- Email: [email protected]
Towards Identifying Biologically Relevant Intermediate Conformational States in Dopamine Transporter
626a
Wednesday, March 2, 2016
interactions between the N-term and the intracellular loops of the transporter molecule. Quantitative analyses of collective motions in the trajectories reveal that these interactions correlate with inward-opening dynamics of hDAT and are allosterically coupled to the known functional sites of the transporter. The observed large-scale motions are enabled by specific reconfiguration of the network of ionic interactions at the intracellular end of the protein. The isomerization to the inward-facing state in hDAT is accompanied by concomitant movements in the extracellular vestibule and results in release of Naþ ion from the Na2 site and destabilization of the substrate dopamine in the primary substrate binding S1 site. The dynamic mechanism emerging from the findings highlights the involvement of the PIP2-regulated interactions between the N-term and the intracellular loop 4 in the functionally relevant conformational transitions that are also similar to those found to underlie state-to-state transitions in the leucine transporter (LeuT), a prototypical bacterial homolog of the NSS. 3090-Pos Board B467 Computational Investigation of the Transport Mechanism of Neurotransmitter Sodium Symporters using a Physiological Ion Gradient Emily M. Benner, Jeffry D. Madura. Duquesne University, Pittsburgh, PA, USA. Neurotransmitter transporter proteins serve a critical role in the synaptic cleft, aiding in the maintenance of synaptic concentrations of neurotransmitters, terminating neurotransmitter effect via a process known as reuptake. The serotonin transporter (SERT) belongs to the larger monoamine transporter (MAT) family, and is responsible for the reuptake of serotonin. The MAT proteins are implicated in several psychological disorders, including depression. All current treatment options for depression are focused on the inhibition of the MAT system, and are often selective serotonin reuptake inhibitors (SSRIs). SSRIs act by binding to the substrate binding site of SERT, blocking the reuptake of serotonin. Our recent work has been focused on the transport mechanism of SERT, where we have employed both a dual bilayer system and a single bilayer system to investigate this phenomenon. We are utilizing the multilevel summation method of evaluating electrostatic forces with a semi-periodic system. Using boundary forces at the minimum and maximum z-values, the ion concentration on either side of the membrane remains constant, keeping the membrane potential near 70 mV, which is resting potential. Understanding more fully how the transport process occurs can aid in drug discovery and design for treatments that better alleviate symptoms of depression and other related disorders. In this poster, results from our ongoing work with the single bilayer SERT system, as well as conformational analyses will be presented. 3091-Pos Board B468 Towards Identifying Biologically Relevant Intermediate Conformational States in Dopamine Transporter Ara M. Abramyan1, Nicholas Taro1, Sebastian Stolzenberg2, Lei Shi1. 1 Molecular Targets and Medications Discovery Branch, National Institute on Drug Abuse-Intramural Research Program, National Institutes of Health, Baltimore, MD, USA, 2Department of Mathematics and Computer Science, Freie Universita¨t Berlin, Berlin, Germany. Psychostimulant abuse leads to debilitating disorders, the treatment of which remains an alarming challenge. Cocaine and amphetamine act on the dopamine transporter (DAT), which belongs to the neurotransmitter:sodium symporter family that terminates neurotransmission by reuptake of neurotransmitters from the synaptic cleft. The reuptake process can be described by the Naþcoupled alternating-access mechanism in which the transporter adopts outward-open, occluded and inward-open conformations. Cocaine is known to inhibit DAT function by trapping the protein in an outward-open conformation. Interestingly, several other DAT inhibitors, such as benztropine, modafinil, and some of their derivatives, appear to have low abuse liability. Compared to cocaine, these atypical DAT inhibitors show a preference for the conformation of DAT that can be stabilized by a mutation at the intracellular gate, Y335A, and were deduced to prefer less outward-open conformations than cocaine. Nevertheless their differential inhibiting mechanisms at the atomistic level is not well understood. We carried out comparatively molecular dynamics simulations of both DATWT and DAT-Y335A constructs, stabilized by a variety of DAT inhibitors, and used Markov State Models to analyze the resulting trajectories. This analysis identifies an ensemble of conformational states exhibited spontaneously by the molecule at local equilibrium, and has the advantage in its ability to represent both thermodynamic and kinetic characteristics of protein conformational changes. Thus we capture the conformational states and kinetics of transitions between outward-occluded and outward-open states
of DAT. Identification and characterization of the intermediate states that are stabilized by the atypical inhibitors and/or Y335A mutation is crucial in understanding the mechanism of action of these inhibitors. This understanding of how these states eventually trigger different downstream cellular effects from cocaine holds new promises to develop targeted treatment of cocaine dependence. 3092-Pos Board B469 Keeping Secondary Transporters under Control: Lessons from a NaD/ Ca2D Exchanger Fabrizio Marinelli, Jose´ Faraldo-Go´mez. National Heart, Lung, and Blood Institute, Bethesda, MD, USA. Secondary-active transporters catalyze the translocation of substances across biological membranes, driven by the electrochemical gradient of one of the transported species, typically Naþ or Hþ. The mechanism of these transporters has been widely rationalized as a conformational cycle (alternating-access model) that exposes substrate binding sites within the protein to one or the other side of the membrane (or none), but not both simultaneously. The electrochemical gradient drives the cycle in one direction but is not required to interconvert between different conformational states (as opposed to voltagegated ion-channels). However, active transport does require that the interconversion between outward and inward facing states occur only upon binding of specific substrates, and in uniquely defined stoichiometries. The physical basis of this mechanism of conformational control, however, remains to be established. Here, we address this central question for a prokaryotic homolog of the cardiac Naþ/Ca2þ exchanger, NCX_Mj, which transports either three Naþ or one Ca2þ across the membrane. Specifically, we determine how Naþ/Ca2þ recognition by the transporter outward-facing state reshapes its conformational free-energy landscape, which we examine via enhancedsampling molecular-dynamics simulations and crystallographic titration experiments. Our results demonstrate that only upon binding of three Naþ or one Ca2þ can the protein adopt a state occluded to both sides of the membrane, which necessarily precedes the transition to the inward-facing conformation. Binding sites depletion and/or Hþ binding, by contrast, eradicates the occluded state population, and induces the opening of hydrated access pathways connecting these sites to the surrounding solution. Interestingly, water in these pathways is markedly polarized, in part explaining the large energetic cost associated with occlusion. This study provides clear evidence that it is by inducing or precluding the formation of occluded, dehydrated states that substrate recognition controls the alternating-access transition in secondary transporters. 3093-Pos Board B470 Combined QM/MM Dynamics Simulations of Proton Transfer in E. coli CLC Chloride/Proton Antiporter Christina Garza. Chemistry, University of Colorado Denver, Denver, CO, USA. The ClC family of transmembrane proteins include both Cl– channels and Cl/ Hþ antiporters, and they play critical roles in many cellular processes, such as extreme acid response in E. coli and acidification of membranes in humans. The E. coli ClC (EcClC) antiporter has been extensively characterized; however, it remains a mystery how the proton is shuttled between the two gating glutamic ˚ . Previacid residues that are separated by a largely hydrophobic gap of ~15 A ous molecular dynamics studies have suggested transient formation of a water wire in this gap that can be the path for proton transport. Here, we aim to elucidate the detailed process of proton transport through EcClC by doing combined QM/MM simulations, where the proton is treated explicitly and the reorganization of the covalent and hydrogen bonds during proton relay is described quantum mechanically. 3094-Pos Board B471 Computational Studies of Elevator-Like Movements in Secondary Transport Cristina Fenollar-Ferrer1, Claudio Anselmi2, Ariela Vergara Jaque3, Hossein Ali Karimi-Verzaneh4, Horacio Poblete-Vilches5, Christopher Mulligan1, Ian C. Forster6, Joseph A. Mindell1, Jose´ D. Faraldo-Go´mez2, Lucy R. Forrest1. 1 National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA, 2National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA, 3Kansas State University, Kansas City, KS, USA, 4Continental, Hannover, Germany, 5Institute of Computational Comparative Medicine, Kansas State University, Kansas City, KS, USA, 6Institute of Physiology and Zurich Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland.
Wednesday, March 2, 2016
interactions between the N-term and the intracellular loops of the transporter molecule. Quantitative analyses of collective motions in the trajectories reveal that these interactions correlate with inward-opening dynamics of hDAT and are allosterically coupled to the known functional sites of the transporter. The observed large-scale motions are enabled by specific reconfiguration of the network of ionic interactions at the intracellular end of the protein. The isomerization to the inward-facing state in hDAT is accompanied by concomitant movements in the extracellular vestibule and results in release of Naþ ion from the Na2 site and destabilization of the substrate dopamine in the primary substrate binding S1 site. The dynamic mechanism emerging from the findings highlights the involvement of the PIP2-regulated interactions between the N-term and the intracellular loop 4 in the functionally relevant conformational transitions that are also similar to those found to underlie state-to-state transitions in the leucine transporter (LeuT), a prototypical bacterial homolog of the NSS. 3090-Pos Board B467 Computational Investigation of the Transport Mechanism of Neurotransmitter Sodium Symporters using a Physiological Ion Gradient Emily M. Benner, Jeffry D. Madura. Duquesne University, Pittsburgh, PA, USA. Neurotransmitter transporter proteins serve a critical role in the synaptic cleft, aiding in the maintenance of synaptic concentrations of neurotransmitters, terminating neurotransmitter effect via a process known as reuptake. The serotonin transporter (SERT) belongs to the larger monoamine transporter (MAT) family, and is responsible for the reuptake of serotonin. The MAT proteins are implicated in several psychological disorders, including depression. All current treatment options for depression are focused on the inhibition of the MAT system, and are often selective serotonin reuptake inhibitors (SSRIs). SSRIs act by binding to the substrate binding site of SERT, blocking the reuptake of serotonin. Our recent work has been focused on the transport mechanism of SERT, where we have employed both a dual bilayer system and a single bilayer system to investigate this phenomenon. We are utilizing the multilevel summation method of evaluating electrostatic forces with a semi-periodic system. Using boundary forces at the minimum and maximum z-values, the ion concentration on either side of the membrane remains constant, keeping the membrane potential near 70 mV, which is resting potential. Understanding more fully how the transport process occurs can aid in drug discovery and design for treatments that better alleviate symptoms of depression and other related disorders. In this poster, results from our ongoing work with the single bilayer SERT system, as well as conformational analyses will be presented. 3091-Pos Board B468 Towards Identifying Biologically Relevant Intermediate Conformational States in Dopamine Transporter Ara M. Abramyan1, Nicholas Taro1, Sebastian Stolzenberg2, Lei Shi1. 1 Molecular Targets and Medications Discovery Branch, National Institute on Drug Abuse-Intramural Research Program, National Institutes of Health, Baltimore, MD, USA, 2Department of Mathematics and Computer Science, Freie Universita¨t Berlin, Berlin, Germany. Psychostimulant abuse leads to debilitating disorders, the treatment of which remains an alarming challenge. Cocaine and amphetamine act on the dopamine transporter (DAT), which belongs to the neurotransmitter:sodium symporter family that terminates neurotransmission by reuptake of neurotransmitters from the synaptic cleft. The reuptake process can be described by the Naþcoupled alternating-access mechanism in which the transporter adopts outward-open, occluded and inward-open conformations. Cocaine is known to inhibit DAT function by trapping the protein in an outward-open conformation. Interestingly, several other DAT inhibitors, such as benztropine, modafinil, and some of their derivatives, appear to have low abuse liability. Compared to cocaine, these atypical DAT inhibitors show a preference for the conformation of DAT that can be stabilized by a mutation at the intracellular gate, Y335A, and were deduced to prefer less outward-open conformations than cocaine. Nevertheless their differential inhibiting mechanisms at the atomistic level is not well understood. We carried out comparatively molecular dynamics simulations of both DATWT and DAT-Y335A constructs, stabilized by a variety of DAT inhibitors, and used Markov State Models to analyze the resulting trajectories. This analysis identifies an ensemble of conformational states exhibited spontaneously by the molecule at local equilibrium, and has the advantage in its ability to represent both thermodynamic and kinetic characteristics of protein conformational changes. Thus we capture the conformational states and kinetics of transitions between outward-occluded and outward-open states
of DAT. Identification and characterization of the intermediate states that are stabilized by the atypical inhibitors and/or Y335A mutation is crucial in understanding the mechanism of action of these inhibitors. This understanding of how these states eventually trigger different downstream cellular effects from cocaine holds new promises to develop targeted treatment of cocaine dependence. 3092-Pos Board B469 Keeping Secondary Transporters under Control: Lessons from a NaD/ Ca2D Exchanger Fabrizio Marinelli, Jose´ Faraldo-Go´mez. National Heart, Lung, and Blood Institute, Bethesda, MD, USA. Secondary-active transporters catalyze the translocation of substances across biological membranes, driven by the electrochemical gradient of one of the transported species, typically Naþ or Hþ. The mechanism of these transporters has been widely rationalized as a conformational cycle (alternating-access model) that exposes substrate binding sites within the protein to one or the other side of the membrane (or none), but not both simultaneously. The electrochemical gradient drives the cycle in one direction but is not required to interconvert between different conformational states (as opposed to voltagegated ion-channels). However, active transport does require that the interconversion between outward and inward facing states occur only upon binding of specific substrates, and in uniquely defined stoichiometries. The physical basis of this mechanism of conformational control, however, remains to be established. Here, we address this central question for a prokaryotic homolog of the cardiac Naþ/Ca2þ exchanger, NCX_Mj, which transports either three Naþ or one Ca2þ across the membrane. Specifically, we determine how Naþ/Ca2þ recognition by the transporter outward-facing state reshapes its conformational free-energy landscape, which we examine via enhancedsampling molecular-dynamics simulations and crystallographic titration experiments. Our results demonstrate that only upon binding of three Naþ or one Ca2þ can the protein adopt a state occluded to both sides of the membrane, which necessarily precedes the transition to the inward-facing conformation. Binding sites depletion and/or Hþ binding, by contrast, eradicates the occluded state population, and induces the opening of hydrated access pathways connecting these sites to the surrounding solution. Interestingly, water in these pathways is markedly polarized, in part explaining the large energetic cost associated with occlusion. This study provides clear evidence that it is by inducing or precluding the formation of occluded, dehydrated states that substrate recognition controls the alternating-access transition in secondary transporters. 3093-Pos Board B470 Combined QM/MM Dynamics Simulations of Proton Transfer in E. coli CLC Chloride/Proton Antiporter Christina Garza. Chemistry, University of Colorado Denver, Denver, CO, USA. The ClC family of transmembrane proteins include both Cl– channels and Cl/ Hþ antiporters, and they play critical roles in many cellular processes, such as extreme acid response in E. coli and acidification of membranes in humans. The E. coli ClC (EcClC) antiporter has been extensively characterized; however, it remains a mystery how the proton is shuttled between the two gating glutamic ˚ . Previacid residues that are separated by a largely hydrophobic gap of ~15 A ous molecular dynamics studies have suggested transient formation of a water wire in this gap that can be the path for proton transport. Here, we aim to elucidate the detailed process of proton transport through EcClC by doing combined QM/MM simulations, where the proton is treated explicitly and the reorganization of the covalent and hydrogen bonds during proton relay is described quantum mechanically. 3094-Pos Board B471 Computational Studies of Elevator-Like Movements in Secondary Transport Cristina Fenollar-Ferrer1, Claudio Anselmi2, Ariela Vergara Jaque3, Hossein Ali Karimi-Verzaneh4, Horacio Poblete-Vilches5, Christopher Mulligan1, Ian C. Forster6, Joseph A. Mindell1, Jose´ D. Faraldo-Go´mez2, Lucy R. Forrest1. 1 National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA, 2National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA, 3Kansas State University, Kansas City, KS, USA, 4Continental, Hannover, Germany, 5Institute of Computational Comparative Medicine, Kansas State University, Kansas City, KS, USA, 6Institute of Physiology and Zurich Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland.