Konstantin S. Mineev


PeriodCountry, cityEducation institutionAdditional info
2001–2007 Russia, Dolgoprudnyi MIPT M.S.
2007–2010 Russia, Dolgoprudnyi MIPT Ph.D. in biophysics

Scientific interests

NMR spectroscopy, membrane mimetics, spatial structures of membrtane proteins, activation mechanisms of receptor tyrosine kinases, protein folding

Structure of low molecular weight compounds, luciferin/luciferase systems

Main scientific results

Determined spatial structures of more than 30 soluble and membrane proteins, elucidated several structures of low-molecular-weight compounds.

Scientific societies’ membership

FEBS member since 2011

Biophysical Society member in 2012-2013

Selected publications

  1. Kot E.F., Arseniev A.S., Mineev K.S. (2018). Behavior of Most Widely Spread Lipids in Isotropic Bicelles. Langmuir 34 (28), 8302–8313 [+]

    Isotropic bicelles are a widely used membrane mimetic for structural studies of membrane proteins and their transmembrane domains. Simple and cheap in preparation, they contain a patch of lipid bilayer that reproduces the native environment of membrane proteins. Despite the obvious power of bicelles in reproducing the various kinds of environments, the vast majority of structural studies employ the single lipid/detergent system. On the other hand, even if the alternative bicelle composition is used, the properties of mixtures are not characterized, and the mere presence of lipid bilayer and discoidal shape of bicelle particles is not confirmed. Here we present an extensive investigation of various bicellar mixtures and describe the behavior of bicelles with lipids other than classical DMPC, namely sphingomyelins (SM), phosphatidylethanolamines (PE), phosphatidylglycerols (PG), phosphatidylserines (PS), and cholesterol. These lipids are rarely used in modern structural biology, but can help a lot in understanding the influence of the membrane composition on the properties of both integral and peripheral membrane proteins. Additionally, the ability of diheptanoylphosphatidylcholine (DH7PC) to serve as a rim-forming agent was investigated. We followed the phase transitions as revealed by P NMR and size of particles measured by H NMR diffusion as the criteria of the proper morphology and structure of bicelles. As an outcome, we state that SM exclusively, and PG/PS in mixtures with zwitterionic lipids can form small isotropic bicelles, which reproduce the key features of lipid behavior in bilayers. Mixtures, containing exclusively the anionic lipids, fail to reveal the lipid phase transition and do not follow the size predicted for the ideal bicelle particles. PE and DH7PC are the unwanted components of bicellar mixtures, and cholesterol can be added to bicelles, however, with certain precautions. In combination with our several most recent works, this study provides a practical guide for the preparation of small isotropic bicelles.

  2. Kuldyushev N.A., Mineev K.S., Berkut A.A., Peigneur S., Arseniev A.S., Tytgat J., Grishin E.V., Vassilevski A.A. (2018). Refined structure of BeM9 reveals arginine hand, an overlooked structural motif in scorpion toxins affecting sodium channels. Proteins , [+]

    Sodium channel alpha-toxins from scorpion venom (α-NaTx) inhibit the inactivation of voltage-gated sodium channels. We used solution NMR to investigate the structure of BeM9 toxin from Mesobuthus eupeus scorpion, a prototype α-NaTx classified as an "α-like" toxin due to its wide spectrum of activity on insect and mammalian channels. We identified a new motif that we named "arginine hand", whereby arginine side chain forms several hydrogen bonds with main chain atoms. The arginine hand was found in the "specificity module", a part of the molecule that dictates toxin selectivity; and just single arginine-to-lysine point mutation drastically changed BeM9 selectivity profile. This article is protected by copyright. All rights reserved.

  3. Kot E.F., Goncharuk S.A., Arseniev A.S., Mineev K.S. (2018). Phase Transitions in Small Isotropic Bicelles. Langmuir 34 (11), 3426–3437 [+]

    Isotropic phospholipid bicelles are one of the most prospective membrane mimetics for the structural studies of membrane proteins in solution. Recent works provided an almost full set of data regarding the properties of isotropic bicelles; however, one major aspect of their behavior is still under consideration: the possible mixing between the lipid and detergent in the bilayer area. This problem may be resolved by studying the lipid phase transitions in bicelle particles. In the present work, we investigate two effects: phase transitions of bilayer lipids and temperature-induced growth of isotropic bicelles using the NMR spectroscopy. We propose an approach to study the phase transitions in isotropic bicelles based on the properties ofP NMR spectra of bilayer-forming lipids. We show that phase transitions in small bicelles are "fractional", particles with the liquid-crystalline and gel bilayers coexist in solution at certain temperatures. We study the effects of lipid fatty chain type and demonstrate that the behavior of various lipids in bilayers is reproduced in the isotropic bicelles. We show that the temperature-induced growth of isotropic bicelles is not related directly to the phase transition but is the result of the reversible fusion of bicelle particles. In accordance with our data, rim detergents also have an impact on phase transitions: detergents that resist the temperature-induced growth provide the narrowest and most expressed transitions at higher temperatures. We demonstrate clearly that phase transitions take place even in the smallest bicelles that are applicable for structural studies of membrane proteins by solution NMR spectroscopy. This last finding, together with other data draws a thick line under the long-lasting argument about the relevance of small isotropic bicelles. We show with certainty that the small bicelles can reproduce the most fundamental property of lipid membranes: the ability to undergo phase transition.

  4. Lesovoy D.M., Mineev K.S., Bragin P.E., Bocharova O.V., Bocharov E.V., Arseniev A.S. (2017). NMR relaxation parameters of methyl groups as a tool to map the interfaces of helix-helix interactions in membrane proteins. J. Biomol. NMR , [+]

    In the case of soluble proteins, chemical shift mapping is used to identify the intermolecular interfaces when the NOE-based calculations of spatial structure of the molecular assembly are impossible or impracticable. However, the reliability of the membrane protein interface mapping based on chemical shifts or other relevant parameters was never assessed. In the present work, we investigate the predictive power of various NMR parameters that can be used for mapping of helix-helix interfaces in dimeric TM domains. These parameters are studied on a dataset containing three structures of helical dimers obtained for two different proteins in various membrane mimetics. We conclude that the amide chemical shifts have very little predictive value, while the methyl chemical shifts could be used to predict interfaces, though with great care. We suggest an approach based on conversion of the carbon NMR relaxation parameters of methyl groups into parameters of motion, and one of such values, the characteristic time of methyl rotation, appears to be a reliable sensor of interhelix contacts in transmembrane domains. The carbon NMR relaxation parameters of methyl groups can be measured accurately and with high sensitivity and resolution, making the proposed parameter a useful tool for investigation of protein-protein interfaces even in large membrane proteins. An approach to build the models of transmembrane dimers based on perturbations of methyl parameters and TMDOCK software is suggested.

  5. Mineev K.S., Goncharuk S.A., Goncharuk M.V., Volynsky P.E., Novikova E.V., Aresinev A.S. (2017). Spatial structure of TLR4 transmembrane domain in bicelles provides the insight into the receptor activation mechanism. Sci Rep 7 (1), 6864 [+]

    Toll-like receptors (TLRs) play a key role in the innate and adaptive immune systems. While a lot of structural data is available for the extracellular and cytoplasmic domains of TLRs, and a model of the dimeric full-length TLR3 receptor in the active state was build, the conformation of the transmembrane (TM) domain and juxtamembrane regions in TLR dimers is still unclear. In the present work, we study the transmembrane and juxtamembrane parts of human TLR4 receptor using solution NMR spectroscopy in a variety of membrane mimetics, including phospholipid bicelles. We show that the juxtamembrane hydrophobic region of TLR4 includes a part of long TM α-helix. We report the dimerization interface of the TM domain and claim that long TM domains with transmembrane charged aminoacids is a common feature of human toll-like receptors. This fact is analyzed from the viewpoint of protein activation mechanism, and a model of full-length TLR4 receptor in the dimeric state has been proposed.

  6. Bozhanova N.G., Baranov M.S., Sarkisyan K.S., Gritcenko R., Mineev K.S., Golodukhina S.V., Baleeva N.S., Lukyanov K.A., Mishin A.S. (2017). Yellow and Orange Fluorescent Proteins with Tryptophan-based Chromophores. ACS Chem. Biol. 12 (7), 1867–1873 [+]

    Rapid development of new microscopy techniques exposed the need for genetically encoded fluorescent tags with special properties. Recent works demonstrated the potential of fluorescent proteins with tryptophan-based chromophores. We applied rational design and random mutagenesis to the monomeric red fluorescent protein FusionRed and found two groups of mutants carrying a tryptophan-based chromophore: with yellow (535 nm) or orange (565 nm) emission. On the basis of the properties of proteins, a model synthetic chromophore, and a computational modeling, we concluded that the presence of a ketone-containing chromophore in different isomeric forms can explain the observed yellow and orange phenotypes.

  7. Logashina Y.A., Solstad R.G., Mineev K.S., Korolkova Y.V., Mosharova I.V., Dyachenko I.A., Palikov V.A., Palikova Y.A., Murashev A.N., Arseniev A.S., Kozlov S.A., Stensvåg K., Haug T., Andreev Y.A. (2017). New Disulfide-Stabilized Fold Provides Sea Anemone Peptide to Exhibit Both Antimicrobial and TRPA1 Potentiating Properties. Toxins (Basel) 9 (5), [+]

    A novel bioactive peptide named τ-AnmTx Ueq 12-1 (short name Ueq 12-1) was isolated and characterized from the sea anemone Urticina eques. Ueq 12-1 is unique among the variety of known sea anemone peptides in terms of its primary and spatial structure. It consists of 45 amino acids including 10 cysteine residues with an unusual distribution and represents a new group of sea anemone peptides. The 3D structure of Ueq 12-1, determined by NMR spectroscopy, represents a new disulfide-stabilized fold partly similar to the defensin-like fold. Ueq 12-1 showed the dual activity of both a moderate antibacterial activity against Gram-positive bacteria and a potentiating activity on the transient receptor potential ankyrin 1 (TRPA1). Ueq 12-1 is a unique peptide potentiator of the TRPA1 receptor that produces analgesic and anti-inflammatory effects in vivo. The antinociceptive properties allow us to consider Ueq 12-1 as a potential analgesic drug lead with antibacterial properties.

  8. Kaskova Z.M., Dörr F.A., Petushkov V.N., Purtov K.V., Tsarkova A.S., Rodionova N.S., Mineev K.S., Guglya E.B., Kotlobay A., Baleeva N.S., Baranov M.S., Arseniev A.S., Gitelson J.I., Lukyanov S., Suzuki Y., Kanie S., Pinto E., DiMascio P., Waldenmaier H.E., Pereira T.A., Carvalho R.P., Oliveira A.G., Oba Y., Bastos E.L., Stevani C.V., Yampolsky I.V. (2017). Mechanism and Color Modulation of Fungal Bioluminescence. Sci Adv 3 (4), e1602847 [+]

    Bioluminescent fungi are spread throughout the globe, but details on their mechanism of light emission are still scarce. Usually, the process involves three key components: an oxidizable luciferin substrate, a luciferase enzyme, and a light emitter, typically oxidized luciferin, and called oxyluciferin. We report the structure of fungal oxyluciferin, investigate the mechanism of fungal bioluminescence, and describe the use of simple synthetic α-pyrones as luciferins to produce multicolor enzymatic chemiluminescence. A high-energy endoperoxide is proposed as an intermediate of the oxidation of the native luciferin to the oxyluciferin, which is a pyruvic acid adduct of caffeic acid. Luciferase promiscuity allows the use of simple α-pyrones as chemiluminescent substrates.

  9. Shenkarev Z.O., Melnikova D.N., Finkina E.I., Sukhanov S.V., Boldyrev I.A., Gizatullina A.K., Mineev K.S., Arseniev A.S., Ovchinnikova T.V. (2017). Ligand Binding Properties of the Lentil Lipid Transfer Protein: Molecular Insight into the Possible Mechanism of Lipid Uptake. Biochemistry 56 (12), 1785–1796 [+]

    The lentil lipid transfer protein, designated as Lc-LTP2, was isolated from Lens culinaris seeds. The protein belongs to the LTP1 subfamily and consists of 93 amino acid residues. Its spatial structure includes four α-helices (H1-H4) and a long C-terminal tail. Here, we report the ligand binding properties of Lc-LTP2. The fluorescent 2-p-toluidinonaphthalene-6-sulfonate binding assay revealed that the affinity of Lc-LTP2 for saturated and unsaturated fatty acids was enhanced with a decrease in acyl-chain length. Measurements of boundary potential in planar lipid bilayers and calcein dye leakage in vesicular systems revealed preferential interaction of Lc-LTP2 with the negatively charged membranes. Lc-LTP2 more efficiently transferred anionic dimyristoylphosphatidylglycerol (DMPG) than zwitterionic dimyristoylphosphatidylcholine. Nuclear magnetic resonance experiments confirmed the higher affinity of Lc-LTP2 for anionic lipids and those with smaller volumes of hydrophobic chains. The acyl chains of the bound lysopalmitoylphosphatidylglycerol (LPPG), DMPG, or dihexanoylphosphatidylcholine molecules occupied the internal hydrophobic cavity, while their headgroups protruded into the aqueous environment between helices H1 and H3. The spatial structure and backbone dynamics of the Lc-LTP2-LPPG complex were determined. The internal cavity was expanded from ∼600 to ∼1000 Å(3) upon the ligand binding. Another entrance into the internal cavity, restricted by the H2-H3 interhelical loop and C-terminal tail, appeared to be responsible for the attachment of Lc-LTP2 to the membrane or micelle surface and probably played an important role in the lipid uptake determining the ligand specificity. Our results confirmed the previous assumption regarding the membrane-mediated antimicrobial action of Lc-LTP2 and afforded molecular insight into its biological role in the plant.

  10. Bocharov E.V., Bragin P.E., Pavlov K.V., Bocharova O.V., Mineev K.S., Polyansky A.A., Volynsky P.E., Efremov R.G., Arseniev A.S. (2017). The Conformation of the Epidermal Growth Factor Receptor Transmembrane Domain Dimer Dynamically Adapts to the Local Membrane Environment. Biochemistry 56 (12), 1697–1705 [+]

    The epidermal growth factor receptor (EGFR) family is an important class of receptor tyrosine kinases, mediating a variety of cellular responses in normal biological processes and in pathological states of multicellular organisms. Different modes of dimerization of the human EGFR transmembrane domain (TMD) in different membrane mimetics recently prompted us to propose a novel signal transduction mechanism based on protein-lipid interaction. However, the experimental evidence for it was originally obtained with slightly different TMD fragments used in the two different mimetics, compromising the validity of the comparison. To eliminate ambiguity, we determined the nuclear magnetic resonance (NMR) structure of the bicelle-incorporated dimer of the EGFR TMD fragment identical to the one previously used in micelles. The NMR results augmented by molecular dynamics simulations confirm the mutual influence of the TMD and lipid environment, as is required for the proposed lipid-mediated activation mechanism. They also reveal the possible functional relevance of a subtle interplay between the concurrent processes in the lipid and protein during signal transduction.

  11. Baranov M.S., Kaskova Z.M., Gritсenko R., Postikova S.G., Ivashkin P.E., Kislukhin A.A., Moskvin D.I., Mineev K.S., Arseniev A.S., Labas Yu.A., Yampolsky I.V. (2017). Synthesis of Panal Terpenoid Core. Synlett 28 (5), 583–588 [+]

    Panal is a natural bicyclic cadalane-type sesquiterpenoid with an unusual combination of stereocenters. It was isolated in 1988 as an alleged biosynthetic precursor of luciferin (a light-emitting molecule) in a bioluminescent fungus Panellus stipticus. Herein we present the first approach to the synthesis of the terpenoid skeleton of panal, which includes construction of five stereocenters, one of which is easily epimerizable. The key steps in the synthetic approach presented are high-pressure Diels–Alder reaction disobeying the ‘endo rule’, Barbier reductive allylation, and cyclization of trans-decalin ring via ring-closing metathesis.

  12. Mineev K.S., Nadezhdin K.D. (2017). Membrane mimetics for solution NMR studies of membrane proteins. Nanotechnology Reviews 6 (1), 15–32 ID:1781
  13. Mineev K.S., Nadezhdin K.D., Goncharuk S.A., Arseniev A.S. (2017). Façade detergents as bicelle rim-forming agents for solution NMR spectroscopy. Nanotechnology Reviews 6 (1), 93–103 [+]

    Out of all membrane mimetics available for solution nuclear magnetic resonance (NMR) spectroscopy, phospholipid bicelles are the most prospective. Unlike lipid-protein nanodiscs their size can be easily controlled over a wide range, and the exchange of matter between the particles can take place. However, recent studies revealed several major drawbacks of conventional 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) and DMPC/3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) bicelles. First, size of such bicelles can increase dramatically upon heating, and, second, rim-forming detergents of bicelles can cause improper folding of the water-soluble globular domains of membrane proteins. In order to avoid these effects, we tested the Façade detergents as possible alternative rim-forming agents for small isotropic bicelles. In the present work we characterized the size of bicelles formed by 3α-hydroxy-7α,12α-di-((O-β-D-maltosyl)-2-hydroxyethoxy)-cholane (Façade-EM) and 3α-hydroxy-7α,12α-di-(((2-(trimethylamino)ethyl)phosphoryl)ethyloxy)-cholane Façade-EPC as a function of temperature and lipid/detergent ratio by 1H NMR diffusion spectroscopy. Additionally, the denaturing effects of these two rim-forming agents were investigated using the junction of the transmembrane and intracellular domains of the p75 neurotrophin receptor (p75NTR) as a model object. We show that the use of Façades allows decreasing the temperature-dependent growth of bicelles. The ability of Façade-EM-based bicelles to support the native structure and soluble state of the p75NTR intracellular domain was also revealed.

  14. Bocharov E.V., Mineev K.S., Pavlov K.V., Akimov S.A., Kuznetsov A.S., Efremov R.G., Arseniev A.S. (2016). Helix-helix interactions in membrane domains of bitopic proteins: Specificity and role of lipid environment. Biochim. Biophys. Acta , [+]

    Interaction between transmembrane helices often determines biological activity of membrane proteins. Bitopic proteins, a broad subclass of membrane proteins, form dimers containing two membrane-spanning helices. Some aspects of their structure-function relationship cannot be fully understood without considering the protein-lipid interaction, which can determine the protein conformational ensemble. Experimental and computer modeling data concerning transmembrane parts of bitopic proteins are reviewed in the present paper. They highlight the importance of lipid-protein interactions and resolve certain paradoxes in the behavior of such proteins. Besides, some properties of membrane organization provided a clue to understanding of allosteric interactions between distant parts of proteins. Interactions of these kinds appear to underlie a signaling mechanism, which could be widely employed in the functioning of many membrane proteins. Treatment of membrane proteins as parts of integrated fine-tuned proteolipid system promises new insights into biological function mechanisms and approaches to drug design.

  15. Mineev K.S., Nadezhdin K.D., Goncharuk S.A., Arseniev A.S. (2016). Characterization of Small Isotropic Bicelles with Various Compositions. Langmuir 32 (26), 6624–37 [+]

    Structural studies of membrane proteins are of great importance and interest, with solution and solid state NMR spectroscopy being very promising tools for that task. However, such investigations are hindered by a number of obstacles, and in the first place by the fact that membrane proteins need an adequate environment that models the cell membrane. One of the most widely used and prospective membrane mimetics is isotropic bicelles. While large anisotropic bicelles are well-studied, the field of small bicelles contains a lot of "white spots". The present work reports the radii of particles and concentration of the detergents in the monomeric state in solutions of isotropic bicelles, formed by 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), and sodium cholate, as a function of lipid/detergent ratio and temperature. These parameters were measured using (1)H NMR diffusion spectroscopy for the bicelles composed of lipids with saturated fatty chains of different length and lipids, containing unsaturated fatty acid residue. The influence of a model transmembrane protein (membrane domain of rat TrkA) on the properties of bicelles and the effect of the bicelle size and composition on the properties of the transmembrane protein were investigated with heteronuclear NMR and nuclear Overhauser effect spectroscopy. We show that isotropic bicelles that are applicable for solution NMR spectroscopy behave as predicted by the theoretical models and are likely to be bicelles rather than mixed micelles. Using the obtained data, we propose a simple approach to control the size of bicelles at low concentrations. On the basis of our results, we compared different rim-forming agents and selected CHAPS as a detergent of choice for structural studies in bicelles, if the deuteration of the detergent is not required.

  16. Nadezhdin K.D., GarcíaCarpio I., Goncharuk S.A., Mineev K.S., Arseniev A.S., Vilar M. (2016). Structural Basis of p75 Transmembrane Domain Dimerization. J. Biol. Chem. 291 (23), 12346–57 [+]

    Dimerization of single span transmembrane receptors underlies their mechanism of activation. p75 neurotrophin receptor plays an important role in the nervous system, but the understanding of p75 activation mechanism is still incomplete. The transmembrane (TM) domain of p75 stabilizes the receptor dimers through a disulfide bond, essential for the NGF signaling. Here we solved by NMR the three-dimensional structure of the p75-TM-WT and the functionally inactive p75-TM-C257A dimers. Upon reconstitution in lipid micelles, p75-TM-WT forms the disulfide-linked dimers spontaneously. Under reducing conditions, p75-TM-WT is in a monomer-dimer equilibrium with the Cys(257) residue located on the dimer interface. In contrast, p75-TM-C257A forms dimers through the AXXXG motif on the opposite face of the α-helix. Biochemical and cross-linking experiments indicate that AXXXG motif is not on the dimer interface of p75-TM-WT, suggesting that the conformation of p75-TM-C257A may be not functionally relevant. However, rather than mediating p75 homodimerization, mutagenesis of the AXXXG motif reveals its functional role in the regulated intramembrane proteolysis of p75 catalyzed by the γ-secretase complex. Our structural data provide an insight into the key role of the Cys(257) in stabilization of the weak transmembrane dimer in a conformation required for the NGF signaling.

  17. Bocharova O.V., Bragin P.E., Bocharov E.V., Mineev K.S., Goncharuk S.A., Arseniev A.S. (2016). Cell Free Expression and Purification of the Fragments of the Receptor Tyrosine Kynases of the EGFR Family, Containing the Transmembrane Domain with the Juxtamembrane Region, for Structural Studies. BIOLOGICHESKIE MEMBRANY 33 (2), 124–132 [+]

    The EGFR/HER receptor family of an epidermal growth factor represents an important class of the receptor tyrosine kinases playing the key role in the control of cell growth and differentiation in mammalian cells, as well as in the development of a number of pathological processes, including oncogenesis. Binding of a ligand to the extracellular domains initiates switching of the EGFR/HER receptor between the alternative dimeric states that causes the allosteric activation of kinase domains in cell cytoplasm. The transmembrane (TM) domain and adjacent flexible regions alternatively interacting with either membrane surface or kinase domains are directly involved in the complex conformational transition in EGFR/HERs. Here we report on a highly efficient system of the cell free production of the EGFR/HER TM domains with functionally important juxtamembrane (JM) regions for the investigation of the molecular basis of biochemical signal transduction across the cell membrane. To increase the efficiency of synthesis of the EGFR/HER TM-JM fragments of the receptors, we used two N-terminal expression tags, which significantly increased the protein yield. In the case of the TM-JM fragments of EGFR (residues 638–692) and HER2 (residues 644–700), the method allowed us to obtain milligram quantities of the 13C,15N-labeled protein for structural and biophysical investigations in the membrane-mimicking environments using high-resolution heteronuclear NMR spectroscopy.

  18. Bragin P.E., Mineev K.S., Bocharova O.V., Volynsky P.E., Bocharov E.V., Arseniev A.S. (2016). HER2 Transmembrane Domain Dimerization Coupled with Self-Association of Membrane-Embedded Cytoplasmic Juxtamembrane Regions. J. Mol. Biol. 428 (1), 52–61 [+]

    Receptor tyrosine kinases of the human epidermal growth factor receptor (HER or ErbB) family transduce biochemical signals across plasma membrane, playing a significant role in vital cellular processes and in various cancers. Inactive HER/ErbB receptors exist in equilibrium between the monomeric and unspecified pre-dimerized states. After ligand binding, the receptors are involved in strong lateral dimerization with proper assembly of their extracellular ligand-binding, single-span transmembrane, and cytoplasmic kinase domains. The dimeric conformation of the HER2 transmembrane domain that is believed to support the cytoplasmic kinase domain configuration corresponding to the receptor active state was previously described in lipid bicelles. Here we used high-resolution NMR spectroscopy in another membrane-mimicking micellar environment and identified an alternative HER2 transmembrane domain dimerization coupled with self-association of membrane-embedded cytoplasmic juxtamembrane region. Such a dimerization mode appears to be capable of effectively inhibiting the receptor kinase activity. This finding refines the molecular mechanism regarding the signal propagation steps from the extracellular to cytoplasmic domains of HER/ErbB receptors.

  19. Melnikova D.N., Mineev K.S., Finkina E.I., Arseniev A.S., Ovchinnikova T.V. (2016). A novel lipid transfer protein from the dill Anethum graveolens L.: isolation, structure, heterologous expression, and functional characteristics. J. Pept. Sci. 22 (1), 59–66 [+]

    A novel lipid transfer protein, designated as Ag-LTP, was isolated from aerial parts of the dill Anethum graveolens L. Structural, antimicrobial, and lipid binding properties of the protein were studied. Complete amino acid sequence of Ag-LTP was determined. The protein has molecular mass of 9524.4 Da, consists of 93 amino acid residues including eight cysteines forming four disulfide bonds. The recombinant Ag-LTP was overexpressed in Escherichia coli and purified. NMR investigation shows that the Ag-LTP spatial structure contains four α-helices, forming the internal hydrophobic cavity, and a long C-terminal tail. The measured volume of the Ag-LTP hydrophobic cavity is equal to ~800 A(3) , which is much larger than those of other plant LTP1s. Ag-LTP has weak antifungal activity and unpronounced lipid binding specificity but effectively binds plant hormone jasmonic acid. Our results afford further molecular insight into biological functions of LTP in plants. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

  20. Mineev K.S., Panova S.V., Bocharova O.V., Bocharov E.V., Arseniev A.S. (2015). The Membrane Mimetic Affects the Spatial Structure and Mobility of EGFR Transmembrane and Juxtamembrane Domains. Biochemistry 54 (41), 6295–8 [+]

    The epidermal growth factor receptor (EGFR) is one of the most extensively studied receptor tyrosine kinases, as it is involved in a wide range of cellular processes and severe diseases. Recent works reveal that the single-helix transmembrane domains and cytoplasmic juxtamembrane regions play an important role in the receptor activation process. Here we present the results of our investigation of the spatial structure and mobility of the EGFR transmembrane domain and juxtamembrane regions in various membranelike environments, which shed light on the effects of the membrane physical properties and composition on the behavior of the juxtamembrane domain.

  21. Meshcheryakova E.A., Mineev K.S., Volynski P.E., Andronova T.M., Ivanov V.T. (2015). GMDP: unusual physico-chemical and biological properties of the anomeriс forms. J. Pept. Sci. 21 (9), 717–22 [+]

    Disaccharide containing unit of peptidoglycan from bacterial cell wall, N-acetyl-d-glucosaminyl-N-acetylmuramyl-l-alanyl-d-glutaminamide (gluсosaminyl-muramyl-dipeptide) registered in Russia as an immunomodulatory drug, is shown to participate in slow equilibrium of α and β anomeric forms. Data of NMR spectra and molecular dynamics indicate that the α-anomer predominantly acquires a folded conformation stabilized by intramolecular hydrogen bond between the alanyl carbonyl and muramyl NH proton. The β-form displays a considerable fraction of extended, non-hydrogen bonded structures. In the standard immunoadjuvant test system, the α-form is practically inactive, and the activity of the equilibrium mixture with α : β = 68 : 32 ratio is due to the presence of β-anomer. Such unique α-β selectivity of biological action must be considered at the design of related immunoactive glycopeptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

  22. Mineev K.S., Goncharuk S.A., Kuzmichev P.K., Vilar M., Arseniev A.S. (2015). NMR Dynamics of Transmembrane and Intracellular Domains of p75NTR in Lipid-Protein Nanodiscs. Biophys. J. 109 (4), 772–82 [+]

    P75NTR is a type I integral membrane protein that plays a key role in neurotrophin signaling. However, structural data for the receptor in various functional states are sparse and controversial. In this work, we studied the spatial structure and mobility of the transmembrane and intracellular parts of p75NTR, incorporated into lipid-protein nanodiscs of various sizes and compositions, by solution NMR spectroscopy. Our data reveal a high level of flexibility and disorder in the juxtamembrane chopper domain of p75NTR, which results in the motions of the receptor death domain being uncoupled from the motions of the transmembrane helix. Moreover, none of the intracellular domains of p75NTR demonstrated a propensity to interact with the membrane or to self-associate under the experimental conditions. The obtained data are discussed in the context of the receptor activation mechanism.

  23. Purtov K.V., Petushkov V.N., Baranov M.S., Mineev K.S., Rodionova N.S., Kaskova Z.M., Tsarkova A.S., Petunin A.I., Bondar V.S., Rodicheva E.K., Medvedeva S.E., Oba Y., Arseniev A.S., Lukyanov S., Gitelson J.I., Yampolsky I.V. (2015). The Chemical Basis of Fungal Bioluminescence. Angew. Chem. Int. Ed. 127 (28), 8242–8246 [+]

    Many species of fungi naturally produce light, a phenomenon known as bioluminescence, however, the fungal substrates used in the chemical reactions that produce light have not been reported. We identified the fungal compound luciferin 3-hydroxyhispidin, which is biosynthesized by oxidation of the precursor hispidin, a known fungal and plant secondary metabolite. The fungal luciferin does not share structural similarity with the other eight known luciferins. Furthermore, it was shown that 3-hydroxyhispidin leads to bioluminescence in extracts from four diverse genera of luminous fungi, thus suggesting a common biochemical mechanism for fungal bioluminescence.

  24. Dubinnyi M.A., Tsarkova A.S., Petushkov V.N., Kaskova Z.M., Rodionova N.S., Kovalchuk S.I., Ziganshin R.H., Baranov M.S., Mineev K.S., Yampolsky I.V. (2015). Novel Peptide Chemistry in Terrestrial Animals: Natural Luciferin Analogues from the Bioluminescent Earthworm Fridericia heliota. Chem. Eur. J. 21 (10), 3942–3947 [+]

    We report isolation and structure elucidation of AsLn5, AsLn7, AsLn11 and AsLn12: novel luciferin analogs from the bioluminescent earthworm Fridericia heliota. They were found to be highly unusual modified peptides, comprising either of the two tyrosine-derived chromophores, CompX or CompY and a set of amino acids, including threonine, gamma-aminobutyric acid, homoarginine, and unsymmetrical N,N-dimethylarginine. These natural compounds represent a unique peptide chemistry found in terrestrial animals and rise novel questions concerning their biosynthetic origin.

  25. Mineev K.S., Goncharuk S.A., Arseniev A.S. (2014). Toll-like receptor 3 transmembrane domain is able to perform various homotypic interactions: An NMR structural study. FEBS Lett. , [+]

    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.

  26. Shenkarev Z.O., Gizatullina A.K., Finkina E.I., Alekseeva E.A., Balandin S.V., Mineev K.S., Arseniev A.S., Ovchinnikova T.V. (2014). Heterologous expression and solution structure of defensin from lentil Lens culinaris. Biochem. Biophys. Res. Commun. 451 (2), 252–7 [+]

    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 conditions. The Lc-def antifungal activity might be mediated through electrostatic interaction with anionic lipid components of fungal membranes.

  27. Manni S., Mineev K.S., Usmanova D., Lyukmanova E.N., Shulepko M.A., Kirpichnikov M.P., Winter J., Matkovic M., Deupi X., Arseniev A.S., BallmerHofer K. (2014). Structural and functional characterization of alternative transmembrane domain conformations in VEGF receptor 2 activation. Structure 22 (8), 1077–89 [+]

    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 rearrangement. A molecular dynamics simulation confirmed the transmembrane helix arrangement of wild-type and mutant TMDs revealed by nuclear magnetic resonance spectroscopy.

  28. Berkut A.A., Usmanova D.R., Peigneur S., Oparin P.B., Mineev K.S., Odintsova T.I., Tytgat J., Arseniev A.S., Grishin E.V., Vassilevski A.A. (2014). Structural similarity between defense peptide from wheat and scorpion neurotoxin permits rational functional design. J. Biol. Chem. 289 (20), 14331–40 [+]

    In this study, we present the spatial structure of the wheat antimicrobial peptide (AMP) Tk-AMP-X2 studied using NMR spectroscopy. This peptide was found to adopt a disulfide-stabilized α-helical hairpin fold and therefore belongs to the α-hairpinin family of plant defense peptides. Based on Tk-AMP-X2 structural similarity to cone snail and scorpion potassium channel blockers, a mutant molecule, Tk-hefu, was engineered by incorporating the functionally important residues from κ-hefutoxin 1 onto the Tk-AMP-X2 scaffold. The designed peptide contained the so-called essential dyad of amino acid residues significant for channel-blocking activity. Electrophysiological studies showed that although the parent peptide Tk-AMP-X2 did not present any activity against potassium channels, Tk-hefu blocked Kv1.3 channels with similar potency (IC50 ∼ 35 μm) to κ-hefutoxin 1 (IC50 ∼ 40 μm). We conclude that α-hairpinins are attractive in their simplicity as structural templates, which may be used for functional engineering and drug design.

  29. Shenkarev Z.O., Lyukmanova E.N., Paramonov A.S., Panteleev P.V., Balandin S.V., Shulepko M.A., Mineev K.S., Ovchinnikova T.V., Kirpichnikov M.P., Arseniev A.S. (2014). Lipid-protein nanodiscs offer new perspectives for structural and functional studies of water-soluble membrane-active peptides. Acta Naturae 6 (2), 84–94 [+]

    Lipid-protein nanodiscs (LPNs) are nanoscaled fragments of a lipid bilayer stabilized in solution by the apolipoprotein or a special membrane scaffold protein (MSP). In this work, the applicability of LPN-based membrane mimetics in the investigation of water-soluble membrane-active peptides was studied. It was shown that a pore-forming antimicrobial peptide arenicin-2 from marine lugworm (charge of +6) disintegrates LPNs containing both zwitterionic phosphatidylcholine (PC) and anionic phosphatidylglycerol (PG) lipids. In contrast, the spider toxin VSTx1 (charge of +3), a modifier of Kv channel gating, effectively binds to the LPNs containing anionic lipids (POPC/DOPG, 3 : 1) and does not cause their disruption. VSTx1 has a lower affinity to LPNs containing zwitterionic lipids (POPC), and it weakly interacts with the protein component of nanodiscs, MSP (charge of -6). The neurotoxin II (NTII, charge of +4) from cobra venom, an inhibitor of the nicotinic acetylcholine receptor, shows a comparatively low affinity to LPNs containing anionic lipids (POPC/DOPG, 3 : 1 or POPC/DOPS, 4 : 1), and it does not bind to LPNs/POPC. The obtained data show that NTII interacts with the LPN/POPC/DOPS surface in several orientations, and that the exchange process among complexes with different topologies proceeds fast on the NMR timescale. Only one of the possible NTII orientations allows for the previously proposed specific interaction between the toxin and the polar head group of phosphatidylserine from the receptor environment (Lesovoy et al., Biophys. J. 2009. V. 97. № 7. P. 2089-2097). These results indicate that LPNs can be used in structural and functional studies of water-soluble membrane-active peptides (probably except pore-forming ones) and in studies of the molecular mechanisms of peptide-membrane interaction.

  30. Gizatullina A.K., Finkina E.I., Mineev K.S., Melnikova D.N., Bogdanov I.V., Telezhinskaya I.N., Balandin S.V., Shenkarev Z.O., Arseniev A.S., Ovchinnikova T.V. (2013). Recombinant production and solution structure of lipid transfer protein from lentil Lens culinaris. Biochem. Biophys. Res. Commun. 439 (4), 427–32 [+]

    Lipid transfer protein, designated as Lc-LTP2, was isolated from seeds of the lentil Lens culinaris. The protein has molecular mass 9282.7Da, consists of 93 amino acid residues including 8 cysteines forming 4 disulfide bonds. Lc-LTP2 and its stable isotope labeled analogues were overexpressed in Escherichia coli and purified. Antimicrobial activity of the recombinant protein was examined, and its spatial structure was studied by NMR spectroscopy. The polypeptide chain of Lc-LTP2 forms four α-helices (Cys4-Leu18, Pro26-Ala37, Thr42-Ala56, Thr64-Lys73) and a long C-terminal tail without regular secondary structure. Side chains of the hydrophobic residues form a relatively large internal tunnel-like lipid-binding cavity (van der Waals volume comes up to ∼600Å(3)). The side-chains of Arg45, Pro79, and Tyr80 are located near an assumed mouth of the cavity. Titration with dimyristoyl phosphatidylglycerol (DMPG) revealed formation of the Lc-LTP2/lipid non-covalent complex accompanied by rearrangements in the protein spatial structure and expansion of the internal cavity. The resultant Lc-LTP2/DMPG complex demonstrates limited lifetime and dissociates within tens of hours.

  31. Mineev K.S., Lesovoy D.M., Usmanova D.R., Goncharuk S.A., Shulepko M.A., Lyukmanova E.N., Kirpichnikov M.P., Bocharov E.V., Arseniev A.S. (2013). NMR-based approach to measure the free energy of transmembrane helix-helix interactions. Biochim. Biophys. Acta 1838 (1PB), 164–172 [+]

    Knowledge of the energetic parameters of transmembrane helix-helix interactions is necessary for the establishment of a structure-energy relationship for α-helical membrane domains. A number of techniques have been developed to measure the free energies of dimerization and oligomerization of transmembrane α-helices, and all of these have their advantages and drawbacks. In this study we propose a methodology to determine the magnitudes of the free energy of interactions between transmembrane helices in detergent micelles. The suggested approach employs solution nuclear magnetic resonance (NMR) spectroscopy to determine the population of the oligomeric states of the transmembrane domains and introduces a new formalism to describe the oligomerization equilibrium, which is based on the assumption that both the dimerization of the transmembrane domains and the dissociation of the dimer can occur only upon the collision of detergent micelles. The technique has three major advantages compared with other existing approaches: it may be used to analyze both weak and relatively strong dimerization/oligomerization processes, it works well for the analysis of complex equilibria, e.g. when monomer, dimer and high-order oligomer populations are simultaneously present in the solution, and it can simultaneously yield both structural and energetic characteristics of the helix-helix interaction under study. The proposed methodology was applied to investigate the oligomerization process of transmembrane domains of fibroblast growth factor receptor 3 (FGFR3) and vascular endothelium growth factor receptor 2 (VEGFR2), and allowed the measurement of the free energy of dimerization of both of these objects. In addition the proposed method was able to describe the multi-state oligomerization process of the VEGFR2 transmembrane domain.

  32. Mineev K.S., Lyukmanova E.N., Krabben L., Serebryakova M.V., Shulepko M.A., Arseniev A.S., Kordyukova L.V., Veit M. (2013). Structural investigation of influenza virus hemagglutinin membrane-anchoring peptide. Protein Eng. Des. Sel. 26 (9), 547–52 [+]

    Hemagglutinin (HA), the trimeric spike of influenza virus, catalyzes fusion of viral and cellular membranes. We have synthesized the anchoring peptide including the linker, transmembrane region and cytoplasmic tail (HA-TMR-CT) in a cell-free system. Furthermore, to mimic the palmitoylation of three conserved cysteines within the CT, we chemically alkylated HA-TMR-CT using hexadecyl-methanethiosulfonate. While the nuclear magnetic resonance spectroscopy showed pure and refolded peptides, the formation of multiple oligomers of higher order impeded further structural analysis. Circular dichroism spectroscopy of both alkylated and non-alkylated HA-TMR-CT revealed an α-helical secondary structure. No major impact of the fatty acids on the secondary structure was detected.

  33. Osmakov D.I., Kozlov S.A., Andreev Y.A., Koshelev S.G., Sanamyan N.P., Sanamyan K.E., Dyachenko I.A., Bondarenko D.A., Murashev A.N., Mineev K.S., Arseniev A.S., Grishin E.V. (2013). Sea Anemone Peptide with Uncommon β-Hairpin Structure Inhibits Acid-sensing Ion Channel 3 (ASIC3) and Reveals Analgesic Activity. J. Biol. Chem. 288 (32), 23116–27 [+]

    Three novel peptides were isolated from the venom of the sea anemone Urticina grebelnyi. All of them are 29 amino acid peptides cross-linked by two disulfide bridges, with a primary structure similar to other sea anemone peptides belonging to structural group 9a. The structure of the gene encoding the shared precursor protein of the identified peptides was determined. One peptide, π-AnmTX Ugr 9a-1 (short name Ugr 9-1), produced a reversible inhibition effect on both the transient and the sustained current of human ASIC3 channels expressed in Xenopus laevis oocytes. It completely blocked the transient component (IC50 10 ± 0.6 μm) and partially (48 ± 2%) inhibited the amplitude of the sustained component (IC50 1.44 ± 0.19 μm). Using in vivo tests in mice, Ugr 9-1 significantly reversed inflammatory and acid-induced pain. The other two novel peptides, AnmTX Ugr 9a-2 (Ugr 9-2) and AnmTX Ugr 9a-3 (Ugr 9-3), did not inhibit the ASIC3 current. NMR spectroscopy revealed that Ugr 9-1 has an uncommon spatial structure, stabilized by two S-S bridges, with three classical β-turns and twisted β-hairpin without interstrand disulfide bonds. This is a novel peptide spatial structure that we propose to name boundless β-hairpin.

  34. Beloglazova I.B., Beabealashvilli R.S.h., Gursky Y.G., Bocharov E.V., Mineev K.S., Parfenova E.V., Tkachuk V.A. (2013). Structural investigations of recombinant urokinase growth factor-like domain. Biochemistry Mosc. 78 (5), 517–30 [+]

    Urokinase-type plasminogen activator (uPA) is a serine protease that converts the plasminogen zymogen into the enzymatically active plasmin. uPA is synthesized and secreted as the single-chain molecule (scuPA) composed of an N-terminal domain (GFD) and kringle (KD) and C-terminal proteolytic (PD) domains. Earlier, the structure of ATF (which consists of GFD and KD) was solved by NMR (A. P. Hansen et al. (1994) Biochemistry, 33, 4847-4864) and by X-ray crystallography alone and in a complex with the soluble form of the urokinase receptor (uPAR, CD87) lacking GPI (C. Barinka et al. (2006) J. Mol. Biol., 363, 482-495). According to these data, GFD contains two β-sheet regions oriented perpendicularly to each other. The area in the GFD responsible for binding to uPAR is localized in the flexible Ω-loop, which consists of seven amino acid residues connecting two strings of antiparallel β-sheet. It was shown by site-directed mutagenesis that shortening of the Ω-loop length by one amino acid residue leads to the inability of GFD to bind to uPAR (V. Magdolen et al. (1996) Eur. J. Biochem., 237, 743-751). Here we show that, in contrast to the above-mentioned studies, we found no sign of the β-sheet regions in GFD in our uPA preparations either free or in a complex with uPAR. The GFD seems to be a rather flexible and unstructured domain, demonstrating in spite of its apparent flexibility highly specific interaction with uPAR both in vitro and in cell culture experiments. Circular dichroism, tryptophan fluorescence during thermal denaturation of the protein, and heteronuclear NMR spectroscopy of ¹⁵N/¹³C-labeled ATF both free and in complex with urokinase receptor were used to judge the secondary structure of GFD of uPA.

  35. Sverdlov E.D., Mineev K. (2013). Mutation rate in stem cells: an underestimated barrier on the way to therapy. Trends Mol Med , [+]

    Stem cells (SCs) are thought to have great therapeutic potential, but due to continuously and stochastically arising new mutations that unpredictably change the composition of a cell population, the large-scale manufacturing of SCs with uniform properties and predictable behavior is a challenge. Quantitative evaluation of the characteristic mutation rate of a given stem cell line could be an important criterion in making the decision to use the line in medical practice. Such an evaluation could provide a new quality standard for newly derived human embryonic stem cell (hESC) lines prior to depositing them in stem cell banks. Here, we substantiate this view with simple calculations showing the effect of the mutation rate on changes in the cell population composition due to amplification. Selection of SCs with low mutation rate could reduce the risk of negative side effects during treatment.

  36. Bocharov E.V., Mineev K.S., Goncharuk M.V., Arseniev A.S. (2012). Structural and thermodynamic insight into the process of "weak" dimerization of the ErbB4 transmembrane domain by solution NMR. Biochim. Biophys. Acta 1818 (9), 2158–70 [+]

    Specific helix-helix interactions between the single-span transmembrane domains of receptor tyrosine kinases are believed to be important for their lateral dimerization and signal transduction. Establishing structure-function relationships requires precise structural-dynamic information about this class of biologically significant bitopic membrane proteins. ErbB4 is a ubiquitously expressed member of the HER/ErbB family of growth factor receptor tyrosine kinases that is essential for the normal development of various adult and fetal human tissues and plays a role in the pathobiology of the organism. The dimerization of the ErbB4 transmembrane domain in membrane-mimicking lipid bicelles was investigated by solution NMR. In a bicellar DMPC/DHPC environment, the ErbB4 membrane-spanning α-helices (651-678)(2) form a right-handed parallel dimer through the N-terminal double GG4-like motif A(655)GxxGG(660) in a fashion that is believed to permit proper kinase domain activation. During helix association, the dimer subunits undergo a structural adjustment (slight bending) with the formation of a network of inter-monomeric polar contacts. The quantitative analysis of the observed monomer-dimer equilibrium provides insights into the kinetics and thermodynamics of the folding process of the helical transmembrane domain in the model environment that may be directly relevant to the process that occurs in biological membranes. The lipid bicelles occupied by a single ErbB4 transmembrane domain behave as a true ("ideal") solvent for the peptide, while multiply occupied bicelles are more similar to the ordered lipid microdomains of cellular membranes and appear to provide substantial entropic enhancement of the weak helix-helix interactions, which may be critical for membrane protein activity.

  37. Oparin P.B., Mineev K.S., Dunaevsky Y.E., Arseniev A.S., Belozersky M.A., Grishin E.V., Egorov T.A., Vassilevski A.A. (2012). Buckwheat trypsin inhibitor with helical hairpin structure belongs to a new family of plant defence peptides. Biochem. J. 446 (1), 69–77 [+]

    A new peptide trypsin inhibitor named BWI-2c was obtained from buckwheat (Fagopyrum esculentum) seeds by sequential affinity, ion exchange and reversed-phase chromatography. The peptide was sequenced and found to contain 41 amino acid residues, with four cysteine residues involved in two intramolecular disulfide bonds. Recombinant BWI-2c identical to the natural peptide was produced in Escherichia coli in a form of a cleavable fusion with thioredoxin. The 3D (three-dimensional) structure of the peptide in solution was determined by NMR spectroscopy, revealing two antiparallel α-helices stapled by disulfide bonds. Together with VhTI, a trypsin inhibitor from veronica (Veronica hederifolia), BWI-2c represents a new family of protease inhibitors with an unusual α-helical hairpin fold. The linker sequence between the helices represents the so-called trypsin inhibitory loop responsible for direct binding to the active site of the enzyme that cleaves BWI-2c at the functionally important residue Arg19. The inhibition constant was determined for BWI-2c against trypsin (1.7×10-10 M), and the peptide was tested on other enzymes, including those from various insect digestive systems, revealing high selectivity to trypsin-like proteases. Structural similarity shared by BWI-2c, VhTI and several other plant defence peptides leads to the acknowledgement of a new widespread family of plant peptides termed α-hairpinins.

  38. Lyukmanova E.N., Shenkarev Z.O., Khabibullina N.F., Kopeina G.S., Shulepko M.A., Paramonov A.S., Mineev K.S., Tikhonov R.V., Shingarova L.N., Petrovskaya L.E., Dolgikh D.A., Arseniev A.S., Kirpichnikov M.P. (2011). Lipid-protein nanodisks for cell-free production of integral membrane proteins in a soluble and folded state: Comparison with detergent micelles, bicelles and liposomes. Biochim. Biophys. Acta , [+]

    Production of integral membrane proteins (IMPs) in a folded state is a key prerequisite for their functional and structural studies. In cell-free (CF) expression systems membrane mimicking components could be added to the reaction mixture that promotes IMP production in a soluble form. Here lipid-protein nanodisks (LPNs) of different lipid compositions (DMPC, DMPG, POPC, POPC/DOPG) have been compared with classical membrane mimicking media such as detergent micelles, lipid/detergent bicelles and liposomes by their ability to support CF synthesis of IMPs in a folded and soluble state. Three model membrane proteins of different topology were used: homodimeric transmembrane (TM) domain of human receptor tyrosine kinase ErbB3 (TM-ErbB3, 1TM); voltage-sensing domain of K(+) channel KvAP (VSD, 4TM); and bacteriorhodopsin from Exiguobacterium sibiricum (ESR, 7TM). Structural and/or functional properties of the synthesized proteins were analyzed. LPNs significantly enhanced synthesis of the IMPs in a soluble form regardless of the lipid composition. A partial disintegration of LPNs composed of unsaturated lipids was observed upon co-translational IMP incorporation. Contrary to detergents the nanodisks resulted in the synthesis of ~80% active ESR and promoted correct folding of the TM-ErbB3. None of the tested membrane mimetics supported CF synthesis of correctly folded VSD, and the protocol of the domain refolding was developed. The use of LPNs appears to be the most promising approach to CF production of IMPs in a folded state. NMR analysis of (15)N-Ile-TM-ErbB3 co-translationally incorporated into LPNs shows the great prospects of this membrane mimetics for structural studies of IMPs produced by CF systems.

  39. Goncharuk M.V., Shulga A.A., Ermoliuk Ia.S., Tkach E.N., Goncharuk S.A., Pustovalova Iu.E., Mineev K.S., Bocharov E.V., Maslennikov I.V., Arsenev A.S., Kirpichnikov M.P. (2011). [Bacterial synthesis, purification, and solubilization of transmembrane segments of ErbB family members]. Mol. Biol. (Mosk.) 45 (5), 892–902 [+]

    A family of epidermal growth factor receptors, ErbB, represents an important class of receptor tyrosine kinases, playing a leading role in cellular growth, development and differentiation. Transmembrane domains of these receptors transduce biochemical signals across plasma membrane via lateral homo- and heterodimerization. Relatively small size of complexes of ErbB transmembrane domains with detergents or lipids allows one to study their detailed spatial structure using three-dimensional heteronuclear high-resolution NMR spectroscopy. Here, we describe the effective expression system and purification procedure for preparative-scale production of transmembrane peptides from four representatives of ErbB family, ErbB1, ErbB2, ErbB3, ErbB4, for structural studies. The recombinant peptides were produced in Escherichia coli BL21(DE3)pLysS as C-terminal extensions of thioredoxin A. The fusion protein cleavage was accomplished with the light subunit of human enterokinase. Several (10-30) milligrams of purified isotope-labeled transmembrane peptides were isolated with the use of a simple and convenient procedure, which consists of consecutive steps of immobilized metal affinity chromatography and cation-exchange chromatography. The purified peptides were reconstituted in lipid/detergent environment (micelles or bicelles) and characterized using dynamic light scattering, CD and NMR spectroscopy. The data obtained indicate that the purified ErbB transmembrane peptides are suitable for structural and dynamic studies of their homo- and heterodimer complexes using high resolution NMR spectroscopy.

  40. Mineev K.S., Khabibullina N.F., Lyukmanova E.N., Dolgikh D.A., Kirpichnikov M.P., Arseniev A.S. (2011). Spatial structure and dimer-monomer equilibrium of the ErbB3 transmembrane domain in DPC micelles. Biochim. Biophys. Acta 1808 (8), 2081–8 [+]

    In present work the interaction of two TM α-helices of the ErbB3 receptor tyrosine kinase from the ErbB or HER family (residues 639-670) was studied by means of NMR spectroscopy in a membrane-mimicking environment provided by the DPC micelles. The ErbB3 TM segment appeared to form a parallel symmetric dimer in a left-handed orientation. The interaction between TM spans is accomplished via the non-standard motif and is supported by apolar contacts of bulky side chains and by stacking of aromatic rings together with π-cation interactions of Phe and Arg side chains. The investigation of the dimer-monomer equilibrium revealed thermodynamic properties of the assembly and the presence of two distinct regimes of the dimerization at low and at high peptide/detergent ratio. It was found that the detergent in case of ErbB3 behaves not as an ideal solvent, thus affecting the dimer-monomer equilibrium. Such behavior may account for the problems occurring with the refolding and stability of multispan helical membrane proteins in detergent solutions. The example of ErbB3 allows us to conclude that the thermodynamic parameters of dimerization, measured in micelles for two different helical pairs, cannot be compared without the investigation of their dependence on detergent concentration.

  41. Mineev K.S., Bocharov E.V., Volynsky P.E., Goncharuk M.V., Tkach E.N., Ermolyuk Y.S., Schulga A.A., Chupin V.V., Maslennikov I.V., Efremov R.G., Arseniev A.S. (2011). Dimeric structure of the transmembrane domain of glycophorin a in lipidic and detergent environments. Acta Naturae 3 (2), 90–8 [+]Specific interactions between transmembrane α-helices, to a large extent, determine the biological function of integral membrane proteins upon normal development and in pathological states of an organism. Various membrane-like media, partially those mimicking the conditions of multicomponent biological membranes, are used to study the structural and thermodynamic features that define the character of oligomerization of transmembrane helical segments. The choice of the composition of the membrane-mimicking medium is conducted in an effort to obtain a biologically relevant conformation of the protein complex and a sample that would be stable enough to allow to perform a series of long-term experiments with its use. In the present work, heteronuclear NMR spectroscopy and molecular dynamics simulations were used to demonstrate that the two most widely used media (detergent DPC micelles and lipid DMPC/DHPC bicelles) enable to perform structural studies of the specific interactions between transmembrane α-helices by the example of dimerizing the transmembrane domain of the bitopic protein glycophorin A. However, a number of peculiarities place lipid bicelles closer to natural lipid bilayers in terms of their physical properties. ID:712
  42. Lyukmanova E.N., Shenkarev Z.O., Shulepko M.A., Mineev K.S., DHoedt D., Kasheverov I.E., Filkin S.Y., Krivolapova A.P., Janickova H., Dolezal V., Dolgikh D.A., Arseniev A.S., Bertrand D., Tsetlin V.I., Kirpichnikov M.P. (2011). NMR structure and action on nicotinic acetylcholine receptors of water-soluble domain of human LYNX1. J. Biol. Chem. 286 (12), 10618–27 [+]

    Discovery of proteins expressed in the central nervous system sharing the three-finger structure with snake α-neurotoxins provoked much interest to their role in brain functions. Prototoxin LYNX1, having homology both to Ly6 proteins and three-finger neurotoxins, is the first identified member of this family membrane-tethered by a GPI anchor, which considerably complicates in vitro studies. We report for the first time the NMR spatial structure for the water-soluble domain of human LYNX1 lacking a GPI anchor (ws-LYNX1) and its concentration-dependent activity on nicotinic acetylcholine receptors (nAChRs). At 5-30 μM, ws-LYNX1 competed with (125)I-α-bungarotoxin for binding to the acetylcholine-binding proteins (AChBPs) and to Torpedo nAChR. Exposure of Xenopus oocytes expressing α7 nAChRs to 1 μM ws-LYNX1 enhanced the response to acetylcholine, but no effect was detected on α4β2 and α3β2 nAChRs. Increasing ws-LYNX1 concentration to 10 μM caused a modest inhibition of these three nAChR subtypes. A common feature for ws-LYNX1 and LYNX1 is a decrease of nAChR sensitivity to high concentrations of acetylcholine. NMR and functional analysis both demonstrate that ws-LYNX1 is an appropriate model to shed light on the mechanism of LYNX1 action. Computer modeling, based on ws-LYNX1 NMR structure and AChBP x-ray structure, revealed a possible mode of ws-LYNX1 binding.

  43. Shenkarev Z.O., Finkina E.I., Nurmukhamedova E.K., Balandin S.V., Mineev K.S., Nadezhdin K.D., Yakimenko Z.A., Tagaev A.A., Temirov Y.V., Arseniev A.S., Ovchinnikova T.V. (2010). Isolation, structure elucidation, and synergistic antibacterial activity of a novel two-component lantibiotic lichenicidin from Bacillus licheniformis VK21. Biochemistry 49 (30), 6462–72 [+]

    A novel synergetic lantibiotic pair, Lchalpha(3249.51 Da) and Lchbeta(3019.36 Da), termed lichenicidin VK21, was isolated from the producer strain Bacillus licheniformis VK21. Chemical and spatial structures of Lchalphaand Lchbeta were determined. Each peptide contains 31 amino acid residues linked by 4 intramolecular thioether bridges and the N-terminal 2-oxobutyryl group. Spatial structures of Lchalpha and Lchbetawere studied by NMR spectroscopy in methanol solution. Lchalpha peptide displays structural homology with mersacidin-like lantibiotics and involves relatively well-structured N- and C-terminal domains connected by a flexible loop stabilized by thioether bridge Ala11-S-Ala21. In contrast, the Lchbetapeptide represents prolonged hydrophobic alpha-helix flanked with more flexible N- and C-terminal domains. A lantibiotic cluster of the Bacillus licheniformis VK21 genome which comprises the structural genes, lchA1 and lchA2, encoding the lantibiotics precursors, as well as the gene of a modifying enzyme lchM1, was amplified and sequenced. The mature peptides, Lchalphaand Lchbetainteract synergistically to possess antibiotic activity against Gram-positive bacteria within a nanomolar concentration range, though the individual peptides were shown to be active at micromolar concentrations. Our results afford molecular insight into mechanism of lichenicidin VK21 action.

  44. Mineev K.S., Bocharov E.V., Pustovalova Y.E., Bocharova O.V., Chupin V.V., Arseniev A.S. (2010). Spatial Structure of the Transmembrane Domain Heterodimer of ErbB1 and ErbB2 Receptor Tyrosine Kinases. J. Mol. Biol. 400 (2), 231–243 [+]

    Growth factor receptor tyrosine kinases of the ErbB family play a significant role in vital cellular processes and various cancers. During signal transduction across plasma membrane, ErbB receptors are involved in lateral homodimerization and heterodimerization with proper assembly of their extracellular single-span transmembrane (TM) and cytoplasmic domains. The ErbB1/ErbB2 heterodimer appears to be the strongest and most potent inducer of cellular transformation and mitogenic signaling compared to other ErbB homodimers and heterodimers. Spatial structure of the heterodimeric complex formed by TM domains of ErbB1 and ErbB2 receptors embedded into lipid bicelles was obtained by solution NMR. The ErbB1 and ErbB2 TM domains associate in a right-handed alpha-helical bundle through their N-terminal double GG4-like motif T(648)G(649)X(2)G(652)A(653) and glycine zipper motif T(652)X(3)S(656)X(3)G(660), respectively. The described heterodimer conformation is believed to support the juxtamembrane and kinase domain configuration corresponding to the receptor active state. The capability for multiple polar interactions, along with hydrogen bonding between TM segments, correlates with the observed highest affinity of the ErbB1/ErbB2 heterodimer, implying an important contribution of the TM helix-helix interaction to signal transduction.

  45. Bocharov E.V., Mayzel M.L., Volynsky P.E., Mineev K.S., Tkach E.N., Ermolyuk Y.S., Schulga A.A., Efremov R.G., Arseniev A.S. (2010). Left-handed dimer of EphA2 transmembrane domain: Helix packing diversity among receptor tyrosine kinases. Biophys. J. 98 (5), 881–9 [+]

    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.

  46. Bocharov E.V., Mineev K.S., Volynsky P.E., Ermolyuk Y.S., Tkach E.N., Sobol A.G., Chupin V.V., Kirpichnikov M.P., Efremov R.G., Arseniev A.S. (2008). Spatial structure of the dimeric transmembrane domain of the growth factor receptor ErbB2 presumably corresponding to the receptor active state. J. Biol. Chem. 283 (11), 6950–6 [+]

    Proper lateral dimerization of the transmembrane domains of receptor tyrosine kinases is required for biochemical signal transduction across the plasma membrane. The spatial structure of the dimeric transmembrane domain of the growth factor receptor ErbB2 embedded into lipid bicelles was obtained by solution NMR, followed by molecular dynamics relaxation in an explicit lipid bilayer. ErbB2 transmembrane segments associate in a right-handed alpha-helical bundle through the N-terminal tandem GG4-like motif Thr652-X3-Ser656-X3-Gly660, providing an explanation for the pathogenic power of some oncogenic mutations.

  47. Volynsky P.E., Bocharov E.V., Nolde D.E., Vereshaga Y.A., Mayzel M.L., Mineev K.S., Mineeva E.V., Pustovalova Yu.E., Gagnidze I.A., Efremov R.G., Arseniev A.S. (2006). Solution of the Spatial Structure of Dimeric Transmembrane Domains of Proteins by Heteronuclear NMR Spectroscopy and Molecular Modeling. Biophysics 51 (S1), S23–S27 [+]

    Membrane proteins play an important role in various biological processes. An approach combining
    NMR spectroscopy with molecular modeling was used to study the spatial structure and intramolecular dynamics of protein transmembrane domains consisting of two interacting α-helices. The approach was tested with model transmembrane domains and yielded detailed atomic-level data on the protein–protein and protein–lipid interactions.

  48. Polyakov V.B., Mineev S.D., Clayton R.N., Hu G., Mineev K.S. (2005). Determination of tin equilibrium isotope fractionation factors from synchrotron radiation experiments. Geochimica et cosmochimica acta 69 (23), 5531 [+]

    A method of determination of the reduced isotopic partition function ratio (beta-factor) from the partial density of state (PDOS) obtained by inelastic nuclear resonant X-ray scattering (INRXS) in synchrotron radiation experiments has been established. The method has been demonstrated by the example of tin isotopes. The tin beta-factors for CaSnO3, SnO2, SnO have been computed from the INRXS-derived PDOSs.

    ln beta(122/116Sn) =(0.390 +/- 0.0076)x - (0.00160 +/- 0.0000242)x(2) + (1.099 +/- 0.0573) - 10(-5)x(3)) for SnO

    ln(beta 122/116Sn) = (0.771 +/- 0.0150)x - (0.00392 +/- 0.000061)x(2) + (3.548 +/- 0.287). 10(-5)x(3) for SnO2

    ln beta(122/116Sn) = (0.776 +/- 0.0157)x - (0.00334 +/- 0.000064)x(2) + (2.561 +/- 0.157). 10(-5)x(3) for CaSnO3

    Equilibrium Sn-122/116 isotope fractionation between di- and tetravalent tin compounds is about 0.4 parts per thousand at 1000 K and about 4.1 parts per thousand at room temperature and can be measured by modern multicollector inductively-coupled plasma mass-spectrometers. Tin beta-factors reveal dependence on oxidation state previously detected for iron isotopes.

    A comparison of the tin beta-factors for SnO2 obtained on the basis of the INRXS-derived PDOS with those obtained by the Mossbauer spectroscopy method shows that both methods give similar results, but application of synchrotron radiation provides more accurate and reliable data.

    Equilibrium stable isotope fractionation of transition metals between different oxidation state compounds is not negligible even for elements as heavy as tin.