Laboratory of structural biology of ion channels

Our laboratory studies membrane receptors and ion channels, and also compounds acting upon them (toxins, endogenous ligands). For studies of structure and molecular mechanisms of action we apply modern methods of structural biology: NMR-spectroscopy and cryo-electron microscopy. At the moment we are studying mechanisms of interaction between toxins and voltage-gated K+ and Na+ channels (Kv and Nav), and also the structures of protein ligands of nicotinic acetylcholine receptor (nAChR). Structural studies of membrane receptors and channels are necessary for development of new methods of diagnostics and therapy of deceases of nervous, cardiovascular and muscular systems. Besides, we study structures and mechanisms of action of antibiotics and defensive peptides of plants and animals. These studies are important for development of new drugs for treatment of infectious deceases.

Fig 1. Structure of complex of human muscle Nav1.4 channel and Hm-3 toxin from Heriaeus melloteei spider venom. Structure was calculated based on NMR data. (A) Complex of Hm-3 (blue/purple) and first voltage-sensing domain (DI) of Nav1.4 channel (pale-yellow/red). Side view from lipid bilayer. (В) Toxin-channel complex. View from outside cell on the membrane surface.

Our laboratory carried out NMR studies of the structure and dynamics of voltage-sensing domains of several K+ and Na+ channels, including Nav1.4 channel from human muscles. Dysfunctions of Nav1.4 channel cause disorders of musculoskeletal system, such as paralysis, myasthenic syndrome and myotonia. For the first time, “unusual” fluctuations in the structure of domains occurring with characteristic times in the μs-ms range, possibly the prototype of the structural rearrangements occurring during voltage-dependent activation, were characterized. It is shown that the domains of Na+ channels have a greater conformational plasticity compared to the domains of K+ channels. This property probably leads to a faster response of the Nav channels when the transmembrane potential changes. We have studied VSTx1 and Hm-3 toxins from spider venom, which by binding to the membrane patch surrounding the ion channel block voltage-dependent activation. Based on the experimental data of NMR spectroscopy, together with the Laboratory of Molecular Instruments for Neurobiology IBCh RAS a model of the toxin complex Hm-3 with Nav1.4 channel of human skeletal muscles was constructed. The obtained structural data opens up the possibility of further pharmacological developments.

Currently, together with the Biological Department of Moscow State University, using electron microscopy methods, we are investigating the spatial structure of full-sized Kv7.1 channel of human cardiac muscle. Dysfunctions of this channel lead to the development of hereditary arrhythmias – a syndrome of an extended QT interval.

Together with the group of Bioengineering of Neuromodulators and Neuroreceptors of IBCh RAS, using the methods of NMR spectroscopy, we determined the structures of several “three-loop” proteins acting on the ion channel of the nicotinic acetylcholine receptor (nAChR). The spatial structures of the WTX toxin from cobra venom, Lynx1 and Lypd6 regulatory proteins from the human nervous system, and SLURP-1 and SLURP-2 proteins produced by human epithelium cells were determined. These molecules have prospects for the development of new drugs aimed at the treatment of neurodegenerative diseases.

ris2_trehpetelnye.png

Fig 2. Spatial NMR structures of three-loop proteins, acting upon nicotinic acetylcholine receptors. WTX – toxin from venom of Naja kaouthia cobra. Lynx1 and Lypd6 – regulating proteins of human nervous system.

Using the methods of NMR spectroscopy, our laboratory together with the Educational and Scientific Center of IBCh RAS examines the structure and mechanism of action of new antibiotics and defensive peptides of plants and animals. Most of the studied molecules exhibit increased affinity for the membranes of bacterial cells and are able to form pores and ion channels, which lead to the death of the target cell. We have determined the spatial structure and dynamics of the following molecules: two-component lantibiotic lichenicidin, antiamoebin antibiotic, an antimicrobial peptide arenicine from marine worm, antimicrobial peptide aurelin from jellyfish, defensin peptide from lentils, a lipid-transporting protein from lentils in complex with lipids.

ris3_arenitsin_dimer.png

Fig 3. Spatial structure and conformational plasticity of antimicrobial peptide arenicin monomer in water and dimer in membrane environment. Formation of toroidal pores in bacterial membranes by arenicin is shown on the right.

The laboratory is also developing new approaches for studying membrane biomolecules. One of the promising areas is the use of nanoparticles based on high-density lipoproteins (nanodisc). We first demonstrated the possibility of using nanodiscs as a medium for NMR studies of membrane proteins and membrane-active peptides, investigated the possibility of using nanodisks in cell-free synthesis systems to obtain functionally active forms of membrane proteins, as well as the possibility of using nanodiscs for folding membrane proteins in vitro.

Ris4_nanodiskibeskletka.png

Fig 4. The addition of preformed nanodiscs to the translational mixture of the cell-free synthesis system makes it possible to obtain functionally active membrane proteins available for studies by NMR spectroscopy.

Our laboratory is developing new approaches to establish the chemical structure and study the mechanisms of transformation into nitrogen-rich heterocyclic compounds. One of the developed approaches is based on measuring and analyzing “small” (amplitude from 1 to 0.01 Hz) 13C-15N and 1H-15N spin-spin coupling constants in compounds selectively labeled with stable 15N isotope. The application of this approach allowed us to study the processes of azido-tetrazole tautomerism in azido-triazines and azido-pyrimidines.

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Fig 5. Analysis of «small» 13С-15N spin-spin coupling constants in compounds labeled with stable 15N isotope, allows to determine the structure of terazole form in azido-triazines and azido-pyrimidines.

Our laboratory works in collaboration with subdivisions of IBCh RAS:

 

Our laboratory collaborates with Russian scientific and educational organizations:

 

Foreign collaborations:

  • Sobolevsky Lab, Department of Biochemistry and Molecular Biophysics, Columbia University (USA) (Head - Alexander Sobolevsky)

 

The group of structural biology of ion channels was founded in 2015 as a part of the program of Presidium RAS “Molecular and Cell Biology” (de facto the group existed since 2008). In 2019 the group was given laboratory status.

 

  1. Structural studies of human K+ and Na+ channels and mechanisms of action of toxins, affecting voltage-dependent activation.
  2. Studies of interconnection between structure and function of "three-loop" proteins – ligands of nicotinic acetylcholine receptor (nAChR).
  3. Studies of sctructure and mechanisms of action of new antibiotics and defensive antimicrobial peptides.
  4. Development of new membrane-mimicking media on the basis of high-density lipoproteins (nanodiscs) for production and ctructural and functional studies of membrane proteins and peptides.
  5. Development of new NMR techniques for determination of structure and mechanisms of transformation of nitrogen-rich heterocyclic compounds.

All publications (show selected)

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Zakhar Shenkarev

Combinatorial selective incorporation of stable13C and 15N isotopes facilitates NMR spectra analysis and allows mapping of the binding interfaces between membrane receptors and their ligands

In collaboration with Group of in silico analysis of membrane proteins structure,  Laboratory of bioengineering of neuromodulators and neuroreceptors,  Laboratory of Molecular Instruments for Neurobiology

Combinatorial incorporation of stable 13C and 15N isotopes into protein molecules can significantly simplify the analysis of NMR spectra. For the first time, the problem was solved and the CombLabel algorithm was developed for calculating combinatorial 13C and 15N  labeling schemes with a minimum price. The application of the program allowed to assign 50% of the NMR signals of the backbone of the second voltage-sensing domain of human sodium channel Nav1.4 (VSD-II). Leak currents through mutant variants of Nav1.4 containing Arg675Gly mutation in VSD-II lead to the development of a hereditary disease – normokalemic periodic paralysis. Hm-3 toxin from the venom of spider Heriaeus melloteei is able to block leak currents in VSD-II. By the means of NMR spectroscopy the interaction interface between VSD-II and Hm-3 toxin was determined. According to the model of the VSD-II/Hm-3 complex, based on the NMR data, the toxin binds to the extracellular S1-S2 loop, destabilizing the state of the domain, at which leak currents are observed. Using the example of the complexes of Hm-3 toxin with VSD-I and VSD-II of the Nav1.4 channel, it has been shown that arachnid toxins can interact differently with different domains within the same sodium channel.

Human Three-finger Proteins Inhibit the Growth of Carcinoma Cells

In collaboration with Laboratory of bioengineering of neuromodulators and neuroreceptors

Nicotinic acetylcholine receptors (nAChR) play an important role in the physiology of epithelial cells, and their activation contributes to the development of carcinomas. Natural modulators of nicotinic acetylcholine receptors may become promising prototypes of new antitumor agents.

Recombinant analogs of human three-finger proteins ws-Lynx1 and rSLURP-1 were shown to inhibit the growth of lung carcinoma and melanoma cells. Ws-Lynx1 in A549 cells stimulates antiproliferative and proapoptotic signaling cascades associated with activation of α7-nAChR. rSLURP-1 inhibits nicotine-induced lung carcinoma cell growth, and also abolishes nicotine-induced increase in the α7-nAChR expression and decrease in the PTEN tumor suppressor gene expression. In addition, rSLURP-1 inhibits the growth of multicellular spheroids from cells of various carcinomas. The combined use of rSLURP-1 with other antitumor drugs (gefitinib, bortezomib, doxorubicin) leads to a complete stop in the growth of spheroids.

Thus, ws-Lynx1 and rSLURP-1 are promising prototypes for the development of new drugs for the cancer treatment.

Interaction of gating modifier toxin Hm-3 with voltage-sensing domains of Nav1.4 sodium channel: structural view on the membrane-mediated binding

In collaboration with Laboratory of bioengineering of neuromodulators and neuroreceptors,  Laboratory of Molecular Instruments for Neurobiology

Voltage-gated Na+ channels (Nav) are essential for the functioning of cardiovascular, muscular, and nervous systems. Certain mutations trigger a leak current through voltage-sensing domains (VSDs) of Nav leading to various diseases. Hypokalemic periodic paralysis (HypoPP) type 2 is caused by mutations in the S4 segments of VSDs in the human skeletal muscle channel NaV1.4. The gating modifier toxin Hm-3 (crab spider Heriaeus melloteei) inhibits leak currents through such mutant channels. To investigate molecular basis of Hm-3 interaction with NaV1.4 channel, we studied isolated VSD-I by NMR spectroscopy in membrane mimicking environment. Hm-3/VSD-I complex was modeled using protein-protein docking guided by NMR restrains. The toxin initially anchors onto the membrane surface and then forms the complex with the S3b-S4 loop of the VSD-I. The Hm-3 binding blocks movement of the voltage-sensor helix S4 and induces some allosteric changes that prevent development of gating-pore currents. Our report is the first NMR study of structural interactions between gating modifier toxins and Nav channels.

Publications

  1. Männikkö R, Shenkarev ZO, Thor MG, Berkut AA, Myshkin MY, Paramonov AS, Kulbatskii DS, Kuzmin DA, Castañeda MS, King L, Wilson ER, Lyukmanova EN, Kirpichnikov MP, Schorge S, Bosmans F, Hanna MG, Kullmann DM, Vassilevski AA (2018). Spider toxin inhibits gating pore currents underlying periodic paralysis. Proc Natl Acad Sci U S A 115 (17), 4495–4500

The human secreted protein SLURP-1, which is expressed in epithelial cells and controls their proliferation and migration, has been found to inhibit the growth of epithelial cancer cells.

The effect of SLURP-1 on cancer cells is characterized by a positive feedback: exogenous (recombinant) SLURP-1 binds to α7 nicotinic acetylcholine receptors on the cell membrane and triggers a cascade of signals that activates secretion of endogenous SLURP-1 from intracellular depot, quickly increasing its concentration in the intercellular space and enhancing antiproliferative action.

Concentrations of SLURP-1, which suppress the growth of cancer cells, do not affect the growth of normal cells.

Efremenko A.V., Sharonov G.V., Feofanov A.V. (Laboratory of optical microscopy and spectroscopy of biomolecules), Lyukmanova E.N., Bychkov M.L., Shulepko M.A., Kulbatskii D.S., Dolgikh D.A., Kirpichnikov M.P. (Group of bioengineering of neuromodulators and neuroreceptors), Shenkarev Z.O. (Group of structural biology of ion channels).

The human secreted protein SLURP-1, which is expressed in epithelial cells and controls their proliferation and migration, has been found to inhibit the growth of epithelial cancer cells. The effect of SLURP-1 on cancer cells is characterized by a positive feedback: exogenous (recombinant) SLURP-1 binds to α7 nicotinic acetylcholine receptors on the cell membrane and triggers a cascade of signals that activates secretion of endogenous SLURP-1 from intracellular depot, quickly increasing its concentration in the intercellular space and enhancing antiproliferative action. Concentrations of SLURP-1, which suppress the growth of cancer cells, do not affect the growth of normal cells.

The structure of the two components of the lipopeptide antibiotic crystallomycin from a sample obtained 60 years ago has been established. The identity of the components of two crystallomycin components to these of aspartocin (the structure of which has been elucidated recently) has been found. The antibiotic exhibits Ca2+ -dependent activity against gram-positive bacteria. The conformations of crystallomycin 2 in solution were investigated using NMR.

The amino acid 4-chloro-L-kinurenin, previously found in natural products only once, was found in the peptide antibiotic INA-5812. We first described the fluorescent properties of 4-chloro-L-kinurenin and its use as an energy donor for the excitation of other fluorophores.

The structure of two new macrolide antibiotics, astolides A and B, has been established using various 2D NMR techniques. Astolide molecules contain simultaneously a membrane-active polyol macrolide and a redox-active naphthoquinone residue as aglycones. The presence of a hydroxyl group at position 18 dramatically changes the spectrum of biological activity in comparison with the known analogues – antifungal activity increases and cytotoxicity reduces.

Publications

  1. Alferova VA, Shuvalov MV, Suchkova TA, Proskurin GV, Aparin IO, Rogozhin EA, Novikov RA, Solyev PN, Chistov AA, Ustinov AV, Tyurin AP, Korshun VA (2018). 4-Chloro-l-kynurenine as fluorescent amino acid in natural peptides. Amino Acids 50 (12), 1697–1705
  2. Alferova VA, Novikov RA, Bychkova OP, Rogozhin EA, Shuvalov MV, Prokhorenko IA, Sadykova VS, Kulko AB, Dezhenkova LG, Stepashkina EA, Efremov MA, Sineva ON, Kudryakova GK, Peregudov AS, Solyev PN, Tkachev YV, Fedorova GB, Terekhova LP, Tyurin AP, Trenin AS, Korshun VA (2018). Astolides A and B, antifungal and cytotoxic naphthoquinone-derived polyol macrolactones from Streptomyces hygroscopicus. Tetrahedron 74 (52), 7442–7449
  3. Jiang ZK, Tuo L, Huang DL, Osterman IA, Tyurin AP, Liu SW, Lukyanov DA, Sergiev PV, Dontsova OA, Korshun VA, Li FN, Sun CH (2018). Diversity, novelty, and antimicrobial activity of endophytic actinobacteria from mangrove plants in Beilun Estuary National Nature Reserve of Guangxi, China. Front Microbiol 9 (MAY), 868
  4. Tyurin AP, Alferova VA, Paramonov AS, Shuvalov MV, Malanicheva IA, Grammatikova NE, Solyev PN, Liu S, Sun C, Prokhorenko IA, Efimenko TA, Terekhova LP, Efremenkova OV, Shenkarev ZO, Korshun VA (2018). Crystallomycin revisited after 60 years: Aspartocins B and C. Medchemcomm 9 (4), 667–675

Spider toxin inhibits aberrant currents in mutant ion channels

In collaboration with Laboratory of bioengineering of neuromodulators and neuroreceptors,  Laboratory of Molecular Instruments for Neurobiology

Toxin from the venom of the crab spider Heriaeus melloteei may serve as a hit in drug discovery for hypokalemic periodic paralysis type 2; there is no reliable medication for all cases of this disease. It is caused by mutations in the gene encoding voltage-gated sodium channels NaV1.4, characteristic of skeletal muscles. As a result of the mutations, these channels conduct aberrant currents, the muscles are unable to respond to the signals of the nervous system, and weakness develops followed by paralysis. Hm-3 toxin was found to be able to selectively inhibit such currents through voltage-sensing domain I of mutant channels. Read more in the press release on the IBCh website.

Publications

  1. Männikkö R, Shenkarev ZO, Thor MG, Berkut AA, Myshkin MY, Paramonov AS, Kulbatskii DS, Kuzmin DA, Castañeda MS, King L, Wilson ER, Lyukmanova EN, Kirpichnikov MP, Schorge S, Bosmans F, Hanna MG, Kullmann DM, Vassilevski AA (2018). Spider toxin inhibits gating pore currents underlying periodic paralysis. Proc Natl Acad Sci U S A 115 (17), 4495–4500

Novel antimicrobial peptides from ancient marine invertebrates

In collaboration with Science-Educational center

As a part of the study of antimicrobial peptides (AMPs) from animal species, conducting at the Science-Educational Centre of the IBCh RAS, novel host-defense cationic peptides from ancient marine invertebrates were found – nicomicin-1 and -2 from the small Arctic polychaeta Nicomache minor and polyphemusin III from the horseshoe crab Limulus polyphemus, and their structural and biological properties were studied. The peptides were expressed in the bacterial system, and their spatial structure was analyzed. Nicomicins are unique among polychaeta AMPs scaffolds, combining an amphipathic N-terminal α-helix and C-terminal extended part with a six-residue loop stabilized by a disulfide bridge. This structural arrangement resembles C-terminal Rana-box motif observed in the α-helical host-defense peptides isolated from frog skin. Nicomicin-1 exhibited strong in vitro antimicrobial activity against Gram-positive bacteria at submicromolar concentrations. The main mechanism of nicomicin-1 action is based on membrane damage but not on the inhibition of bacterial translation. The structural analysis of prepronicomicins reveals that the BRICHOS domain does not exclusively participate in biosynthesis of β-hairpin polychaeta AMPs, but could also be a part of precursor of α-helical AMPs, namely nicomicins. Polyphemusin III is β-hairpin AMP that caused fast permeabilization of the cytoplasmic membrane of human leukemia cells HL-60. Flow cytometry experiments for annexin V-FITC / propidium iodide double staining revealed that the caspase inhibitor, Z-VAD-FMK, did not abrogate disruption of the plasma membrane by polyphemusin III. Our data suggest that polyphemusin III disrupts the plasma membrane integrity and induces cell death that is apparently not related to apoptosis. In comparison to known polyphemusins and tachyplesins, polyphemusin III demonstrates a similar or lower antibacterial effect, but significantly higher cytotoxicity against human cancer and transformed cells in vitro.

Secondary structure and dynamics of the voltage-sensing domain of second pseudosubunit of human skeletal muscle sodium channel Nav1.4

In collaboration with Laboratory of biomolecular NMR-spectroscopy,  Laboratory of biomolecular modeling,  Laboratory of bioengineering of neuromodulators and neuroreceptors

Voltage-gated Na+ channels are essential for the functioning of cardiovascular, muscular, and nervous systems. The α-subunit of eukaryotic Na+ channel consists of ~2000 amino acid residues. This complexity significantly impedes structural studies of full-sized Na+ channels. The isolated voltage-sensing domain (VSD-II) of human skeletal muscle Nav1.4 channel was studied by NMR in membrane mimicking environment. Secondary structure of VSD-II showed similarity with the bacterial Na+ channels. Fragment of S4 helix between the first and second conserved Arg residues probably adopts 3/10-helical conformation. 15N-relaxation data revealed characteristic pattern of μs-ms time scale motions in the VSD-II regions sharing expected interhelical contacts. VSD-II demonstrated enhanced mobility at ps-ns time scale as compared to isolated VSDs of K+ channels.

The mechanism of lipid binding and transfer by plant lipid-transfer proteins

In collaboration with Laboratory of biomolecular NMR-spectroscopy,  Science-Educational center

The lentil lipid transfer protein, designated as Lc-LTP2, was isolated from the Lens culinaris seeds. The protein belongs to LTP1 subfamily and consists of 93 amino acid residues. Its spatial structure contains four α-helices (H1-H4) and a long C-terminal tail. Here, we report the ligand-binding properties of Lc-LTP2. The fluorescent TNS binding assay revealed that the Lc-LTP2 affinity 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 dimyristoyl-phosphatidylglycerol (DMPG) than zwitterionic dimyristoyl-phosphatidylcholine (DMPC). NMR experiments confirmed the higher affinity of Lc-LTP2 for anionic lipids and the ones with smaller volumes of hydrophobic chains. The acyl chains of the bound lyso-palmitoyl-phosphatidylglycerol (LPPG), DMPG, or dihexanoyl-phosphatidylcholine molecules occupied the internal hydrophobic cavity, while their head groups protruded into aqueous environment between H1 and H3 helices. The spatial structure and backbone dynamics of the Lc-LTP2/LPPG complex were determined. The internal cavity was expanded from ~600 to ~1000 А3 upon the protein ligation. Another entrance into the internal cavity, restricted by the H2-H3 interhelical loop and C-terminal tail, appeared to be responsible for the Lc-LTP2 attachment to the membrane or micelle surface and probably played an important role in the lipid uptake determining the ligand specificity. Our results confirmed previous assumption regarding the membrane-mediated antimicrobial action of Lc-LTP2 and afforded molecular insight into its biological role in the plant.