Denmark-Japan Joint Mini Workshop on Ion Transport Proteins

Transcription

1 Denmark-Japan Joint Mini Workshop on Ion Transport Proteins Confirmed Speakers: Nobuyuki Uozumi (Tohoku Univerity) Kazuhiro Abe (Nagoya University) Ryoung Shin (RIKEN) Himanshu Khandelia (University of Southern Denmark) Date & Venue: September 2, 2015, 13:30-17:30 Room Number C218/220 (Central building 2F) RIKEN, Suehiro-cho, Tsurumi-ku, Yokohama City, Kanagawa, , Japan No registration fee Contact: Organized by: Funding: Himanshu Khandelia, Ryoung Shin, University of Southern Denmark RIKEN, Japan Ministry of Higher Education and Science, Denmark

2 Workshop schedule Time Speaker Title 1:30-1:40 Opening 1:40-2:15 Prof. Nobuyuki Uozumi Comparative analysis of K transport system 2:15-2:50 Dr. Himanshu Khandelia Molecular determinants of external K + selectivity in the Na + /K + ATPase 2:50-3:05 Dr. Minwoo Han Role of protons in transmembrane ion transport of the Na + /K + ATPase: multiscale computational analysis 3:05-3:20 Dr. Eri Adams CsTolen A, a chemical capable of binding cesium, inhibits its entry and enhances tolerance in plants 3:20-3:40 Coffee break 3:40-4:15 Dr. Kazuhiro Abe Structural physiology of gastric proton pump 4:15-4:30 Dr. Wojciech Kopec Interactions of K + congeners with the E1P conformation of the Na + /K + ATPase 4:30-5:05 Dr. Ryoung Shin Molecular regulation of an Arabidopsis potassium high affinity transporter, AtHAK5 5:05-5:20 Dr. Jinsoo Yi Self-assembled cell system with in vitro expressed single membrane protein 5:20-5:30 closing

3 Comparative analysis of K transport system Nobuyuki UOZUMI Tohoku University, Graduate School of Engineering, Department of Biomolecular Engineering K is the most abundant cations in all living cells. Animal cells use the Na motive force to energize other transport processes. Na ions accumulating in the cytosol are transported to the extracellular space by the Na/K exchanger, which leads to supply K to maintain the high content of K in the cytosol. In contrast, Na/K exchanger is missing in bacteria and plant cells. They use the H motive force to take up essential molecules and the cells have other special K uptake systems in their membrane; four classes of K uptake transport systems have been identified in the organisms. (References) Sato, et al. J. Biochem. 155, (2014) Hamamoto, et al. Curr. Opin. Biotechnol. 32, (2015) Nanatani, et al. J.Bacteriol. 197, (2015) Molecular determinants of external K + selectivity in the Na + /K + ATPase Himanshu Khandelia The Na + /K + ATPase is an essential membrane protein that exports 3 Na+ and imports 2 K +, both against their concentration gradients, by burning one molecule of ATP. When importing K +, the protein is exposed to a 30-fold higher concentration of Na +, and yet binds and transports only K +. I will talk about the molecular motifs that impart to the protein its remarkable ion selectivity, discovered using molecular simulations.

4 Role of protons in transmembrane ion transport of the Na + /K + ATPase: multiscale computational analysis Minwoo Han Na + /K + ATPase (NKA) is the one of the most significant ion-translocating ATPase which exchanges three Na ions and two K ions through membrane. In recently, it have been revealed that not only the pump exchanges the ions, but also it develops a selective pathway for protons to cross the cell membrane during every cycle. The function as a proton channel gave us great scientific impact since the protons are the key substrate for several cellular proteins in many biological systems. In order to catch the multiscale phenomena of the protons in the pump, we performed molecular dynamics (MD) and quantum mechanical (QM) calculation for complementation. By using the MD, we could obtain reliable Na + binding structures of different protonation states of the NKA and construct model system of the three Na + binding sites for QM calculation. Since the Na + binding sites contain six essential charged residues : Glu and Asp, the QM calculations were thoroughly performed for each different protonation schemes to determine the Na + binding energy of each site and charge distributions around the ion binding site were closely analyzed. The result gave us the three clues of role of proton in the NKA ion binding : (1) As increasing the number of protonated residue, Na + binding energy of each site is decreasing (2) The binding energy of each site is closely related to each other when one of residue is protonated. (3) The correlations among three Na + binding energies governed by the distance between each Na + binding site and number of oxygen atoms around Na +. CsTolen A, a chemical capable of binding cesium, inhibits its entry and enhances tolerance in plants Eri Adams RIKEN Center for Sustainable Resource Science, Regulatory Network Research Unit The accident at the Fukushima nuclear power plant in Japan following the great earthquake in 2011 caused the spread of radiocesium over the surrounding areas including farmland. Therefore, the techniques to remediate the land and ensure security of agricultural products using plants came to be the focus of attention. In order to select small compounds which either enhance plant tolerance to cesium or increase cesium

5 uptake ability in plants, chemical library screening was performed. Of 20,000 chemicals tested, 34 chemicals were found to alter response to or accumulation of cesium in plants. One of these chemicals, named CsTolen A, was confirmed to enhance cesium tolerance in plants through reduction of cesium accumulation. Physiological experiments together with theoretical modelling have revealed that CsTolen A specifically binds to cesium and inhibits it from going into plant cells. Application of CsTolen A to soil-grown plants also reduced cesium accumulation and improved plant performance. Structural physiology of gastric proton pump Kazuhiro ABE Cellular and Structural Physiology Institute (CeSPI), Department of Medicinal Science, Graduate School of Pharmaceutical Science, Nagoya University, CREST, JST We are occasionally reminded of the gastric proton pump acidifying the gastric juice as an unpleasant symptom of heartburn. In a worse case, the painful conditions of dyspepsia, such as gastric ulcer or gastroesophageal-reflux disease, cause a significantly reduced health of those affected. Fortunately, proton pump inhibitors targeted the gastric H +,K + -ATPase provide for very efficient cures, making this proton pump one of the most prominent drug targets of all in terms of annual sales. Besides its significant interest as a drug target, gastric H +,K + -ATPase faces a remarkable task of pumping protons against a million-fold gradient ranging from approximately ph 7 in the parietal cell to 1 in the stomach. Maintaining a potent concentration gradient of six orders of magnitude is hardly met by any other membrane pump in nature. A key requirement is to ensure a tight membrane that keeps the gradient intact, and as a consequence the gastric proton pump should go one way pumping proton out of the cell, and certainly not the other way. So how does the proton pump generate, and manage to resist such a steep H + gradient and never go in reverse? By determining medium-resolution structures of gastric H +,K + -ATPase employing electroncrystallography of two-dimensional crystals, we have found unique structural features relevant to its physiological functions. Taking advantage of conventional biochemical analyses based on the determined structures, our proposed transport model describes an example of how the energetic challenges that membrane pumps are confronted with are met in living system.

6 Interactions of K + congeners with the E1P conformation of the Na + /K + ATPase Wojciech Kopec, The sodium-potassium pump (Na + /K + -ATPase) is an essential transmembrane protein of the P-type pump family and serves as a primary active ion transporter in all eukaryotic cells. Na + /K + -ATPase exchanges three cytoplasmic Na + ions for two extracellular K + ions, thereby maintaining proper ion gradients across the cell plasma membrane. Two organic cations - acetamidinium (Acet + ) and formamidinium (Form + ) - have been shown to replace extracellular K + ions in the Na + /K + -ATPase reaction cycle and permeate through the pump to the cytoplasm. Here, we show that these cations are unable to replace cytoplasmic Na + ions. Using all-atom Molecular Dynamics (MD) simulations of the recently crystallised Na + /K + -ATPase in the sodium-bound E1P state, we first propose the protonation scheme of the acidic ion binding residues. Subsequently, we replace bound Na + with Acet + and Form +, showing that i) they differently distort ion binding sites ii) the hydroxymethyl group of T772 rotates to stabilise bound Form + together with water molecules and iii) this rotamer transition is mediated by water entering the binding sites. These results suggest a catalytic role of water in Na + /K + -ATPase function and indicate a backbone-independent but a side chain-dependent ion selectivity mechanism. Molecular regulation of an Arabidopsis potassium high affinity transporter, AtHAK5 Ryoung Shin RIKEN Center for Sustainable Resource Science Potassium (K + ) is an essential plant macronutrient which exerts various functions on all over the plant throughout its life. The process of sensing K + availability in soil and transmitting signals through the downstream cascades are essential for plant survival. It has been known high affinity K + transporters are main players for transporting K + from soil to plants when K + is limited. In this talk, K + deficient signaling will be summarized focusing on the regulation of an Arabidopsis high affinity K + transporter, AtHAK5. To elucidate regulatory components of high affinity K + transport, genetic, K + molecular and biochemical approaches along with high-throughput omics-type approaches are being used. Results

7 from multiple studies show that in Arabidopsis thaliana, phytohormones and ROS are important contributing factors for activating K + uptake and increasing tolerance to low potassium and for altering root hair and primary root growth. In addition, for identifying the direct regulators of AtHAK5, FOX (Full length cdna OverExpressor)/TF (Transcription Factor) FOX transformed into AtHAK5 promoter::luciferase plants and the candidates which activated AtHAK5 expressions were selected analyzed. Altogether, AtHAK5 dependent low K + signal pathway will be summarized. Self-assembled cell system with in vitro expressed single membrane protein Jinsoo Yi Transmembrane proteins including ion transporters are usually huge macromolecules and very hydrophobic. Therefore, those proteins are difficult to purify and reconstitute in the proteoliposomes. To investigate the exact functionality of the proteins, we have suggested a self-assembled artificial cell system, encapsulating in vitro protein expression solutions in micro-giant vesicles employing wateroil emulsion method. The final goal is reconstruction of a single membrane protein incorporated proteolipsome to investigate the ion transportation through the single transmembrane protein.