Group of structural biology of ion channels

Our group 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 group 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 group 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 group 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 group 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.

ris5_himiya15N.png

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 group works in collaboration with subdivisions of IBCh RAS:

 

Our group collaborates with Russian scientific and educational organizations:

Foreign collaborations:

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

 

Our group was founded in 2015 as a part of the program of Presidium RAS “Molecular and Cell Biology” (de facto the group exists since 2008).

In the picture from left to right: Mikhail Yu. Myshkin, Andrey V. Tsarev, Zakhar O. Shenkarev, Alexander S. Paramonov.

  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.

Selected publications (show all)

Loading...

Zakhar Shenkarev

Interaction of gating modifier toxin Hm-3 with voltage-sensing domains of Nav1.4 sodium channel: structural view on the membrane-mediated binding (2018-12-03)

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

Secondary structure and dynamics of the voltage-sensing domain of second pseudosubunit of human skeletal muscle sodium channel Nav1.4 (2017-11-28)

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.

отчёт 2016

В мембраномоделирующих средах на основе мицелл детергентов методами ЯМР-спектроскопии проведено исследование потенциалочувствительного домена (ПЧД) канала Kv2.1 человека. Разработана новая методика отнесения сигналов ЯМР основной цепи белка, основанная на методе комбинаторного введения изотопов в бесклеточной системе синтеза (система сопряженной транскрипции-трансляции in vitro). Разработан новый программный комплекс позволяющий рассчитывать оптимальную схему введения изотопов. С использованием нового метода охарактеризована вторичная структура и внутримолекулярная динамика ПЧД-Kv2.1. Разработан протокол ренатурации рекомбинантного аналога токсина паука, действующего на ПЧД-Kv2.1. Разработанные протоколы делают возможными структурные исследования лиганд-рецепторных взаимодействий токсин-домен. Кроме того за отчетный период проведены структурные исследования ряда токсинов, защитных пептидов и токсин-подобных белков человека, действующих на ионные каналы и мембраны клеток.