Гончарук Сергей Александрович

Образование

Период обученияСтрана, городУчебное заведениеДополнительная информация
1999–2005 Россия, Долгопрудный Московский Физико-Технический Институт (Государственный Университет) (МФТИ) Бакалавр. Магистр.
2002–2005 Россия, Москва Институт Биоорганической химии имени академиков М.М. Шемякина и Ю.А. Овчинникова РАН, Учебно Научный Центр.
2005–2008 Россия, Долгопрудный Московский Физико-Технический Институт (Государственный Университет) (МФТИ) Аспирант.
2008 Россия, Москва Московский государственный университет им. М.В. Ломоносова, кафедра биоинженерии биологического факультета Защита кандидатской диссертации по биологическим наукам (специальность 03.00.02 Биофизика)

Научные интересы

Молекулярная биология, биофизика, генетика, биохимия, эволюция (биологическая), эволюция вселенной.

Членство в научных обществах

FEBS

Избранные публикации

  1. 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.

    ID:1913
  2. 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.

    ID:1782
  3. 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.

    ID:1569
  4. 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.

    ID:1571
  5. 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.

    ID:1312
  6. 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.

    ID:1107
  7. Bocharov E.V., Lesovoy D.M., Goncharuk S.A., Goncharuk M.V., Hristova K., Arseniev A.S. (2013). Structure of FGFR3 Transmembrane Domain Dimer: Implications for Signaling and Human Pathologies. Structure 21 (11), 2087–93 [+]

    Fibroblast growth factor receptor 3 (FGFR3) transduces biochemical signals via lateral dimerization in the plasma membrane, and plays an important role in human development and disease. Eight different pathogenic mutations, implicated in cancers and growth disorders, have been identified in the FGFR3 transmembrane segment. Here, we describe the dimerization of the FGFR3 transmembrane domain in membrane-mimicking DPC/SDS (9/1) micelles. In the solved NMR structure, the two transmembrane helices pack into a symmetric left-handed dimer, with intermolecular stacking interactions occurring in the dimer central region. Some pathogenic mutations fall within the helix-helix interface, whereas others are located within a putative alternative interface. This implies that although the observed dimer structure is important for FGFR3 signaling, the mechanism of FGFR3-mediated transduction across the membrane is complex. We propose an FGFR3 signaling mechanism that is based on the solved structure, available structures of isolated soluble FGFR domains, and published biochemical and biophysical data.

    ID:968
  8. 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.

    ID:967
  9. Goncharuk S.A., Goncharuk M.V., Mayzel M.L., Lesovoy D.M., Chupin V.V., Bocharov E.V., Arseniev A.S., Kirpichnikov M.P. (2011). Bacterial Synthesis and Purification of Normal and Mutant Forms of Human FGFR3 Transmembrane Segment. Acta Naturae 3 (3), 77–84 [+]

    The fibroblast growth factor receptor 3 (FGFR3) is a protein belonging to the family of receptor tyrosine kinases. FGFR3 plays an important role in human skeletal development. Mutations in this protein, including Gly380Arg or Ala391Glu substitutions in the transmembrane (TM) region, can cause different disorders in bone development. The determination of the spatial structure of the FGFR3 TM domain in a normal protein and in a protein with single Gly380Arg and Ala391Glu mutations is essential in order to understand the mechanisms that control dimerization and signal transduction by receptor tyrosine kinases. The effective system of expression of eukaryotic genes in bacteria and the purification protocol for the production of milligram amounts of both normal TM fragments of FGFR3 and those with single pathogenic mutations Gly380Arg and Ala391Glu, as well as their(15)N- and [(15)N,(13)C]-isotope-labelled derivatives, were described. Each peptide was produced inEscherichia coliBL21(DE3)pLysS cells as a C-terminal extension of thioredoxin A. The purification protocol involved immobilized metal affinity chromatography and cation- and anion-exchange chromatography, as well as the fusion protein cleavage with the light subunit of human enterokinase. The efficiency of the incorporation of target peptides into DPC/SDS and DPC/DPG micelles was confirmed using NMR spectroscopy. The described methodology of production of the native FGFR3 TM domain in norma and with single Gly380Arg and Ala391Glu mutations enables one to study their spatial structure using high-resolution heteronuclear NMR spectroscopy.

    ID:966
  10. 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 [+]

    Описан эффективный способ получения мембранного белка KCNE3, а также его изотопно-меченых производных (15N-, 13C-), в количествах, достаточных для проведения структурно-функциональных исследований. Очищенный белковый препарат в составе мицелл различных детергентов охарактеризован методами динамического светорассеяния, КД-спектроскопии и ЯМР-спектроскопии. Показано, что в мицеллах DPC/LDAO белок находится в мономерной форме и принимает преимущественно альфа-спиральную конформацию. Наличие кросс-пиков от всех глицинов в 15N-HSQC–спектре ЯМР, а также относительно небольшая ширина линий (~20 Гц) подтверждают высокое качество полученного образца и возможность получения структурно-динамической информации о KCNE3 методом гетероядерной спектроскопии ЯМР высокого разрешения.

    ID:273