Laboratory of protein engineering

Bioengineering department

Head: Dmitriy Dolgikh, D.Sc
dolgikh@nmr.ru+7(495)336-80-11, +7(495)995-55-57#2503

The Laboratory is engaged in design and study of different recombinant proteins, first of all for medicine and biotechnology. In particular, researchers study toxins, antibodies, cytokines, membrane proteins (receptors, transporters, ion channels, etc.).

Ekaterina Lyukmanova’s group studies three-finger proteins of the Ly6 / uPAR family of human and snake origin. The main focus is on the molecular mechanisms of interaction between the studied proteins with their targets. The group owns advanced technologies of the recombinant production of the three-finger proteins that are not available from natural sources. It had allowed for the first time to investigate the spatial structure and perform a series of studies of functional human proteins such as Lynx1 (neuroplasticity regulation factor), as well as SLURP-1 and SLURP-2 (auto/paracrine regulators of epithelial cells). In the future these proteins may be used as a prototype for drugs that enhance cognitive function, anticancer drugs, and drugs aimed at the treatment of several skin diseases. Today the group actively studies other little-known three-finger proteins Lypd6 and Lypd6. Moreover the group collaborates with the group of the Structural Biology of Ion Channels of the IBCh RAS headed by Zakhar Shenkarev to study of voltage-gated ion channels.

Teymur Aliyev’s group is engaged in the design and improvement (humanization, affinity increase) of recombinant monoclonal antibodies, development of methods for the expression of recombinant antibodies in mammalian cells. Such antibodies may be used for the therapy and diagnosis of autoimmune, infectious and oncological diseases.

The Laboratory produces IgA antibodies against influenza A virus in an attempt to create a universal prophylactic drug against all subtypes of virus. Moreover, researchers produce unique antibodies to create instruments for the prevention and treatment at the early stages of Ebola disease.

Apart from it, the Laboratory creates artificial binding proteins with antibody functions. For the selection of such proteins derived from 10th human fibronectin domain, the cell surface display systems are being constructed. In particular, for these purposes the outer membrane protein of the microorganism isolated from permafrost – autotransporter from Psychrobacter cryohalolentis was expressed and characterized. Another interesting object is a proteorhodopsin from Exiguobacterium sibiricum, discovered and studied by researchers of the Laboratory. In collaboration with the Institute of Structural Biology (Grenoble, France), they determined the spatial structure of the protein.

Another group led by Marine Gasparian developed and received a unique receptor – a selective mutant variant of the antitumor cytokine TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand) – DR5-B. DR5-B kills tumor cells significantly more efficiently than wildtype TRAIL as alone as in combination with chemotherapy. DR5-B drug shows no toxicity in preclinical studies, and therefore it can be regarded as an effective way for the treatment of tumor diseases of various origins.

The Rita Chertkova’s group studies the functional activities of the electron transfer protein – cytochrome c from the different organisms. Using mutagenesis, the amino acid residues which are responsible for the activity of this protein in apoptosis were revealed, as well as residues responsible for transfer of electrons to the respiratory chain. The studies of mechanisms of inter- and intramolecular electron transfer from cytochrome, which results can be used to produce electronic devices with the protein molecules in a monolayer on the conductive surfaces.

Over the years the Laboratory creates and studies artificial proteins based albebetin, the first de novo protein created by a given structure. As a result albebetin variants were produced, which have antiviral, antiproliferative and insulin-like activity. Furthermore, albebetin and biologically active forms were used as a model protein to study amyloid fibril formation patterns underlying Alzheimer's disease.

Research is conducted in collaboration with the Faculty of Biology of Moscow State University, Research Institute of Influenza, Russian Research Center of Molecular Diagnostics and Therapy, Institute of Protein Research RAS, Institute of Physical-Chemical and Biological Problems of Soil Science RAS, V.V. Dokuchaev Soil Science Institute, University of Copenhagen, University of California at Irvine, and others.

The Laboratory of Protein Engineering was established in 2002 as an independent research structural unit of the Institute of Bioorganic Chemistry on the basis of the laboratory of Gene Chemistry and the Protein Engineering group of the laboratory of Spectral Analysis. The Laboratory was founded by Academician Mikhail Petrovich Kirpichnikov, who is now the Dean of the Faculty of Biology in the Lomonosov Moscow State University and the Head of the Department of Bioengineering of the Institute of Bioorganic Chemistry. For pioneering work on the design of artificial proteins with a given structure and biological activity, initiating the development of molecular bioengineering in our country, M.P. Kirpichnikov and D.A. Dolgikh were awarded the State Prize of the Russian Federation in the field of Science and Technology for 1999 (together with O.B. Ptitsin and A.V. Finkelstein of the Institute of Protein Research RAS).

Now the Laboratory directed by Professor Dmitry Dolgikh. He specializes in structural and functional studies, design and bioengineering of proteins. Under his leadership, the Laboratory carried out fundamental studies of the structure and function of a number of proteins that are of primary scientific and practical interest, including cytokines, bioactive artificial proteins, neurotoxins, cytochrome c, ion channels, antibodies.

The main achievements of the Laboratory of Protein Engineering since its inception include the following works:

- Based on the de novo protein albebetin with a given structure were constructed artificial proteins simulated human antiviral binding site of alfa2-interferon and having pronounced antiviral activity comparable with the activity of alfa2-interferon.

- Was created a unique high-yield expression systems to produce snake neurotoxins for structural and functional studies, and proposed models of the interaction of some neurotoxins with their receptors.

- Mutant variants of cytochrome c with abolished apoptotic activity, but retaining the function of an electron carrier in the respiratory chain and antioxidant properties of cytochrome c were designed.

- A new cold-active esterase from Gram-negative bacteria Psychrobacter cryohalolentis, isolated from permafrost, was produced and characterized. Its spatial structure and functional properties were studied.

- A unique retinal protein from Exiguobacterium sibiricum which conducts a transmembrane proton transfer, was expressed and characterized. We have identified the main stages of this process, determined its spatial structure and showed that its proton-acceptor site includes residues His57 and Asp85, and a proton donor for the Schiff base is a lysine residue.

- Recombinant analogues of neuromodulators Lynx1 and SLURPs – unique proteins regulating the nicotinic acetylcholine receptor were produced, and their physical-chemical and functional properties were studied.

- Cell-free expression system for production of integral membrane proteins based on the use of membrane mimetics (micelles of detergents, bicelles, liposomes, and lipid-protein nanodiscs), which allow to receive structured and functionally active membrane proteins, was developed. This system opens new perspectives for high-yield production of membrane proteins for medicine and biotechnology.

NamePositionContacts
Dmitriy Dolgikh, D.Scdepart. dir.dolgikh@nmr.ru+7(495)336-80-11, +7(495)995-55-57#2503
Andrey Wul'fson, Ph.D.l. r. f.andreywulfson@mail.ru+7(495)330-72-74
Aleksej Nekrasov, Ph.D.s. r. f.an_nekrasov@mail.ru
Marine Gasparian, Ph.D.s. r. f.marine_gasparian@yahoo.com+7(495)335-28-88
Rita Chertkova, Ph.D.s. r. f.cherita@inbox.ru+7(495)335-28-88
Ivan Litvinov, Ph.D.s. r. f.litvinov@mail.ibch.ru+7(495)3307265
Lada Petrovskaya, Ph.D.s. r. f.lpetr65@yahoo.com+7(495)330-69-83
Ljudmila Shingarova, Ph.D.s. r. f.lshing@mx.ibch.ru+7(495)330-69-83
Elena Boldyrevar. f.+7(495)330-66-29
Anna Paninar. f.+7()3306638
Anna Yagolovich, Ph.D.r. f.anne-gor2002@yandex.ru+7(926)3780155
Viktorija Toporovaj. r. f.toporova-viktorija@rambler.ru+7(495)330-69-83
Artem Artykovj. r. f.art.al.artykov@gmail.com+7(915)4695747
Dmitry Balabashinj. r. f.
Sultan Gapizovj. r. f.gsultan3@gmail.com
Maria Larinaj. r. f.willis03@yandex.ru
Tat'jana Markovat. q. - lab. as.+7()3307265
Faina Zhuravlevares. eng.

Former members:

Mikhail Kolosov, member of the academy of sciencesdepart. dir.
Ekaterina Lyukmanova, Ph.D.s. r. f.ekaterina-lyukmanova@yandex.ru
Aleksandr Shul'gas. r. f.
Aleksej Shul'ga, Ph.D.s. r. f.schulga@gmail.com
Mikhail Shulepko, Ph.D.r. f.mikhailshulepko@gmail.com
Irina Zyrianova, Ph.D.r. f.zyrianova@yandex.ru
Marina Goncharuk, Ph.D.r. f.m.s.goncharuk@gmail.com
Sergey Goncharuk, Ph.D.r. f.ms.goncharuk@gmail.com
Dmitrii Kulbatskiij. r. f.d.kulbatskiy@gmail.com
Tatyana Bryantcevaj. r. f.tato-tato@yandex.ru
Ya KimPhD stud.
Maxim BychkovPhD stud.mlb@live.ru
Ivan Okhrimenkostud.i.s.okhrimenko@yandex.ru
Mikhail Nureeveng.mnureev@mail.ru
Tat'jana Dobryninares. eng.

Selected publications

  1. Dubovskii P.V., Dubinnyi M.A., Konshina A.G., Kazakova E.D., Sorokoumova G.M., Ilyasova T.M., Shulepko M.A., Chertkova R.V., Lyukmanova E.N., Dolgikh D.A., Arseniev A.S., Efremov R.G. (2017). Structural and Dynamic "Portraits" of Recombinant and Native Cytotoxin I from Naja oxiana: How Close Are They? Biochemistry 56 (34), 4468–4477 [+]

    Today, recombinant proteins are quite widely used in biomedical and biotechnological applications. At the same time, the question about their full equivalence to the native analogues remains unanswered. To gain additional insight into this problem, intimate atomistic details of a relatively simple protein, small and structurally rigid recombinant cardiotoxin I (CTI) from cobra Naja oxiana venom, were characterized using nuclear magnetic resonance (NMR) spectroscopy and atomistic molecular dynamics (MD) simulations in water. Compared to the natural protein, it contains an additional Met residue at the N-terminus. In this work, the NMR-derived spatial structure of uniformly (13)C- and (15)N-labeled CTI and its dynamic behavior were investigated and subjected to comparative analysis with the corresponding data for the native toxin. The differences were found in dihedral angles of only a single residue, adjacent to the N-terminal methionine. Microsecond-long MD traces of the toxins reveal an increased flexibility in the residues spatially close to the N-Met. As the detected structural and dynamic changes of the two CTI models do not result in substantial differences in their cytotoxicities, we assume that the recombinant protein can be used for many purposes as a reasonable surrogate of the native one. In addition, we discuss general features of the spatial organization of cytotoxins, implied by the results of the current combined NMR and MD study.

    ID:1923
  2. Chertkova R.V., Brazhe N.A., Bryantseva T.V., Nekrasov A.N., Dolgikh D.A., Yusipovich A.I., Sosnovtseva O., Maksimov G.V., Rubin A.B., Kirpichnikov M.P. (2017). New insight into the mechanism of mitochondrial cytochrome c function. PLoS ONE 12 (5), e0178280 [+]

    We investigate functional role of the P76GTKMIFA83 fragment of the primary structure of cytochrome c. Based on the data obtained by the analysis of informational structure (ANIS), we propose a model of functioning of cytochrome c. According to this model, conformational rearrangements of the P76GTKMIFA83 loop fragment have a significant effect on conformational mobility of the heme. It is suggested that the conformational mobility of cytochrome c heme is responsible for its optimal orientation with respect to electron donor and acceptor within ubiquinol-cytochrome c oxidoreductase (complex III) and cytochrome c oxidase (complex IV), respectively, thus, ensuring electron transfer from complex III to complex IV. To validate the model, we design several mutant variants of horse cytochrome c with multiple substitutions of amino acid residues in the P76GTKMIFA83 sequence that reduce its ability to undergo conformational rearrangements. With this, we study the succinate-cytochrome c reductase and cytochrome c oxidase activities of rat liver mitoplasts in the presence of mutant variants of cytochrome c. The electron transport activity of the mutant variants decreases to different extent. Resonance Raman spectroscopy (RRS) and surface-enhanced Raman spectroscopy (SERS) data demonstrate, that all mutant cytochromes possess heme with the higher degree of ruffling deformation, than that of the wild-type (WT) cytochrome c. The increase in the ruffled deformation of the heme of oxidized cytochromes correlated with the decrease in the electron transport rate of ubiquinol-cytochrome c reductase (complex III). Besides, all mutant cytochromes have lower mobility of the pyrrol rings and methine bridges, than WT cytochrome c. We show that a decrease in electron transport activity in the mutant variants correlates with conformational changes and reduced mobility of heme porphyrin. This points to a significant role of the P76GTKMIFA83 fragment in the electron transport function of cytochrome c.

    ID:1979
  3. Valieva M.E., Armeev G.A., Kudryashova K.S., Gerasimova N.S., Shaytan A.K., Kulaeva O.I., McCullough L.L., Formosa T., Georgiev P.G., Kirpichnikov M.P., Studitsky V.M., Feofanov A.V. (2016). Large-scale ATP-independent nucleosome unfolding by a histone chaperone. Nat. Struct. Mol. Biol. , [+]

    DNA accessibility to regulatory proteins is substantially influenced by nucleosome structure and dynamics. The facilitates chromatin transcription (FACT) complex increases the accessibility of nucleosomal DNA, but the mechanism and extent of its nucleosome reorganization activity are unknown. Here we determined the effects of FACT from the yeast Saccharomyces cerevisiae on single nucleosomes by using single-particle Förster resonance energy transfer (spFRET) microscopy. FACT binding results in dramatic ATP-independent, symmetrical and reversible DNA uncoiling that affects at least 70% of the DNA within a nucleosome, occurs without apparent loss of histones and proceeds via an 'all-or-none' mechanism. A mutated version of FACT is defective in uncoiling, and a histone mutation that suppresses phenotypes caused by this FACT mutation in vivo restores the uncoiling activity in vitro. Thus, FACT-dependent nucleosome unfolding modulates the accessibility of nucleosomal DNA, and this activity is an important function of FACT in vivo.

    ID:1625
  4. Feldman T.B., Smitienko O.A., Shelaev I.V., Gostev F.E., Nekrasova O.V., Dolgikh D.A., Nadtochenko V.A., Kirpichnikov M.P., Ostrovsky M.A. (2016). Femtosecond spectroscopic study of photochromic reactions of bacteriorhodopsin and visual rhodopsin. J. Photochem. Photobiol. B, Biol. 164, 296–305 [+]

    Photochromic ultrafast reactions of bacteriorhodopsin (H. salinarum) and bovine rhodopsin were conducted with a femtosecond two-pump probe pulse setup with the time resolution of 20-25fs. The dynamics of the forward and reverse photochemical reactions for both retinal-containing proteins was compared. It is demonstrated that when retinal-containing proteins are excited by femtosecond pulses, dynamics pattern of the vibrational coherent wave packets in the course of the reaction is different for bacteriorhodopsin and visual rhodopsin. As shown in these studies, the low-frequencies that form a wave packets experimentally observed in the dynamics of primary products formation as a result of retinal photoisomerization have different intensities and are clearer for bovine rhodopsin. Photo-reversible reactions for both retinal proteins were performed from the stage of the relatively stable photointermediates that appear within 3-5ps after the light pulse impact. It is demonstrated that the efficiency of the reverse phototransition K-form→bacteriorhodopsin is almost five-fold higher than that of the Batho-intermediate→visual rhodopsin phototransition. The results obtained indicate that in the course of evolution the intramolecular mechanism of the chromophore-protein interaction in visual rhodopsin becomes more perfect and specific. The decrease in the probability of the reverse chromophore photoisomerization (all-trans→11-cis retinal) in primary photo-induced rhodopsin products causes an increase in the efficiency of the photoreception process.

    ID:1612
  5. Siletsky S.A., Mamedov M.D., Lukashev E.P., Balashov S.P., Dolgikh D.A., Rubin A.B., Kirpichnikov M.P., Petrovskaya L.E. (2016). Electrogenic steps of light-driven proton transport in ESR, a retinal protein from Exiguobacterium sibiricum. Biochim. Biophys. Acta 1857 (11), 1741–1750 [+]

    A retinal protein from Exiguobacterium sibiricum (ESR) functions as a light-driven proton pump. Unlike other proton pumps, it contains Lys96 instead of a usual carboxylic residue in the internal proton donor site. Nevertheless, the reprotonation of the Schiff base occurs fast, indicating that Lys96 facilitates proton transfer from the bulk. In this study we examined kinetics of light-induced transmembrane electrical potential difference, ΔΨ, generated in proteoliposomes reconstituted with ESR. We show that total magnitude of ΔΨ is comparable to that produced by bacteriorhodopsin but its kinetic components and their pH dependence are substantially different. The results are in agreement with the earlier finding that proton uptake precedes reprotonation of the Schiff base in ESR, suggesting that Lys96 is unprotonated in the initial state and gains a proton transiently in the photocycle. The electrogenic phases and the photocycle transitions related to proton transfer from the bulk to the Schiff base are pH dependent. At neutral pH, they occur with τ 0.5ms and 4.5ms. At alkaline pH, the fast component ceases and Schiff base reprotonation slows. At pH8.4, a spectrally silent electrogenic component with τ 0.25ms is detected, which can be attributed to proton transfer from the bulk to Lys96. At pH5.1, the amplitude of ΔΨ decreases 10 fold, reflecting a decreased yield and rate of proton transfer, apparently from protonation of the acceptor (Asp85-His57 pair) in the initial state. The features of the photoelectric potential generation correlate with the ESR structure and proposed mechanism of proton transfer.

    ID:1662
  6. Shulepko M.A., Lyukmanova E.N., Shenkarev Z.O., Dubovskii P.V., Astapova M.V., Feofanov A.V., Arseniev A.S., Utkin Y.N., Kirpichnikov M.P., Dolgikh D.A. (2016). Towards universal approach for bacterial production of three-finger Ly6/uPAR proteins: Case study of cytotoxin I from cobra N. oxiana. Protein Expr. Purif. 130, 13–20 [+]

    Cytotoxins or cardiotoxins is a group of polycationic toxins from cobra venom belonging to the 'three-finger' protein superfamily (Ly6/uPAR family) which includes small β-structural proteins (60-90 residues) with high disulfide bond content (4-5 disulfides). Due to a high cytotoxic activity for cancer cells, cytotoxins are considered as potential anticancer agents. Development of the high-throughput production methods is required for the prospective applications of cytotoxins. Here, efficient approach for bacterial production of recombinant analogue of cytotoxin I from N. oxiana containing additional N-terminal Met-residue (rCTX1) was developed. rCTX1 was produced in the form of E. coli inclusion bodies. Refolding in optimized conditions provided ∼6 mg of correctly folded protein from 1 L of bacterial culture. Cytotoxicity of rCTX1 for C6 rat glioma cells was found to be similar to the activity of wild type CTX1. The milligram quantities of (13)C,(15)N-labeled rCTX1 were obtained. NMR study confirmed the similarity of the spatial structures of recombinant and wild-type toxins. Additional Met residue does not perturb the overall structure of the three-finger core. The analysis of available data for different Ly6/uPAR proteins of snake and human origin revealed that efficiency of their folding in vitro is correlated with the number of proline residues in the third loop and the surface area of hydrophobic residues buried within the protein interior. The obtained data indicate that hydrophobic core is important for the folding of proteins with high disulfide bond content. Developed expression method opens new possibilities for structure-function studies of CTX1 and other related three-finger proteins.

    ID:1599
  7. Thomsen M.S., Arvaniti M., Jensen M.M., Shulepko M.A., Dolgikh D.A., Pinborg L.H., Härtig W., Lyukmanova E.N., Mikkelsen J.D. (2016). Lynx1 and Aβ1-42 bind competitively to multiple nicotinic acetylcholine receptor subtypes. Neurobiol. Aging 46, 13–21 [+]

    Lynx1 regulates synaptic plasticity in the brain by regulating nicotinic acetylcholine receptors (nAChRs). It is not known to which extent Lynx1 can bind to endogenous nAChR subunits in the brain or how this interaction is affected by Alzheimer's disease pathology. We apply affinity purification to demonstrate that a water-soluble variant of human Lynx1 (Ws-Lynx1) isolates α3, α4, α5, α6, α7, β2, and β4 nAChR subunits from human and rat cortical extracts, and rat midbrain and olfactory bulb extracts, suggesting that Lynx1 forms complexes with multiple nAChR subtypes in the human and rodent brain. Incubation with Ws-Lynx1 decreases nicotine-mediated extracellular signal-regulated kinase phosphorylation in PC12 cells and striatal neurons, indicating that binding of Ws-Lynx1 is sufficient to inhibit signaling downstream of nAChRs. The effect of nicotine in PC12 cells is independent of α7 or α4β2 nAChRs, suggesting that Lynx1 can affect the function of native non-α7, non-α4β2 nAChR subtypes. We further show that Lynx1 and oligomeric β-amyloid1-42 compete for binding to several nAChR subunits, that Ws-Lynx1 prevents β-amyloid1-42-induced cytotoxicity in cortical neurons, and that cortical Lynx1 levels are decreased in a transgenic mouse model with concomitant β-amyloid and tau pathology. Our data suggest that Lynx1 binds to multiple nAChR subtypes in the brain and that this interaction might have functional and pathophysiological implications in relation to Alzheimer's disease.

    ID:1667
  8. Nekrasova O.V., Volyntseva A.D., Kudryashova K.S., Novoseletsky V.N., Lyapina E.A., Illarionova A.V., Yakimov S.A., Korolkova Y.V., Shaitan K.V., Kirpichnikov M.P., Feofanov A.V. (2016). Complexes of Peptide Blockers with Kv1.6 Pore Domain: Molecular Modeling and Studies with KcsA-Kv1.6 Channel. J Neuroimmune Pharmacol , [+]

    Potassium voltage-gated Kv1.6 channel, which is distributed primarily in neurons of central and peripheral nervous systems, is of significant physiological importance. To date, several high-affinity Kv1.6-channel blockers are known, but the lack of selective ones among them hampers the studies of tissue localization and functioning of Kv1.6 channels. Here we present an approach to advanced understanding of interactions of peptide toxin blockers with a Kv1.6 pore. It combines molecular modeling studies and an application of a new bioengineering system based on a KcsA-Kv1.6 hybrid channel for the quantitative fluorescent analysis of blocker-channel interactions. Using this system we demonstrate that peptide toxins agitoxin 2, kaliotoxin1 and OSK1 have similar high affinity to the extracellular vestibule of the K(+)-conducting pore of Kv1.6, hetlaxin is a low-affinity ligand, whereas margatoxin and scyllatoxin do not bind to Kv1.6 pore. Binding of toxins to Kv1.6 pore has considerable inverse dependence on the ionic strength. Model structures of KcsA-Kv1.6 and Kv1.6 complexes with agitoxin 2, kaliotoxin 1 and OSK1 were obtained using homology modeling and molecular dynamics simulation. Interaction interfaces, which are formed by 15-19 toxin residues and 10 channel residues, are described and compared. Specific sites of Kv1.6 pore recognition are identified for targeting of peptide blockers. Analysis of interactions between agitoxin 2 derivatives with point mutations (S7K, S11G, L19S, R31G) and KcsA-Kv1.6 confirms reliability of the calculated complex structure.

    ID:1611
  9. Lyukmanova E.N., Shulepko M.A., Shenkarev Z.O., Kasheverov I.E., Chugunov A.O., Kulbatskii D.S., Myshkin M.Y., Utkin Y.N., Efremov R.G., Tsetlin V.I., Arseniev A.S., Kirpichnikov M.P., Dolgikh D.A. (2016). Central loop of non-conventional toxin WTX from Naja kaouthia is important for interaction with nicotinic acetylcholine receptors. Toxicon 119, 274–9 [+]

    'Three-finger' toxin WTX from Naja kaouthia interacts with nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs). Mutagenesis and competition experiments with (125)I-α-bungarotoxin revealed that Arg31 and Arg32 residues from the WTX loop II are important for binding to Torpedo californica and human α7 nAChRs. Computer modeling suggested that loop II occupies the orthosteric binding site at α7 nAChR. The similar toxin interface was previously described as a major determinant of allosteric interactions with mAChRs.

    ID:1598
  10. Lyukmanova E.N., Shulepko M.A., Shenkarev Z.O., Bychkov M.L., Paramonov A.S., Chugunov A.O., Kulbatskii D.S., Arvaniti M., Dolejsi E., Schaer T., Arseniev A.S., Efremov R.G., Thomsen M.S., Dolezal V., Bertrand D., Dolgikh D.A., Kirpichnikov M.P. (2016). Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors. Sci Rep 6, 30698 [+]

    Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3β2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure and pharmacology of a recombinant analogue of SLURP-2. Nuclear magnetic resonance spectroscopy revealed a 'three-finger' fold of SLURP-2 with a conserved β-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the α3, α4, α5, α7, β2, and β4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at α4β2 and α3β2-nAChRs (IC50 ~0.17 and >3 μM, respectively) expressed in Xenopus oocytes. In contrast, at α7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations <1 μM but induces inhibition at higher concentrations. SLURP-2 allosterically interacts with human M1 and M3 muscarinic acetylcholine receptors (mAChRs) that are overexpressed in CHO cells. SLURP-2 was found to promote the proliferation of human oral keratinocytes via interactions with α3β2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the 'classical' orthosteric agonist/antagonist binding sites at α7 and α3β2-nAChRs.

    ID:1597
  11. Pletneva N.V., Pletnev S., Pakhomov A.A., Chertkova R.V., Martynov V.I., Muslinkina L., Dauter Z., Pletnev V.Z. (2016). Crystal structure of the fluorescent protein from Dendronephthya sp. in both green and photoconverted red forms. Acta Crystallogr D Struct Biol 72 (Pt 8), 922–32 [+]

    The fluorescent protein from Dendronephthya sp. (DendFP) is a member of the Kaede-like group of photoconvertible fluorescent proteins with a His62-Tyr63-Gly64 chromophore-forming sequence. Upon irradiation with UV and blue light, the fluorescence of DendFP irreversibly changes from green (506 nm) to red (578 nm). The photoconversion is accompanied by cleavage of the peptide backbone at the C(α)-N bond of His62 and the formation of a terminal carboxamide group at the preceding Leu61. The resulting double C(α)=C(β) bond in His62 extends the conjugation of the chromophore π system to include imidazole, providing the red fluorescence. Here, the three-dimensional structures of native green and photoconverted red forms of DendFP determined at 1.81 and 2.14 Å resolution, respectively, are reported. This is the first structure of photoconverted red DendFP to be reported to date. The structure-based mutagenesis of DendFP revealed an important role of positions 142 and 193: replacement of the original Ser142 and His193 caused a moderate red shift in the fluorescence and a considerable increase in the photoconversion rate. It was demonstrated that hydrogen bonding of the chromophore to the Gln116 and Ser105 cluster is crucial for variation of the photoconversion rate. The single replacement Gln116Asn disrupts the hydrogen bonding of Gln116 to the chromophore, resulting in a 30-fold decrease in the photoconversion rate, which was partially restored by a further Ser105Asn replacement.

    ID:1587
  12. Petrovskaya L.E., NovototskayaVlasova K.A., Spirina E.V., Durdenko E.V., Lomakina G.Y., Zavialova M.G., Nikolaev E.N., Rivkina E.M. (2016). Expression and characterization of a new esterase with GCSAG motif from a permafrost metagenomic library. FEMS Microbiol. Ecol. 92 (5), fiw046 [+]

    As a result of construction and screening of a metagenomic library prepared from a permafrost-derived microcosm, we have isolated a novel gene coding for a putative lipolytic enzyme that belongs to the hormone-sensitive lipase family. It encodes a polypeptide of 343 amino acid residues whose amino acid sequence displays maximum likelihood with uncharacterized proteins from Sphingomonas species. A putative catalytic serine residue of PMGL2 resides in a new variant of a recently discovered GTSAG sequence in which a Thr residue is replaced by a Cys residue (GCSAG). The recombinant PMGL2 was produced in Escherichia coli cells and purified by Ni-affinity chromatography. The resulting protein preferably utilizes short-chain p-nitrophenyl esters (C4 and C8) and therefore is an esterase. It possesses maximum activity at 45°C in slightly alkaline conditions and has limited thermostability at higher temperatures. Activity of PMGL2 is stimulated in the presence of 0.25-1.5 M NaCl indicating the good salt tolerance of the new enzyme. Mass spectrometric analysis demonstrated that N-terminal methionine in PMGL2 is processed and cysteine residues do not form a disulfide bond. The results of the study demonstrate the significance of the permafrost environment as a unique genetic reservoir and its potential for metagenomic exploration.

    ID:1668
  13. Lyukmanova E.N., Shulepko M.A., Kudryavtsev D., Bychkov M.L., Kulbatskii D.S., Kasheverov I.E., Astapova M.V., Feofanov A.V., Thomsen M.S., Mikkelsen J.D., Shenkarev Z.O., Tsetlin V.I., Dolgikh D.A., Kirpichnikov M.P. (2016). Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor. PLoS ONE 11 (2), e0149733 [+]

    SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,-non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that the antiproliferative activity of SLURP-1 is related to 'metabotropic' signaling pathway through α7-nAChR, that activates intracellular signaling cascades without opening the receptor channel.

    ID:1420
  14. Kuzmenkov A.I., Nekrasova O.V., Kudryashova K.S., Peigneur S., Tytgat J., Stepanov A.V., Kirpichnikov M.P., Grishin E.V., Feofanov A.V., Vassilevski A.A. (2016). Fluorescent protein-scorpion toxin chimera is a convenient molecular tool for studies of potassium channels. Sci Rep 6, 33314 [+]

    Ion channels play a central role in a host of physiological and pathological processes and are the second largest target for existing drugs. There is an increasing need for reliable tools to detect and visualize particular ion channels, but existing solutions suffer from a number of limitations such as high price, poor specificity, and complicated protocols. As an alternative, we produced recombinant chimeric constructs (FP-Tx) consisting of fluorescent proteins (FP) fused with potassium channel toxins from scorpion venom (Tx). In particular, we used two FP, eGFP and TagRFP, and two Tx, OSK1 and AgTx2, to create eGFP-OSK1 and RFP-AgTx2. We show that these chimeras largely retain the high affinity of natural toxins and display selectivity to particular ion channel subtypes. FP-Tx are displaced by other potassium channel blockers and can be used as an imaging tool in ion channel ligand screening setups. We believe FP-Tx chimeras represent a new efficient molecular tool for neurobiology.

    ID:1561
  15. Lyukmanova E.N., Shenkarev Z.O., Shulepko M.A., Paramonov A.S., Chugunov A.O., Janickova H., Dolejsi E., Dolezal V., Utkin Y.N., Tsetlin V.I., Arseniev A.S., Efremov R.G., Dolgikh D.A., Kirpichnikov M.P. (2015). Structural Insight into Specificity of Interactions between Nonconventional Three-finger Weak Toxin from Naja kaouthia (WTX) and Muscarinic Acetylcholine Receptors. J. Biol. Chem. 290 (39), 23616–30 [+]

    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.

    ID:1394
  16. Gasparian M.E., Bychkov M.L., Yagolovich A.V., Dolgikh D.A., Kirpichnikov M.P. (2015). Mutations Enhancing Selectivity of Antitumor Cytokine TRAIL to DR5 Receptor Increase Its Cytotoxicity against Tumor Cells. Biochemistry Mosc. 80 (8), 1080–91 [+]

    Tumor necrosis factor superfamily cytokine TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) induces apoptosis in tumor cells by binding to death receptors DR4 and DR5 without affecting normal cells. However, the therapeutic use of TRAIL is limited, because many tumor cells are resistant to it. The resistance is partially related to interaction of TRAIL with the decoy receptors DcR1 and DcR2, which do not trigger the apoptotic signal and inhibit signaling of death receptors. Previously, we designed a unique DR5-specific TRAIL mutant variant DR5-B, which binds to DR5 receptor as effectively as the original cytokine, but has practically no interaction with DR4 and DcR1 receptors, and its affinity for DcR2 is reduced 400-fold. In the present work, the cytotoxity of TRAIL and DR5-B was analyzed on 12 different tumor cell lines and two types of normal cells. In nine of 12 tumor cell lines, DR5-B killed 1.5-5.0 times more tumor cells than TRAIL, and it did not exhibit toxicity towards normal cells. Chemotherapeutic drugs such as doxorubicin, paclitaxel, and bortezomib augmented the effect of both TRAIL variants, and the enhancing effect was more pronounced for DR5-B. Half-maximal effective concentrations (EC50) for DR5-B in combination with chemotherapeutic agents were 1.5-10.0 times lower than for wild-type TRAIL. Thus, DR5-B is a promising candidate both for monotherapy and in combination with chemotherapy for treatment of TRAIL-resistant tumors.

    ID:1482
  17. Bychkov M.L., Gasparian M.E., Dolgikh D.A., Kirpichnikov M.P. (2014). Combination of TRAIL with bortezomib shifted apoptotic signaling from DR4 to DR5 death receptor by selective internalization and degradation of DR4. PLoS ONE 9 (10), e109756 [+]

    TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) mediates apoptosis in cancer cells through death receptors DR4 and DR5 preferring often one receptor over another in the cells expressing both receptors. Receptor selective mutant variants of TRAIL and agonistic antibodies against DR4 and DR5 are highly promising anticancer agents. Here using DR5 specific mutant variant of TRAIL - DR5-B we have demonstrated for the first time that the sensitivity of cancer cells can be shifted from one TRAIL death receptor to another during co-treatment with anticancer drugs. First we have studied the contribution of DR4 and DR5 in HCT116 p53+/+ and HCT116 p53−/− cells and demonstrated that in HCT116 p53+/+ cells the both death receptors are involved in TRAIL-induced cell death while in HCT116 p53−/− cells prevailed DR4 signaling. The expression of death (DR4 and DR5) as well as decoy (DcR1 and DcR2) receptors was upregulated in the both cell lines either by TRAIL or by bortezomib. However, combined treatment of cells with two drugs induced strong time-dependent and p53-independent internalization and further lysosomal degradation of DR4 receptor. Interestingly DR5-B variant of TRAIL which do not bind with DR4 receptor also induced elimination of DR4 from cell surface in combination with bortezomib indicating the ligand-independent mechanism of the receptor internalization. Eliminatory internalization of DR4 resulted in activation of DR5 receptor thus DR4-dependent HCT116 p53−/− cells became highly sensitive to DR5-B in time-dependent manner. Internalization and degradation of DR4 receptor depended on activation of caspases as well as of lysosomal activity as it was completely inhibited by Z-VAD-FMK, E-64 and Baf-A1. In light of our findings, it is important to explore carefully which of the death receptors is active, when sensitizing drugs are combined with agonistic antibodies to the death receptors or receptor selective variants of TRAIL to enhance cancer treatment efficiency.

    ID:1296
  18. 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.

    ID:1104
  19. Shenkarev Z.O., Lyukmanova E.N., Butenko I.O., Petrovskaya L.E., Paramonov A.S., Shulepko M.A., Nekrasova O.V., Kirpichnikov M.P., Arseniev A.S. (2013). Lipid-protein nanodiscs promote in vitro folding of transmembrane domains of multi-helical and multimeric membrane proteins. Biochim. Biophys. Acta 1828 (2), 776–84 [+]

    Production of helical integral membrane proteins (IMPs) in a folded state is a necessary prerequisite for their functional and structural studies. In many cases large-scale expression of IMPs in cell-based and cell-free systems results in misfolded proteins, which should be refolded in vitro. Here using examples of the bacteriorhodopsin ESR from Exiguobacterium sibiricum and full-length homotetrameric K(+) channel KcsA from Streptomyces lividans we found that the efficient in vitro folding of the transmembrane domains of the polytopic and multimeric IMPs could be achieved during the protein encapsulation into the reconstructed high-density lipoprotein particles, also known as lipid-protein nanodiscs. In this case the self-assembly of the IMP/nanodisc complexes from a mixture containing apolipoprotein, lipids and the partially denatured protein solubilized in a harsh detergent induces the folding of the transmembrane domains. The obtained folding yields showed significant dependence on the properties of lipids used for nanodisc formation. The largest recovery of the spectroscopically active ESR (~60%) from the sodium dodecyl sulfate (SDS) was achieved in the nanodiscs containing anionic saturated lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPG) and was approximately twice lower in the zwitterionic DMPC lipid. The reassembly of tetrameric KcsA from the acid-dissociated monomer solubilized in SDS was the most efficient (~80%) in the nanodiscs containing zwitterionic unsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The charged and saturated lipids provided lower tetramer quantities, and the lowest yield (<20%) was observed in DMPC. The overall yield of the ESR and KcsA folding was mainly restricted by the efficiency of the protein encapsulation into the nanodiscs.

    ID:802
  20. Lyukmanova E.N., Shenkarev Z.O., Khabibullina N.F., Kulbatskiy D.S., Shulepko M.A., Petrovskaya L.E., Arseniev A.S., Dolgikh D.A., Kirpichnikov M.P. (2012). N-terminal fusion tags for effective production of g-protein-coupled receptors in bacterial cell-free systems. Acta Naturae 4 (4), 58–64 [+]

    G-protein-coupled receptors (GPCR) constitute one of the biggest families of membrane proteins. In spite of the fact that they are highly relevant to pharmacy, they have remained poorly explored. One of the main bottlenecks encountered in structural-functional studies of GPCRs is the difficulty to produce sufficient amounts of the proteins. Cell-free systems based on bacterial extracts fromE. colicells attract much attention as an effective tool for recombinant production of membrane proteins. GPCR production in bacterial cell-free expression systems is often inefficient because of the problems associated with the low efficiency of the translation initiation process. This problem could be resolved if GPCRs were expressed in the form of hybrid proteins with N-terminal polypeptide fusion tags. In the present work, three new N-terminal fusion tags are proposed for cell-free production of the human β2-adrenergic receptor, human M1 muscarinic acetylcholine receptor, and human somatostatin receptor type 5. It is demonstrated that the application of an N-terminal fragment (6 a.a.) of bacteriorhodopsin fromExiguobacterium sibiricum(ESR-tag), N-terminal fragment (16 а.о.) of RNAse A (S-tag), and Mistic protein fromB. subtilisallows to increase the CF synthesis of the target GPCRs by 5-38 times, resulting in yields of 0.6-3.8 mg from 1 ml of the reaction mixture, which is sufficient for structural-functional studies.

    ID:801
  21. Ostapchenko V.G., Gasparian M.E., Kosinsky Y.A., Efremov R.G., Dolgikh D.A., Kirpichnikov M.P. (2012). Dissecting structural basis of the unique substrate selectivity of human enteropeptidase catalytic subunit. J. Biomol. Struct. Dyn. 30 (1), 62–73 [+]

    Enteropeptidase is a key enzyme in the digestion system of higher animals. It initiates enzymatic cascade cleaving trypsinogen activation peptide after a unique sequence DDDDK. Recently, we have found specific activity of human enteropeptidase catalytic subunit (L-HEP) being significantly higher than that of its bovine ortholog (L-BEP). Moreover, we have discovered that L-HEP hydrolyzed several nonspecific peptidic substrates. In this work, we aimed to further characterize species-specific enteropeptidase activities and to reveal their structural basis. First, we compared hydrolysis of peptides and proteins lacking DDDDK sequence by L-HEP and L-BEP. In each case human enzyme was more efficient, with the highest hydrolysis rate observed for substrates with a large hydrophobic residue in P2-position. Computer modeling suggested enzyme exosite residues 96 (Arg in L-HEP, Lys in L-BEP) and 219 (Lys in L-HEP, Gln in L-BEP) to be responsible for these differences in enteropeptidase catalytic activity. Indeed, human-to-bovine mutations Arg96Lys, Lys219Gln shifted catalytic properties of L-HEP toward those of L-BEP. This effect was amplified in case of the double mutation Arg96Lys/Lys219Gln, but still did not cover the full difference in catalytic activities of human and bovine enzymes. To find a missing link, we studied monopeptide benzyl-arginine-β-naphthylamide hydrolysis. L-HEP catalyzed it with an order lower K (m) than L-BEP, suggesting the monopeptide-binding S1 site input into catalytic distinction between two enteropeptidase species. Together, our findings suggest structural basis of the unique catalytic properties of human enteropeptidase and instigate further studies of its tentative physiological and pathological roles.

    ID:809
  22. 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.

    ID:541
  23. 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.

    ID:320
  24. Volynsky P.E., Mineeva E.A., Goncharuk M.V., Ermolyuk Y.S., Arseniev A.S., Efremov R.G. (2010). Computer simulations and modeling-assisted ToxR screening in deciphering 3D structures of transmembrane alpha-helical dimers: ephrin receptor A1. Phys Biol 7, 16014 [+]

    Membrane-spanning segments of numerous proteins (e.g. receptor tyrosine kinases) represent a novel class of pharmacologically important targets, whose activity can be modulated by specially designed artificial peptides, the so-called interceptors. Rational construction of such peptides requires understanding of the main factors driving peptide-peptide association in lipid membranes. Here we present a new method for rapid prediction of the spatial structure of transmembrane (TM) helix-helix complexes. It is based on computer simulations in membrane-like media and subsequent refinement/validation of the results using experimental studies of TM helix dimerization in a bacterial membrane by means of the ToxR system. The approach was applied to TM fragments of the ephrin receptor A1 (EphA1). A set of spatial structures of the dimer was proposed based on Monte Carlo simulations in an implicit membrane followed by molecular dynamics relaxation in an explicit lipid bilayer. The resulting models were employed for rational design of wild-type and mutant genetic constructions for ToxR assays. The computational and the experimental data are self-consistent and provide an unambiguous spatial model of the TM dimer of EphA1. The results of this work can be further used to develop new biologically active 'peptide interceptors' specifically targeting membrane domains of proteins.

    ID:312
  25. Goncharuk S.A., Shulga A.A., Ermolyuk Y.S., Kuzmichev P.K., Sobol V.A., Bocharov E.V., Chupin V.V., Arseniev A.S., Kirpichnikov M.P. (2009). Bacterial synthesis, purification, and solubilization of membrane protein KCNE3, a regulator of voltage-gated potassium channels. Biochemistry Mosc. 74 (12), 1344–9 [+]

    An efficient method is described for production of membrane protein KCNE3 and its isotope labeled derivatives ((15)N-, (15)N-/13C-) in amounts sufficient for structural-functional investigations. The purified protein preparation within different detergent micelles was characterized using dynamic light scattering, CD spectroscopy, and NMR spectroscopy. It is shown that within DPC/LDAO micelles the protein is in monomeric form and acquires mainly alpha-helical conformation. The existence of cross-peaks for all glycines of the (15)N-HSQC NMR spectra as well as relatively small line widths (~20 Hz) confirm the high quality of the preparation and the possibility of obtaining structural-dynamic information on KCNE3 by high resolution heteronuclear NMR spectroscopy.

    ID:273
  26. Lesovoy D.M., Bocharov E.V., Lyukmanova E.N., Kosinsky Y.A., Shulepko M.A., Dolgikh D.A., Kirpichnikov M.P., Efremov R.G., Arseniev A.S. (2009). Specific membrane binding of neurotoxin II can facilitate its delivery to acetylcholine receptor. Biophys. J. 97 (7), 2089–97 [+]

    The action of three-finger snake alpha-neurotoxins at their targets, nicotinic acetylcholine receptors (nAChR), is widely studied because of its biological and pharmacological relevance. Most such studies deal only with ligands and receptor models; however, for many ligand/receptor systems the membrane environment may affect ligand binding. In this work we focused on binding of short-chain alpha-neurotoxin II (NTII) from Naja oxiana to the native-like lipid bilayer, and the possible role played by the membrane in delivering the toxin to nAChR. Experimental (NMR and mutagenesis) and molecular modeling (molecular-dynamics simulation) studies revealed a specific interaction of the toxin molecule with the phosphatidylserine headgroup of lipids, resulting in the proper topology of NTII on lipid bilayers favoring the attack of nAChR. Analysis of short-chain alpha-neurotoxins showed that most of them possess a high positive charge and sequence homology in the lipid-binding motif of NTII, implying that interaction with the membrane surrounding nAChR may be common for the toxin family.

    ID:319
  27. Schulga A.A., Mechev P.V., Kirpichnikov M.P., Skryabin K.G., Deyev S.M. (2009). Construction of the plasmid-free strain for human growth hormone production. Biochimie 128-129, 148–53 [+]

    The E. coli strain, overproducing human growth hormone (hGH) was made by integration of the hGH gene under the control of T7 promoter into the chromosomal LacZ gene of BL21(DE3) via lambda Red recombineering. The strain gave higher productivity (50 mg·L(-1)·OD550(-1)) and better growth characteristics than the corresponding strain in which the same hGH expression cassette was placed in a plasmid. The protein produced by the plasmid-free strain was purified and characterized to be hGH. The results demonstrates that a plasmid-free recombinant strain having a single-copy gene expression cassette in the chromosome could provide better gene activity regulation, higher productivity, superior growth characteristics, as well as more stringent control of the gene sequence invariance than a plasmid-based strain.

    ID:1669
  28. Bocharov E.V., Mayzel M.L., Volynsky P.E., Goncharuk M.V., Ermolyuk Y.S., Schulga A.A., Artemenko E.O., Efremov R.G., Arseniev A.S. (2008). Spatial structure and pH-dependent conformational diversity of dimeric transmembrane domain of the receptor tyrosine kinase EphA1. J. Biol. Chem. 283 (43), 29385–95 [+]

    Eph receptors are found in a wide variety of cells in developing and mature tissues and represent the largest family of receptor tyrosine kinases, regulating cell shape, movements, and attachment. The receptor tyrosine kinases conduct biochemical signals across plasma membrane via lateral dimerization in which their transmembrane domains play an important role. Structural-dynamic properties of the homodimeric transmembrane domain of the EphA1 receptor were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics in explicit lipid bilayer. EphA1 transmembrane segments associate in a right-handed parallel alpha-helical bundle, region (544-569)(2), through the N-terminal glycine zipper motif A(550)X(3)G(554)X(3)G(558). Under acidic conditions, the N terminus of the transmembrane helix is stabilized by an N-capping box formed by the uncharged carboxyl group of Glu(547), whereas its deprotonation results in a rearrangement of hydrogen bonds, fractional unfolding of the helix, and a realignment of the helix-helix packing with appearance of additional minor dimer conformation utilizing seemingly the C-terminal GG4-like dimerization motif A(560)X(3)G(564). This can be interpreted as the ability of the EphA1 receptor to adjust its response to ligand binding according to extracellular pH. The dependence of the pK(a) value of Glu(547) and the dimer conformational equilibrium on the lipid head charge suggests that both local environment and membrane surface potential can modulate dimerization and activation of the receptor. This makes the EphA1 receptor unique among the Eph family, implying its possible physiological role as an "extracellular pH sensor," and can have relevant physiological implications.

    ID:317
  29. 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.

    ID:314

Dmitriy Dolgikh

  • Russia, Moscow, Ul. Miklukho-Maklaya 16/10 — On the map
  • IBCh RAS, build. 33, office. 424
  • Phone: +7(495)336-80-11
  • E-mail: dolgikh@nmr.ru

Fragment (76)PGTKMIFA(83) of horse cytochrome c plays a key role in its electron transport activity (2017-11-28)

The influence of conformation of the loop fragment P76GTKMIFA83 of horse cytochrome c on its electron transport activity was studied. Based on the information structure analysis, a number of variants of cytochrome c with multiple substitutions in the site P76GTKMIFA83, were obtained, aimed at lowering its conformational mobility. Succinate:cytochrome c-reductase and cytochrome c-oxidase activity of rat liver mitoplasts in the presence of cytochrome c variants was studied. It was shown that the ability to transfer electron of mutant variants has been decreased to different extent. According to the Resonance Raman spectroscopy (RRS) and surface-enhanced Raman spectroscopy (SERS) data, a decrease in the electron transport activity in the mutant variants correlates with conformational changes and reduced mobility of heme porphyrin. This points to a significant role of the P76GTKMIFA83 fragment in the electron transport function of cytochrome c.

Publications

  1. Chertkova R.V., Brazhe N.A., Bryantseva T.V., Nekrasov A.N., Dolgikh D.A., Yusipovich A.I., Sosnovtseva O., Maksimov G.V., Rubin A.B., Kirpichnikov M.P. (2017). New insight into the mechanism of mitochondrial cytochrome c function. PLoS ONE 12 (5), e0178280 [+]

    We investigate functional role of the P76GTKMIFA83 fragment of the primary structure of cytochrome c. Based on the data obtained by the analysis of informational structure (ANIS), we propose a model of functioning of cytochrome c. According to this model, conformational rearrangements of the P76GTKMIFA83 loop fragment have a significant effect on conformational mobility of the heme. It is suggested that the conformational mobility of cytochrome c heme is responsible for its optimal orientation with respect to electron donor and acceptor within ubiquinol-cytochrome c oxidoreductase (complex III) and cytochrome c oxidase (complex IV), respectively, thus, ensuring electron transfer from complex III to complex IV. To validate the model, we design several mutant variants of horse cytochrome c with multiple substitutions of amino acid residues in the P76GTKMIFA83 sequence that reduce its ability to undergo conformational rearrangements. With this, we study the succinate-cytochrome c reductase and cytochrome c oxidase activities of rat liver mitoplasts in the presence of mutant variants of cytochrome c. The electron transport activity of the mutant variants decreases to different extent. Resonance Raman spectroscopy (RRS) and surface-enhanced Raman spectroscopy (SERS) data demonstrate, that all mutant cytochromes possess heme with the higher degree of ruffling deformation, than that of the wild-type (WT) cytochrome c. The increase in the ruffled deformation of the heme of oxidized cytochromes correlated with the decrease in the electron transport rate of ubiquinol-cytochrome c reductase (complex III). Besides, all mutant cytochromes have lower mobility of the pyrrol rings and methine bridges, than WT cytochrome c. We show that a decrease in electron transport activity in the mutant variants correlates with conformational changes and reduced mobility of heme porphyrin. This points to a significant role of the P76GTKMIFA83 fragment in the electron transport function of cytochrome c.

    ID:1979

Preclinical studies of the cytokine TRAIL mutant variant possessing high selectivity to the “death receptor” DR5 (2016-03-29)

Gasparian M.E., Bychkov M.L., Yagolovich A.V., Dolgikh D.A., Kirpichnikov M.P. (Institute of Bioorganic Chemistry);

Nalobin D.S., Kalabushev S.N., Ahaev D.N. (Biological Faculty, MSU)

A preclinical study of the mutant variant of the antitumor cytokine TRAIL, which has a higher selectivity for the death receptor DR5, has been done. Variant DR5-B differs from the wild-type TRAIL by six substitutions of amino acid residues; it binds to the apoptosis conductive signal receptor DR5 as efficiently as the wild-type protein, but its affinity to apoptosis inhibitory decoy receptors DcR1 and DcR2 is significantly lower in comparison to TRAIL. Our experiments in cell cultures demonstrated that DR5-B kills tumor cells of different origin (either alone or in combination with chemotherapy) 2-10 times more effectively than wild-type TRAIL, which has recently passed clinical trials in the United States and has shown very limited therapeutic effect. Our results suggest that the mutant version of TRAIL (DR5-B) is not toxic and can be apply for an effective treatment of different types of cancer in which there is increased expression of DR5 receptor. (In collaboration with the Laboratory of prototyping and testing of biotechnological development of the Biological Faculty of Moscow State University).

Bibliography

M.E.Gasparian, M.L.Bychkov, A.V.Yagolovich, D.A.Dolgikh, M.P.Kirpichnikov. Mutations enhancing selectivity of antitumor cytokine TRAIL to DR5 receptor increase its cytotoxicity against tumor cells. Biochemistry (Moscow), 2015, 80: 1080–1091.

Bychkov M.L., Gasparian, M.E., Dolgikh, D.A., Kirpichnikov, M.P. Combination of TRAIL with bortezomib shifted apoptotic signaling from DR4 to DR5 death receptor by selective internalization and degradation of DR4. PLoS One, 2014, 9, e109756.

Publications

  1. Gasparian M.E., Bychkov M.L., Yagolovich A.V., Dolgikh D.A., Kirpichnikov M.P. (2015). Mutations Enhancing Selectivity of Antitumor Cytokine TRAIL to DR5 Receptor Increase Its Cytotoxicity against Tumor Cells. Biochemistry Mosc. 80 (8), 1080–91 [+]

    Tumor necrosis factor superfamily cytokine TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) induces apoptosis in tumor cells by binding to death receptors DR4 and DR5 without affecting normal cells. However, the therapeutic use of TRAIL is limited, because many tumor cells are resistant to it. The resistance is partially related to interaction of TRAIL with the decoy receptors DcR1 and DcR2, which do not trigger the apoptotic signal and inhibit signaling of death receptors. Previously, we designed a unique DR5-specific TRAIL mutant variant DR5-B, which binds to DR5 receptor as effectively as the original cytokine, but has practically no interaction with DR4 and DcR1 receptors, and its affinity for DcR2 is reduced 400-fold. In the present work, the cytotoxity of TRAIL and DR5-B was analyzed on 12 different tumor cell lines and two types of normal cells. In nine of 12 tumor cell lines, DR5-B killed 1.5-5.0 times more tumor cells than TRAIL, and it did not exhibit toxicity towards normal cells. Chemotherapeutic drugs such as doxorubicin, paclitaxel, and bortezomib augmented the effect of both TRAIL variants, and the enhancing effect was more pronounced for DR5-B. Half-maximal effective concentrations (EC50) for DR5-B in combination with chemotherapeutic agents were 1.5-10.0 times lower than for wild-type TRAIL. Thus, DR5-B is a promising candidate both for monotherapy and in combination with chemotherapy for treatment of TRAIL-resistant tumors.

    ID:1482
  2. Bychkov M.L., Gasparian M.E., Dolgikh D.A., Kirpichnikov M.P. (2014). Combination of TRAIL with bortezomib shifted apoptotic signaling from DR4 to DR5 death receptor by selective internalization and degradation of DR4. PLoS ONE 9 (10), e109756 [+]

    TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) mediates apoptosis in cancer cells through death receptors DR4 and DR5 preferring often one receptor over another in the cells expressing both receptors. Receptor selective mutant variants of TRAIL and agonistic antibodies against DR4 and DR5 are highly promising anticancer agents. Here using DR5 specific mutant variant of TRAIL - DR5-B we have demonstrated for the first time that the sensitivity of cancer cells can be shifted from one TRAIL death receptor to another during co-treatment with anticancer drugs. First we have studied the contribution of DR4 and DR5 in HCT116 p53+/+ and HCT116 p53−/− cells and demonstrated that in HCT116 p53+/+ cells the both death receptors are involved in TRAIL-induced cell death while in HCT116 p53−/− cells prevailed DR4 signaling. The expression of death (DR4 and DR5) as well as decoy (DcR1 and DcR2) receptors was upregulated in the both cell lines either by TRAIL or by bortezomib. However, combined treatment of cells with two drugs induced strong time-dependent and p53-independent internalization and further lysosomal degradation of DR4 receptor. Interestingly DR5-B variant of TRAIL which do not bind with DR4 receptor also induced elimination of DR4 from cell surface in combination with bortezomib indicating the ligand-independent mechanism of the receptor internalization. Eliminatory internalization of DR4 resulted in activation of DR5 receptor thus DR4-dependent HCT116 p53−/− cells became highly sensitive to DR5-B in time-dependent manner. Internalization and degradation of DR4 receptor depended on activation of caspases as well as of lysosomal activity as it was completely inhibited by Z-VAD-FMK, E-64 and Baf-A1. In light of our findings, it is important to explore carefully which of the death receptors is active, when sensitizing drugs are combined with agonistic antibodies to the death receptors or receptor selective variants of TRAIL to enhance cancer treatment efficiency.

    ID:1296