhibiting similar membrane activity. This indicates that the hydrophobic pattern of the Ltc1 helix and related antimicrobial peptides specifies their activity mechanism. The latter assumes the formation of variable-sized lesions, which depend upon the potential across the membrane.

Biochemical Journal, 2005

The CTs (cytotoxins) I and II are positively charged three-finger folded proteins from venom of N... more The CTs (cytotoxins) I and II are positively charged three-finger folded proteins from venom of Naja oxiana (the Central Asian cobra). They belong to S- and P-type respectively based on Ser-28 and Pro-30 residues within a putative phospholipid bilayer binding site. Previously, we investigated the interaction of CTII with multilamellar liposomes of dipalmitoylphosphatidylglycerol by wide-line 31P-NMR spectroscopy. To compare interactions of these proteins with phospholipids, we investigated the interaction of CTI with the multilamellar liposomes of dipalmitoylphosphatidylglycerol analogously. The effect of CTI on the chemical shielding anisotropy and deformation of the liposomes in the magnetic field was determined at different temperatures and lipid/protein ratios. It was found that both the proteins do not affect lipid organization in the gel state. In the liquid crystalline state of the bilayer they disturb lipid packing. To get insight into the interactions of the toxins with mem...

Theoretical Chemistry Accounts: Theory, Computation, and Modeling (Theoretica Chimica Acta), 2001

A heterogeneous implicit membrane-mimetic model is applied to simulations of membrane proteins. T... more A heterogeneous implicit membrane-mimetic model is applied to simulations of membrane proteins. The model employs atomic solvation parameters for gas± water and gas±cyclohexane transfer. It is used to analyze structure, energetics, and orientation with respect to the bilayer of two polypeptides with dierent modes of membrane binding ± hydrophobic segment of human glycophorin A (GpA) and cytotoxin II from Naja naja oxiana snake venom (CTX). The native state of GpA represents a transmembrane (TM) a helix, while CTX is a water-soluble protein, which is able to interact with the cell membrane. The conformational space of the polypeptides was explored in Monte Carlo simulations. The results show that the most stable conformers of GpA represent a TM a helix. They are additionally stabilized by an applied TM voltage. The results also show that CTX inserts with its three loops, does not cross the hydrophobic layer, and stays partially immersed in the membrane. This agrees well with the experimental data, thus con®rming the validity of the solvation model.

Biophysical Journal, 2002

Incorporation of ␤-sheet proteins into membrane is studied theoretically for the first time, and ... more Incorporation of ␤-sheet proteins into membrane is studied theoretically for the first time, and the results are validated by the direct experimental data. Using Monte Carlo simulations with implicit membrane, we explore spatial structure, energetics, polarity, and mode of insertion of two cardiotoxins with different membrane-destabilizing activity. Both proteins, classified as P-and S-type cardiotoxins, are found to retain the overall "three-finger" fold interacting with membrane core and lipid/water interface by the tips of the "fingers" (loops). The insertion critically depends upon the structure, hydrophobicity, and electrostatics of certain regions. The simulations reveal apparently distinct binding modes for S-and P-type cardiotoxins via the first loop or through all three loops, respectively. This rationalizes an earlier empirical classification of cardiotoxins into S-and P-type, and provides a basis for the analysis of experimental data on their membrane affinities. Accomplished with our previous simulations of membrane ␣-helices, the computational method may be used to study partitioning of proteins with diverse folds into lipid bilayers.

FEBS letters, Jan 3, 2014

Toll-like receptors (TLRs) take part in both the innate and adaptive immune systems. The role of ... more Toll-like receptors (TLRs) take part in both the innate and adaptive immune systems. The role of the transmembrane domain in TLR signaling is still elusive, while its importance for the TLR activation was clearly demonstrated. In the present study the ability of the TLR3 transmembrane domain to form dimers and trimers in detergent micelles was shown by solution NMR spectroscopy. Spatial structures and free energy magnitudes were determined for the TLR3 transmembrane domain in dimeric and trimeric states, and two possible surfaces that may be used for the helix-helix interaction by the full-length TLR3 were revealed.

Biochemical and biophysical research communications, Jan 22, 2014

A new defensin Lc-def, isolated from germinated seeds of the lentil Lens culinaris, has molecular... more A new defensin Lc-def, isolated from germinated seeds of the lentil Lens culinaris, has molecular mass 5440.4Da and consists of 47 amino acid residues. Lc-def and its (15)N-labeled analog were overexpressed in Escherichia coli. Antimicrobial activity of the recombinant protein was examined, and its spatial structure, dynamics, and interaction with lipid vesicles were studied by NMR spectroscopy. It was shown that Lc-def is active against fungi, but does not inhibit the growth of Gram-positive and Gram-negative bacteria. The peptide is monomeric in aqueous solution and contains one α-helix and triple-stranded β-sheet, which form cysteine-stabilized αβ motif (CSαβ) previously found in other plant defensins. The sterically neighboring loop1 and loop3 protrude from the defensin core and demonstrate significant mobility on the μs-ms timescale. Lc-def does not bind to the zwitterionic lipid (POPC) vesicles but interacts with the partially anionic (POPC/DOPG, 7:3) membranes under low-salt ...

Structure (London, England : 1993), Jan 5, 2014

Transmembrane signaling by receptor tyrosine kinases (RTKs) entails ligand-mediated dimerization ... more Transmembrane signaling by receptor tyrosine kinases (RTKs) entails ligand-mediated dimerization and structural rearrangement of the extracellular domains. RTK activation also depends on the specific orientation of the transmembrane domain (TMD) helices, as suggested by pathogenic, constitutively active RTK mutants. Such mutant TMDs carry polar amino acids promoting stable transmembrane helix dimerization, which is essential for kinase activation. We investigated the effect of polar amino acids introduced into the TMD of vascular endothelial growth factor receptor 2, regulating blood vessel homeostasis. Two mutants showed constitutive kinase activity, suggesting that precise TMD orientation is mandatory for kinase activation. Nuclear magnetic resonance spectroscopy revealed that TMD helices in activated constructs were rotated by 180° relative to the interface of the wild-type conformation, confirming that ligand-mediated receptor activation indeed results from transmembrane helix r...

Journal of Biological Chemistry, 2014

We present a structural and functional study of a sodium channel activation inhibitor from crab s... more We present a structural and functional study of a sodium channel activation inhibitor from crab spider venom. Hm-3 is an insecticidal peptide toxin consisting of 35 amino acid residues from the spider Heriaeus melloteei (Thomisidae). We produced Hm-3 recombinantly in Escherichia coli and determined its structure by NMR spectroscopy. Typical for spider toxins, Hm-3 was found to adopt the so-called "inhibitor cystine knot" or "knottin" fold stabilized by three disulfide bonds. Its molecule is amphiphilic with a hydrophobic ridge on the surface enriched in aromatic residues and surrounded by positive charges. Correspondingly, Hm-3 binds to both neutral and negatively charged lipid vesicles. Electrophysiological studies showed that at a concentration of 1 μm Hm-3 effectively inhibited a number of mammalian and insect sodium channels. Importantly, Hm-3 shifted the dependence of channel activation to more positive voltages. Moreover, the inhibition was voltage-dependent, and strong depolarizing prepulses attenuated Hm-3 activity. The toxin is therefore concluded to represent the first sodium channel gating modifier from an araneomorph spider and features a "membrane access" mechanism of action. Its amino acid sequence and position of the hydrophobic cluster are notably different from other known gating modifiers from spider venom, all of which are described from mygalomorph species. We hypothesize parallel evolution of inhibitor cystine knot toxins from Araneomorphae and Mygalomorphae suborders.

Theoretical Chemistry Accounts: Theory, Computation, and Modeling (Theoretica Chimica Acta), 2001

A heterogeneous implicit membrane-mimetic model is applied to simulations of membrane proteins. T... more A heterogeneous implicit membrane-mimetic model is applied to simulations of membrane proteins. The model employs atomic solvation parameters for gas± water and gas±cyclohexane transfer. It is used to analyze structure, energetics, and orientation with respect to the bilayer of two polypeptides with dierent modes of membrane binding ± hydrophobic segment of human glycophorin A (GpA) and cytotoxin II from Naja naja oxiana snake venom (CTX). The native state of GpA represents a transmembrane (TM) a helix, while CTX is a water-soluble protein, which is able to interact with the cell membrane. The conformational space of the polypeptides was explored in Monte Carlo simulations. The results show that the most stable conformers of GpA represent a TM a helix. They are additionally stabilized by an applied TM voltage. The results also show that CTX inserts with its three loops, does not cross the hydrophobic layer, and stays partially immersed in the membrane. This agrees well with the experimental data, thus con®rming the validity of the solvation model.

FEBS Journal, 2006

Antimicrobial peptides (AMPs) are ubiquitous agents that play a crucial role in the host defense ... more Antimicrobial peptides (AMPs) are ubiquitous agents that play a crucial role in the host defense systems of bacteria, fungi, plants and animals . In higher vertebrates, these peptides work in synergy with the adaptive immune system and form the basis of 'so-called' innate immunity . At the same time, AMPs of plants provide one of the major barriers for invading pathogens and significantly enhance the Keywords cystine knot; divalent cation-binding site; natural abundance 13 C-NMR spectroscopy; knottin; quantitative J-correlation Correspondence A. S. Arseniev, Shemyakin-Ovchinnikov

FEBS Journal, 2006

alpha-Conotoxins are small peptides from cone snail venoms that function as nicotinic acetylcholi... more alpha-Conotoxins are small peptides from cone snail venoms that function as nicotinic acetylcholine receptor (nAChR)-competitive antagonists differentiating between nAChR subtypes. Current understanding about the mechanism of these selective interactions is based largely on mutational analyses, which identify amino acids in the toxin and nAChR that determine the energetics of ligand binding. To identify regions of the nAChR involved in alpha-conotoxin binding by use of photoactivated cross-linking, two benzoylphenylalanine (Bpa) analogs of alpha-conotoxin GI, GI(Bpa12) and GI(Bpa4), were synthesized by replacing the respective residues with Bpa, and their (1)H-NMR structures were determined. Both analogs preserved the GI conformation, but only GI(Bpa12) displaced (125)I-labeled GI from the Torpedo californica nAChR. (125)I-labeled GI(Bpa12) bound to two sites on the receptor (K(d) 13 and 1800 nM), and on UV irradiation specifically photolabeled the alpha, gamma and delta subunits. Photolabeling sites were mapped by selective proteolysis and enzymatic deglycosylation, combined with SDS/PAGE, HPLC and Edman degradation. In the alpha subunit, cobratoxin-inhibited incorporation was limited to the 22-kDa fragment beginning at alphaSer173 and containing the agonist-binding site segment C. In the gamma subunit, radioactivity was localized to two distinct peptides containing agonist-binding site segments F and D: nonglycosylated 24-kDa and glycosylated 13-kDa fragments starting at gammaAla167 and gammaAla49, respectively. The labeling of these fragments is discussed in terms of a model of GI(Bpa12) bound to the extracellular domain of the Torpedo nAChR homology model derived from the cryo-electron microscopy structure of Torpedo marmorata nAChR and X-ray crystal structures of snail acetylcholine-binding protein complexes with agonists and antagonists.

Journal of Biological Chemistry, 2014

Journal of Biological Chemistry, 2014

Background: Protein folds differ in size and complexity and hence in their utility for engineerin... more Background: Protein folds differ in size and complexity and hence in their utility for engineering purposes. Results: The three-dimensional structure of wheat antifungal peptide Tk-AMP-X2 was investigated, and a new functionality was engineered based on its ␣-hairpin scaffold. Conclusion: ␣-Hairpinins are an attractive simple structural template for functional engineering and drug design. Significance: The repertoire of available scaffolds for protein engineering is broadened.

Current Medicinal Chemistry, 2007

Hydrophobic interactions play a key role in the folding and maintenance of the 3-dimensional stru... more Hydrophobic interactions play a key role in the folding and maintenance of the 3-dimensional structure of proteins, as well as in the binding of ligands (e.g. drugs) to protein targets. Therefore, quantitative assessment of spatial hydrophobic (lipophilic) properties of these molecules is indispensable for the development of efficient computational methods in drug design. One possible solution to the problem lies in application of a concept of the 3-dimensional molecular hydrophobicity potential (MHP). The formalism of MHP utilizes a set of atomic physicochemical parameters evaluated from octanol-water partition coefficients (log P) of numerous chemical compounds. It permits detailed assessment of the hydrophobic and/or hydrophilic properties of various parts of molecules and may be useful in analysis of protein-protein and protein-ligand interactions. This review surveys recent applications of MHP-based techniques to a number of biologically relevant tasks. Among them are: (i) Detailed assessment of hydrophobic/hydrophilic organization of proteins; (ii) Application of this data to the modeling of structure, dynamics, and function of globular and membrane proteins, membrane-active peptides, etc. (iii) Employment of the MHP-based criteria in docking simulations for ligands binding to receptors. It is demonstrated that the application of the MHP-based techniques in combination with other molecular modeling tools (e.g. Monte Carlo and molecular dynamics simulations, docking, etc.) permits significant improvement to the standard computational approaches, provides additional important insights into the intimate molecular mechanisms driving protein assembling in water and in biological membranes, and helps in the computer-aided drug discovery process.

Chemistry & Biodiversity, 2007

Antiamoebin I (Aam-I) is a membrane-active peptaibol antibiotic isolated from fungal species belo... more Antiamoebin I (Aam-I) is a membrane-active peptaibol antibiotic isolated from fungal species belonging to the genera Cephalosporium, Emericellopsis, Gliocladium, and Stilbella. Antiamoebin I has the amino acid sequence: Ac-Phe 1 -Aib-Aib-Aib-Iva-Gly-Leu-Aib 8 -Aib-Hyp-Gln-Iva-Hyp-Aib-Pro-Phl 16 . By using the uniformly 13 C, 15 N-labeled sample of Aam-I, the set of conformationally dependent J couplings and 3h J NC couplings through H-bonds were measured. Analysis of these data along with the data on magnetic nonequivalence of the 13 C b nuclei (Dd( 13 C b )) in Aib and Iva residues allowed us to draw the univocal conclusion that the N-terminal part (Phe 1 -Gly 6 ) of Aam-I in MeOH solution is in fast exchange between the right-handed and left-handed 3 10 -helical conformations, with an approximately equal population of both states. An additional conformational exchange process was found at the Aib 8 residue. The 15 N-NMR-relaxation and CD-spectroscopy measurements confirmed these findings. Molecular modeling and Monte Carlo simulations revealed that both exchange processes are correlated and coupled with significant hinge-bending motions around the Aib 8 residue. Our results explain relatively low activity of Aam-I with respect to other 15-amino acid residue peptaibols (for example, zervamicin) in functional and biological tests. The high dynamic propensity possibly prevents both initial binding of the antiamoebin to the membrane and subsequent formation of stable ionic channels according to the barrel-stave mechanism. ¼ 139.4 ms). Peaks are labeled by residue numbers and subdivided in four classes: I denotes intraresidual connectivity through 2 J NC coupling constant; S denotes sequential connectivity through 1 J NC coupling constant, H denotes long-range connectivity through 3h J NC (across H-bond) coupling constant, B denotes possible bifurcated H-bond connectivity. The low intensity (invisible under drawing threshold) cross-peaks are encircled. b)

Biophysical Journal, 2011

proteins, even alpha-helical ones, because the contribution of the backbone conformation is subtr... more proteins, even alpha-helical ones, because the contribution of the backbone conformation is subtracted from our final measurements. We were also able to use OMPLA to measure how the energetics of side-chain insertion vary with depth in the bilayer. Both Arginine and Leucine have their most extreme insertion energies when they are closest to the middle of the hydrophobic region of OMPLA. Further, we carried out a double mutant cycle with Arginines and discovered that the insertion of a second Arginine is aided by the insertion of the first. This result is particularly relevant for understanding the function of the voltage sensing domains of some ion channels, which may involve multiple Arginines penetrating the lipid bilayer.