- Email: [email protected]
Direct Visualization of Glutamate Transporter Transport Cycle
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Monday, February 29, 2016
HBV capsid assembly can inhibit the virus growth. The HBV capsid proteins assemble via tetramer intermediates, with two dimer subunits and a pocket between them. Several compounds can bind to this pocket and alter orientation of the two subunits, which in turn inhibits virus growth by either accelerating the capsid assembly, or causing the proteins to assemble into non-capsid linear polymers. However, a greater therapeutic potential could be achieved by rationally designing such compounds. Here, we propose HBV capsid inhibitors from combined molecular dynamics and docking methodologies. We targeted the limiting step in the capsid formation of HBV, which is formation of a hexamer from a tetramer. Microsecondlong tetramer simulations showed slow motions of the two dimer subunits and large fluctuations in the size of the pocket between them. Based on principal component analysis of the tetramer motion, we selected a structure that was unfavorable for trimer formation and had a larger pocket volume compared to the assembled structure. In order to find potential inhibitors we docked over 100,000 compounds to the pocket in the selected structure. The top candidate compounds were selected based on docking score and surface area. The drug-protein complexes were further evaluated with molecular dynamics simulations. 890-Plat Holliday Junction Thermodynamics and Structure: Comparisons of Coarse-Grained Simulations and Experiments Francis W. Starr1, Wujie Wang1, Laura M. Nocka2, Brianne Z. Wiemann2, Daniel M. Hinckley3, Ishita Mukerji2. 1 Physics, Wesleyan University, Middletown, CT, USA, 2Molecular Biology & Biochemistry, Wesleyan University, Middletown, CT, USA, 3Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI, USA. The DNA Holliday junction plays a vital role in genetic recombination and DNA repair. All-atom simulations of DNA junctions have proved challenging, owing to the substantial size and time scales required to examine conformational changes or junction melting. Here, we combine simulations and experiments to evaluate the possibility of using the 3SPN.2 model, a coarse-grained molecular representation designed to mimic B-DNA, to reproduce the properties of a DNA four-way Holliday junction. We find that the model reproduces our experimental measurements of the junction melting temperature dependence on salt concentration to within 3%. The model reproduces the bias between open and stacked conformations, as well as the relative population of stacked isomers at high salt concentration. We further predict the population of the iso-I and iso-II forms is nearly independent of salt concentration. We directly observe proposed tetrahedral intermediate sub-states implicated in conformational transitions, suggesting that these conformations, rather than planar open states, facilitate these transitions. Finally, we compare our results for inter-duplex angle (IDA) with previous fluorescence experiments and allatom calculations based on the AMBER force field. We find a close correspondence between the average IDA of our all-atom and coarse-grained simulations, but find that the ‘‘arms’’ of the junction in the 3SPN.2 model exhibit larger fluctuations, apparently due to the implicit nature of solvent and ion interactions. Our findings demonstrate that the 3SPN.2 model captures junction properties that are not presently accessible to all-atom studies, opening the possibilities to simulate complex aspects of junction behavior.
Platform: Membrane Pumps, Transporters, and Exchangers 891-Plat An Outward-Facing Open Conformational State in a CLC Transporter Sherwin J. Abraham1, Tanmay Chavan1, Ricky C. Cheng1, Cristina Fenollar-Ferrer2, Wei Han3, Shahidul M. Islam4, Tao Jiang3, Chandra M. Khantwal1, Irimpan I. Mathews5, Richard A. Stein6, Benoit Roux4, Lucy R. Forrest2, Hassane S. Mchaourab6, Emad Tajkhorshid3, Merritt Maduke1. 1 Department of Molecular & Cellular Physiology, Stanford University, Stanford, CA, USA, 2Computational Structural Biology Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA, 3Department of Biochemistry, Center for Biophysics and Computational Biology, and Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, IL, USA, 4Department of Biochemistry, University of Chicago, Chicago, IL, USA, 5Stanford Synchrotron Radiation Lightsource, Stanford University, Stanford, CA, USA, 6Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Vanderbilt University, Nashville, TN, USA. As secondary active transporters, CLCs harness energy stored in one ion concentration gradient (Cl- or Hþ) to pump the other ion against its electrochemical
gradient. This occurs through tight coupling of protein conformational changes to ion binding, unbinding, and translocation events. Crystal structures suggest that the conformational change from occluded to outward-facing states is unusually simple, involving only the rotation of a conserved glutamate (Gluex) upon its protonation. Here, we combine spectroscopy, cross-linking studies, crystallography, and computation to evaluate this simple model. Using 19F NMR and double electron-electron resonance (DEER) spectroscopy, we show that as [Hþ] is increased to enrich the outward-facing state, residues distant from Gluex move. Consistent with the functional relevance of this motion, a cross-link designed to inhibit the motions reduces transport. Molecular dynamics simulations indicate that the cross-link dampens extracellular gateopening motions. Together, the results indicate the existence of a previously uncharacterized ‘‘outward-facing open’’ conformational state. A computational model based on the asymmetry exchange hypothesis for transporters with inverted-topology repeats (Forrest et al. 2008, PNAS) predicts such a state. To test the computational model, we monitored Hþ-induced structural changes using DEER spectroscopy on >20 sets of doubly labeled ClC-ec1 protein samples. Experimental distance distributions are compared with those derived from the ClC-ec1 X-ray structure and the model using restrained-ensemble molecular dynamics simulations (Roux et al. 2013, J Phys Chem B). Preliminary results show Hþ-dependent structural changes consistent with the computational model that can also be used to refine it. These results highlight the relevance of global structural changes in CLC function and provide the necessary foundation for understanding the Cl-/Hþ coupling mechanism. 892-Plat Transport Mechanism of the EIIC Glucose Superfamily of Transporters Zhenning Ren1, Jason G. McCoy1, Vitali Stanevich1, Jumin Lee2, Sharmistha Mitra1, Elena J. Levin1, Sebastien Poget3, Matthias Quick4, Wonpil Im2, Ming Zhou1. 1 Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA, 2Molecular Biosciences and Center for Computational Biology, University of Kansas, Lawrence, KS, USA, 3Chemistry, College of Staten Island, Staten Island, NY, USA, 4Psychiatry and Center for Molecular Recognition, Columbia University, New York, NY, USA. Carbohydrate transporters of the phosphoenolpyruvate:carbohydrate phosphotransferase system are crucial for sugar uptake in bacteria; however, little is known concerning how these proteins recognize and transport carbohydrates ˚ structure and functional across the cell membrane. Here we report the 2.55 A characterization of a maltose transporter, bcMalT. The bcMalT structure is in an outward-facing conformation, in contrast with the previous structure of an N-diacetylchitobiose transporter in an inward-facing conformation. These structures identified a mobile transport domain that may undergo a roughly ˚ rigid-body movement to provide alternating access to the bound substrate 20 A from either side of the membrane. Crosslinking of pairs of cysteine residues that are distant in the crystal structures but are predicted to move close to each other in the alternate conformation provides further support for the large-scale movement of the transport domain. Potential pathways for substrate release from the inward- and outward-facing conformations are also described, including the movement of a conserved cytoplasmic loop that may control substrate access from the cytoplasmic side. These results provide a mechanistic framework for understanding substrate recognition and translocation. 893-Plat Direct Visualization of Glutamate Transporter Transport Cycle Yi Ruan1, Atsushi Miyagi1, Xiaoyu Wang2, Mohamed Chami3, Henning Stahlberg3, Olga Boudker2, Simon Scheuring1. 1 U1006, INSERM / Aix-Marseille Universite´, Marseille, France, 2 Department of Physiology and Biophysics, Weill Cornell Medical College, New York, NY, USA, 3C-CINA, Biozentrum, University of Basel, Basel, Switzerland. Glutamate transporters are essential for recovering the neurotransmitter glutamate from the synaptic cleft after a synaptic stimulation. Crystal structures in the outward-facing (1) and inward-facing (2) conformations of a prokaryotic homolog of glutamate transporters revealed the molecular basis of the transporter cycle. So far, however, dynamics studies of the transport mechanism are sparse and indirect, based on fluorescent microscopy with its limitations (3). Here we present first high speed atom force microscopy (HS-AFM(4,5)) observations of membrane reconstituted glutamate transporters at work. The HS-AFM movies provide an unprecedented real-space and real-time visualization of the transport dynamics and permit quantitative comparison to the transport rates. Our results show transport mediated by large amplitude (~2nm) ‘‘elevator’’ movements of the transport domains. Furthermore, individual monomers within the trimeric complex possess full independence to realize substrate transport.
Monday, February 29, 2016 References: 1) Yernool, D., Boudker, O., Jin, Y. & Gouaux, E. Structure of a glutamate transporter homologue from pyrococcus horikoshii. Nature 431, 811-818, (2004). 2) Reyes, N., Ginter, C. & Boudker, O. Transport mechanism of a bacterial homologue of glutamate transporters. Nature 462, 880-885, (2009). 3) Akyuz, N., Altman, R. B., Blanchard, S. C. & Boudker, O. Transport dynamics in a glutamate transporter homologue. Nature 502, 114-118, (2013). 4) Ando, T., Kodera, N., Takai, E., Maruyama, D., Saito, K. & Toda, A. A highspeed atomic force microscope for studying biological macromolecules. Proceedings of the National Academy of Sciences 98, 12468-12472, (2001). 5) Ando, T., Uchihashi, T. & Scheuring, S. Filming biomolecular processes by high-speed atomic force microscopy. Chem Rev 114, 3120-3188, (2014). 894-Plat Resolving Active Ion Transport at the Single Molecule Level for the First Time Salome Veshaguri1, Sune M. Christensen1, Gerdi C. Kemmer2, Mads P. Møller1, Garima Ghale1, Christina Lohr1, Andreas L. Christensen1, Bo H. Justesen2, Ida L. Jørgensen2, Ju¨rgen Schiller3, Nikos S. Hatzakis1, Michael Grabe4, Thomas Gu¨nther Pomorski2, Dimitrios Stamou1. 1 Department of Chemistry, University of Copenhagen, Copenhagen, Denmark, 2Department of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, Denmark, 3Institute of Medical Physics and Biophysics, Leipzig, Germany, 4Department of Pharmaceutical Chemistry, Cardiovascular Research Institute, San Francisco, CA, USA. Electrochemical gradients across cellular membranes control a plethora of vital biological processes. These gradients are generated by primary active transporters and are subsequently used to drive the exchange of other solutes through secondary active transporters and to facilitate signaling via ion channels. The macroscopic biological phenomena that channels and transporters give rise to are intimately connected to how they function at the single molecule level. For decades, patch clamp recording has been used to observe the functional dynamics of single ion channels revealing discrete on and off states, subconductance states, and other mechanistically important features that macroscopic experiments cannot probe(1). Currently, there aren’t any techniques available to investigate transporter function at the single molecule level, thus they are only studied using ensemble biochemical methods. For a decade we have been developing quantitative fluorescence microscopy based assays of arrayed proteoliposomes for investigating membrane proteins(2-6). Here we extended the platform to monitor the single-molecule activity and the regulation of a prototypic P-type ATPase, Arabidopsis thaliana Hþ-ATPase (AHA2). For the first time we have shown that individual proton pumps are not active continuously but rather transitioning between active and inactive states separated by a large activation barrier (kJMole1). We found that the dynamics of these states form the basis of the regulation of the macroscopic activity either by regulatory R-domain, pH gradients, or ATP. Like for ion channels we often found that regulatory inputs do not affect the intrinsic pumping rates but rather active probabilities. References: 1. Neher & Sajmann, Nature, (1976) 2. Mathiasen et al., Nature Methods, (2014) 3. Larsen et al., Nature Chemical Biology, (2015) 4. Christensen et al., Nature Nanotechnology, (2012) 5. Hatzakis et al., Nature Chemical Biology, (2009) 6. Bendix et al., PNAS, (2009) 895-Plat Protein Interactions that Enable Safe and Efficient Copper Ion Transport in the Human Cytoplasm Pernilla Wittung-Stafshede. Biology and Bioengineering, Chalmers University of Technology, Gothenburg, Sweden. Although copper (Cu) is an essential metal for most living organisms, high levels and free such ions are toxic. In humans, after cellular uptake via the membrane-bound importer Ctr1, Cu is transported to targets by cytoplasmic Cu chaperones: Atox1 delivers Cu to membrane-bound P1B-type ATPases (i.e., ATP7B or Wilson disease protein) in the Golgi (secretory path; here, most Cu-dependent enzymes are loaded with Cu) whereas CCS delivers Cu specifically to cytoplasmic superoxide dismutase. In contrast to bacterial and yeast homologs, ATP7B has six similar metal-binding domains protruding into the cytoplasm: possibly, conformational changes among these regulate overall ATP7B activity. To reveal underlying molecular mechanisms as well as thermodynamic and kinetic driving forces for human Cu transport - from the cell membrane to the Golgi - our strategy involves a range of complementary biophysical experiments on purified proteins, domain constructs and engi-
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neered variants. From our studies, we have discovered that (a) the cytoplasmic C-terminus of Ctr1 binds Cu through its HCH motif with a moderate affinity that allows for Cu delivery to Atox1, (b) Atox1 can interact with CCS and exchange Cu implying cross-reactivity between cytoplasmic chaperones, (c) transfer of Cu from Atox1 to metal-binding domains in ATP7B proceeds through Cu-bridged hetero-protein dimers displaying enthalpy-entropy compensation, (d) conformational changes and domain-domain interactions within ATP7B depend on Cu loading status and minute changes in solvent conditions, and (e), in addition to its cytoplasmic chaperone activity, Atox1 may have functionality in the nucleus as it interacted with several DNA-binding proteins in a yeast two-hybrid screen. 896-Plat Dissecting the Catalytic Cycle of the Serotonin Transporter Peter S. Hasenhuetl, Michael Freissmuth, Harald H. Sitte, Klaus Schicker, Yang Li, Walter Sandtner. Department of Pharmacology, Medical Univeristy of Vienna, Vienna, Austria. The plasmalemmal serotonin transporter (SERT) regulates serotonin homeostasis and signaling by its reuptake from the extracellular space. In accordance with this prominent role, it is a target of several psychoactive substances, ranging from inhibitors (e.g. cocaine) to substrates (e.g. amphetamines). SERT belongs to the solute carrier 6 family. Hence, it utilizes the electrochemical gradient of co-substrates for the uphill transport of serotonin into the cell. Interestingly, the stoichiometry of this secondary-active transport mechanism has been matter of debate - despite decades of investigation. In addition, the order and kinetics of (co-)substrate binding to SERT have remained enigmatic. Here, we utilized the high temporal resolution of the whole-cell patch-clamp technique to decipher the kinetic determinants of selective ligand recognition, and of (co-)substrate binding and transport - in both, the forward transport and substrate exchange mode of SERT. Based on our electrophysiological data, we provide a comprehensive kinetic model of SERT that accounts for kinetics, stoichiometry, and order of (co-)substrate binding and translocation. We find that, Cl- does not participate in coupling of serotonin transport, but is required for substrate binding. In addition, our data suggest that two Naþ ions bind to SERT in a sequential order (Naþ-serotonin-Naþ). These findings are incompatible with an electroneutral stoichiometry. Our data shall provide a mechanistic framework for future attempts to integrate functional data with available structural information. 897-Plat Translocase Activity and Asymmetric Model Membranes Probed by Neutron Scattering Allison M. Whited, Frederick A. Heberle, Robert F. Standaert, Jonathan David Nickels, Xiaolin Cheng, John Katsaras, Alexander Johs. Oak Ridge National Lab, Oak Ridge, TN, USA. Biological membranes are almost universally asymmetric with individual leaflets containing different lipid species at varying concentrations. This asymmetry may play a key role in biological functions such as protein-membrane interactions, membrane trafficking, and cellular signaling. Traditional model membrane systems are comprised of symmetric membrane leaflets and though progress has been made in developing asymmetric model membranes, these systems are unstable and easily contaminated. We have developed an actively asymmetric biomembrane model system by purifying and reconstituting the well-characterized E. coli phospholipid ABC transporter MsbA into proteoliposomes that mimic the phospholipid composition of a physiological bacterial membrane. Subsequent MsbA-mediated phospholipid translocation and resultant membrane asymmetry was studied using small-angle neutron scattering (SANS). SANS is exceptionally well suited for studying the phospholipid distribution perpendicular to the bilayer plane due to its nondestructive nature and sub-nanometer resolution. Additionally, the hydrogen/deuterium (H/D) neutron scattering contrast variation from isotopic phospholipid labeling enables us to track the translocation of lipid membrane components without introducing bulky fluorescence or spin labels. In this study, the MsbA-mediated translocation of the phospholipid phosphatidylethamolamine (PE) between proteoliposome leaflets and the formation of asymmetric bilayers was studied using SANS for the first time. The sustained asymmetry in these model protocells will be central for future studies on biomimetic membrane systems and their role in diverse membrane-directed biological processes. 898-Plat Functional Characterization of Calcium-activated Phospholipid Scramblase Activity of nhTMEM16 Tao Jiang1,2, Sundar Thangapandian1,2, Emad Tajkhorshid1,2. 1 Department of Biochemistry, Center for Biophysics and Computational Biology, University of Illinois at Urbana Champaign, Urbana, IL, USA, 2 Beckman Institute for Advanced Science and Technology, Urbana, IL, USA.
Monday, February 29, 2016
HBV capsid assembly can inhibit the virus growth. The HBV capsid proteins assemble via tetramer intermediates, with two dimer subunits and a pocket between them. Several compounds can bind to this pocket and alter orientation of the two subunits, which in turn inhibits virus growth by either accelerating the capsid assembly, or causing the proteins to assemble into non-capsid linear polymers. However, a greater therapeutic potential could be achieved by rationally designing such compounds. Here, we propose HBV capsid inhibitors from combined molecular dynamics and docking methodologies. We targeted the limiting step in the capsid formation of HBV, which is formation of a hexamer from a tetramer. Microsecondlong tetramer simulations showed slow motions of the two dimer subunits and large fluctuations in the size of the pocket between them. Based on principal component analysis of the tetramer motion, we selected a structure that was unfavorable for trimer formation and had a larger pocket volume compared to the assembled structure. In order to find potential inhibitors we docked over 100,000 compounds to the pocket in the selected structure. The top candidate compounds were selected based on docking score and surface area. The drug-protein complexes were further evaluated with molecular dynamics simulations. 890-Plat Holliday Junction Thermodynamics and Structure: Comparisons of Coarse-Grained Simulations and Experiments Francis W. Starr1, Wujie Wang1, Laura M. Nocka2, Brianne Z. Wiemann2, Daniel M. Hinckley3, Ishita Mukerji2. 1 Physics, Wesleyan University, Middletown, CT, USA, 2Molecular Biology & Biochemistry, Wesleyan University, Middletown, CT, USA, 3Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI, USA. The DNA Holliday junction plays a vital role in genetic recombination and DNA repair. All-atom simulations of DNA junctions have proved challenging, owing to the substantial size and time scales required to examine conformational changes or junction melting. Here, we combine simulations and experiments to evaluate the possibility of using the 3SPN.2 model, a coarse-grained molecular representation designed to mimic B-DNA, to reproduce the properties of a DNA four-way Holliday junction. We find that the model reproduces our experimental measurements of the junction melting temperature dependence on salt concentration to within 3%. The model reproduces the bias between open and stacked conformations, as well as the relative population of stacked isomers at high salt concentration. We further predict the population of the iso-I and iso-II forms is nearly independent of salt concentration. We directly observe proposed tetrahedral intermediate sub-states implicated in conformational transitions, suggesting that these conformations, rather than planar open states, facilitate these transitions. Finally, we compare our results for inter-duplex angle (IDA) with previous fluorescence experiments and allatom calculations based on the AMBER force field. We find a close correspondence between the average IDA of our all-atom and coarse-grained simulations, but find that the ‘‘arms’’ of the junction in the 3SPN.2 model exhibit larger fluctuations, apparently due to the implicit nature of solvent and ion interactions. Our findings demonstrate that the 3SPN.2 model captures junction properties that are not presently accessible to all-atom studies, opening the possibilities to simulate complex aspects of junction behavior.
Platform: Membrane Pumps, Transporters, and Exchangers 891-Plat An Outward-Facing Open Conformational State in a CLC Transporter Sherwin J. Abraham1, Tanmay Chavan1, Ricky C. Cheng1, Cristina Fenollar-Ferrer2, Wei Han3, Shahidul M. Islam4, Tao Jiang3, Chandra M. Khantwal1, Irimpan I. Mathews5, Richard A. Stein6, Benoit Roux4, Lucy R. Forrest2, Hassane S. Mchaourab6, Emad Tajkhorshid3, Merritt Maduke1. 1 Department of Molecular & Cellular Physiology, Stanford University, Stanford, CA, USA, 2Computational Structural Biology Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA, 3Department of Biochemistry, Center for Biophysics and Computational Biology, and Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, IL, USA, 4Department of Biochemistry, University of Chicago, Chicago, IL, USA, 5Stanford Synchrotron Radiation Lightsource, Stanford University, Stanford, CA, USA, 6Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Vanderbilt University, Nashville, TN, USA. As secondary active transporters, CLCs harness energy stored in one ion concentration gradient (Cl- or Hþ) to pump the other ion against its electrochemical
gradient. This occurs through tight coupling of protein conformational changes to ion binding, unbinding, and translocation events. Crystal structures suggest that the conformational change from occluded to outward-facing states is unusually simple, involving only the rotation of a conserved glutamate (Gluex) upon its protonation. Here, we combine spectroscopy, cross-linking studies, crystallography, and computation to evaluate this simple model. Using 19F NMR and double electron-electron resonance (DEER) spectroscopy, we show that as [Hþ] is increased to enrich the outward-facing state, residues distant from Gluex move. Consistent with the functional relevance of this motion, a cross-link designed to inhibit the motions reduces transport. Molecular dynamics simulations indicate that the cross-link dampens extracellular gateopening motions. Together, the results indicate the existence of a previously uncharacterized ‘‘outward-facing open’’ conformational state. A computational model based on the asymmetry exchange hypothesis for transporters with inverted-topology repeats (Forrest et al. 2008, PNAS) predicts such a state. To test the computational model, we monitored Hþ-induced structural changes using DEER spectroscopy on >20 sets of doubly labeled ClC-ec1 protein samples. Experimental distance distributions are compared with those derived from the ClC-ec1 X-ray structure and the model using restrained-ensemble molecular dynamics simulations (Roux et al. 2013, J Phys Chem B). Preliminary results show Hþ-dependent structural changes consistent with the computational model that can also be used to refine it. These results highlight the relevance of global structural changes in CLC function and provide the necessary foundation for understanding the Cl-/Hþ coupling mechanism. 892-Plat Transport Mechanism of the EIIC Glucose Superfamily of Transporters Zhenning Ren1, Jason G. McCoy1, Vitali Stanevich1, Jumin Lee2, Sharmistha Mitra1, Elena J. Levin1, Sebastien Poget3, Matthias Quick4, Wonpil Im2, Ming Zhou1. 1 Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA, 2Molecular Biosciences and Center for Computational Biology, University of Kansas, Lawrence, KS, USA, 3Chemistry, College of Staten Island, Staten Island, NY, USA, 4Psychiatry and Center for Molecular Recognition, Columbia University, New York, NY, USA. Carbohydrate transporters of the phosphoenolpyruvate:carbohydrate phosphotransferase system are crucial for sugar uptake in bacteria; however, little is known concerning how these proteins recognize and transport carbohydrates ˚ structure and functional across the cell membrane. Here we report the 2.55 A characterization of a maltose transporter, bcMalT. The bcMalT structure is in an outward-facing conformation, in contrast with the previous structure of an N-diacetylchitobiose transporter in an inward-facing conformation. These structures identified a mobile transport domain that may undergo a roughly ˚ rigid-body movement to provide alternating access to the bound substrate 20 A from either side of the membrane. Crosslinking of pairs of cysteine residues that are distant in the crystal structures but are predicted to move close to each other in the alternate conformation provides further support for the large-scale movement of the transport domain. Potential pathways for substrate release from the inward- and outward-facing conformations are also described, including the movement of a conserved cytoplasmic loop that may control substrate access from the cytoplasmic side. These results provide a mechanistic framework for understanding substrate recognition and translocation. 893-Plat Direct Visualization of Glutamate Transporter Transport Cycle Yi Ruan1, Atsushi Miyagi1, Xiaoyu Wang2, Mohamed Chami3, Henning Stahlberg3, Olga Boudker2, Simon Scheuring1. 1 U1006, INSERM / Aix-Marseille Universite´, Marseille, France, 2 Department of Physiology and Biophysics, Weill Cornell Medical College, New York, NY, USA, 3C-CINA, Biozentrum, University of Basel, Basel, Switzerland. Glutamate transporters are essential for recovering the neurotransmitter glutamate from the synaptic cleft after a synaptic stimulation. Crystal structures in the outward-facing (1) and inward-facing (2) conformations of a prokaryotic homolog of glutamate transporters revealed the molecular basis of the transporter cycle. So far, however, dynamics studies of the transport mechanism are sparse and indirect, based on fluorescent microscopy with its limitations (3). Here we present first high speed atom force microscopy (HS-AFM(4,5)) observations of membrane reconstituted glutamate transporters at work. The HS-AFM movies provide an unprecedented real-space and real-time visualization of the transport dynamics and permit quantitative comparison to the transport rates. Our results show transport mediated by large amplitude (~2nm) ‘‘elevator’’ movements of the transport domains. Furthermore, individual monomers within the trimeric complex possess full independence to realize substrate transport.
Monday, February 29, 2016 References: 1) Yernool, D., Boudker, O., Jin, Y. & Gouaux, E. Structure of a glutamate transporter homologue from pyrococcus horikoshii. Nature 431, 811-818, (2004). 2) Reyes, N., Ginter, C. & Boudker, O. Transport mechanism of a bacterial homologue of glutamate transporters. Nature 462, 880-885, (2009). 3) Akyuz, N., Altman, R. B., Blanchard, S. C. & Boudker, O. Transport dynamics in a glutamate transporter homologue. Nature 502, 114-118, (2013). 4) Ando, T., Kodera, N., Takai, E., Maruyama, D., Saito, K. & Toda, A. A highspeed atomic force microscope for studying biological macromolecules. Proceedings of the National Academy of Sciences 98, 12468-12472, (2001). 5) Ando, T., Uchihashi, T. & Scheuring, S. Filming biomolecular processes by high-speed atomic force microscopy. Chem Rev 114, 3120-3188, (2014). 894-Plat Resolving Active Ion Transport at the Single Molecule Level for the First Time Salome Veshaguri1, Sune M. Christensen1, Gerdi C. Kemmer2, Mads P. Møller1, Garima Ghale1, Christina Lohr1, Andreas L. Christensen1, Bo H. Justesen2, Ida L. Jørgensen2, Ju¨rgen Schiller3, Nikos S. Hatzakis1, Michael Grabe4, Thomas Gu¨nther Pomorski2, Dimitrios Stamou1. 1 Department of Chemistry, University of Copenhagen, Copenhagen, Denmark, 2Department of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, Denmark, 3Institute of Medical Physics and Biophysics, Leipzig, Germany, 4Department of Pharmaceutical Chemistry, Cardiovascular Research Institute, San Francisco, CA, USA. Electrochemical gradients across cellular membranes control a plethora of vital biological processes. These gradients are generated by primary active transporters and are subsequently used to drive the exchange of other solutes through secondary active transporters and to facilitate signaling via ion channels. The macroscopic biological phenomena that channels and transporters give rise to are intimately connected to how they function at the single molecule level. For decades, patch clamp recording has been used to observe the functional dynamics of single ion channels revealing discrete on and off states, subconductance states, and other mechanistically important features that macroscopic experiments cannot probe(1). Currently, there aren’t any techniques available to investigate transporter function at the single molecule level, thus they are only studied using ensemble biochemical methods. For a decade we have been developing quantitative fluorescence microscopy based assays of arrayed proteoliposomes for investigating membrane proteins(2-6). Here we extended the platform to monitor the single-molecule activity and the regulation of a prototypic P-type ATPase, Arabidopsis thaliana Hþ-ATPase (AHA2). For the first time we have shown that individual proton pumps are not active continuously but rather transitioning between active and inactive states separated by a large activation barrier (kJMole1). We found that the dynamics of these states form the basis of the regulation of the macroscopic activity either by regulatory R-domain, pH gradients, or ATP. Like for ion channels we often found that regulatory inputs do not affect the intrinsic pumping rates but rather active probabilities. References: 1. Neher & Sajmann, Nature, (1976) 2. Mathiasen et al., Nature Methods, (2014) 3. Larsen et al., Nature Chemical Biology, (2015) 4. Christensen et al., Nature Nanotechnology, (2012) 5. Hatzakis et al., Nature Chemical Biology, (2009) 6. Bendix et al., PNAS, (2009) 895-Plat Protein Interactions that Enable Safe and Efficient Copper Ion Transport in the Human Cytoplasm Pernilla Wittung-Stafshede. Biology and Bioengineering, Chalmers University of Technology, Gothenburg, Sweden. Although copper (Cu) is an essential metal for most living organisms, high levels and free such ions are toxic. In humans, after cellular uptake via the membrane-bound importer Ctr1, Cu is transported to targets by cytoplasmic Cu chaperones: Atox1 delivers Cu to membrane-bound P1B-type ATPases (i.e., ATP7B or Wilson disease protein) in the Golgi (secretory path; here, most Cu-dependent enzymes are loaded with Cu) whereas CCS delivers Cu specifically to cytoplasmic superoxide dismutase. In contrast to bacterial and yeast homologs, ATP7B has six similar metal-binding domains protruding into the cytoplasm: possibly, conformational changes among these regulate overall ATP7B activity. To reveal underlying molecular mechanisms as well as thermodynamic and kinetic driving forces for human Cu transport - from the cell membrane to the Golgi - our strategy involves a range of complementary biophysical experiments on purified proteins, domain constructs and engi-
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neered variants. From our studies, we have discovered that (a) the cytoplasmic C-terminus of Ctr1 binds Cu through its HCH motif with a moderate affinity that allows for Cu delivery to Atox1, (b) Atox1 can interact with CCS and exchange Cu implying cross-reactivity between cytoplasmic chaperones, (c) transfer of Cu from Atox1 to metal-binding domains in ATP7B proceeds through Cu-bridged hetero-protein dimers displaying enthalpy-entropy compensation, (d) conformational changes and domain-domain interactions within ATP7B depend on Cu loading status and minute changes in solvent conditions, and (e), in addition to its cytoplasmic chaperone activity, Atox1 may have functionality in the nucleus as it interacted with several DNA-binding proteins in a yeast two-hybrid screen. 896-Plat Dissecting the Catalytic Cycle of the Serotonin Transporter Peter S. Hasenhuetl, Michael Freissmuth, Harald H. Sitte, Klaus Schicker, Yang Li, Walter Sandtner. Department of Pharmacology, Medical Univeristy of Vienna, Vienna, Austria. The plasmalemmal serotonin transporter (SERT) regulates serotonin homeostasis and signaling by its reuptake from the extracellular space. In accordance with this prominent role, it is a target of several psychoactive substances, ranging from inhibitors (e.g. cocaine) to substrates (e.g. amphetamines). SERT belongs to the solute carrier 6 family. Hence, it utilizes the electrochemical gradient of co-substrates for the uphill transport of serotonin into the cell. Interestingly, the stoichiometry of this secondary-active transport mechanism has been matter of debate - despite decades of investigation. In addition, the order and kinetics of (co-)substrate binding to SERT have remained enigmatic. Here, we utilized the high temporal resolution of the whole-cell patch-clamp technique to decipher the kinetic determinants of selective ligand recognition, and of (co-)substrate binding and transport - in both, the forward transport and substrate exchange mode of SERT. Based on our electrophysiological data, we provide a comprehensive kinetic model of SERT that accounts for kinetics, stoichiometry, and order of (co-)substrate binding and translocation. We find that, Cl- does not participate in coupling of serotonin transport, but is required for substrate binding. In addition, our data suggest that two Naþ ions bind to SERT in a sequential order (Naþ-serotonin-Naþ). These findings are incompatible with an electroneutral stoichiometry. Our data shall provide a mechanistic framework for future attempts to integrate functional data with available structural information. 897-Plat Translocase Activity and Asymmetric Model Membranes Probed by Neutron Scattering Allison M. Whited, Frederick A. Heberle, Robert F. Standaert, Jonathan David Nickels, Xiaolin Cheng, John Katsaras, Alexander Johs. Oak Ridge National Lab, Oak Ridge, TN, USA. Biological membranes are almost universally asymmetric with individual leaflets containing different lipid species at varying concentrations. This asymmetry may play a key role in biological functions such as protein-membrane interactions, membrane trafficking, and cellular signaling. Traditional model membrane systems are comprised of symmetric membrane leaflets and though progress has been made in developing asymmetric model membranes, these systems are unstable and easily contaminated. We have developed an actively asymmetric biomembrane model system by purifying and reconstituting the well-characterized E. coli phospholipid ABC transporter MsbA into proteoliposomes that mimic the phospholipid composition of a physiological bacterial membrane. Subsequent MsbA-mediated phospholipid translocation and resultant membrane asymmetry was studied using small-angle neutron scattering (SANS). SANS is exceptionally well suited for studying the phospholipid distribution perpendicular to the bilayer plane due to its nondestructive nature and sub-nanometer resolution. Additionally, the hydrogen/deuterium (H/D) neutron scattering contrast variation from isotopic phospholipid labeling enables us to track the translocation of lipid membrane components without introducing bulky fluorescence or spin labels. In this study, the MsbA-mediated translocation of the phospholipid phosphatidylethamolamine (PE) between proteoliposome leaflets and the formation of asymmetric bilayers was studied using SANS for the first time. The sustained asymmetry in these model protocells will be central for future studies on biomimetic membrane systems and their role in diverse membrane-directed biological processes. 898-Plat Functional Characterization of Calcium-activated Phospholipid Scramblase Activity of nhTMEM16 Tao Jiang1,2, Sundar Thangapandian1,2, Emad Tajkhorshid1,2. 1 Department of Biochemistry, Center for Biophysics and Computational Biology, University of Illinois at Urbana Champaign, Urbana, IL, USA, 2 Beckman Institute for Advanced Science and Technology, Urbana, IL, USA.