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The Energy Landscape of Human Chromosomes
Wednesday, February 15, 2017 nanobook, and a nanorobot. These multidomain assemblies were stabilized via short-ranged nucleobase stacking bonds that compete against electrostatic repulsion between the components’ interfaces. Building on these techniques, in the present study we report on progress with the hierarchical self-assembly of discrete three-dimensional finite size objects whose shapes are encoded in the geometry of its building blocks. We created gear-like objects that form from self-limiting oligomerization of V-shaped building blocks. The diameter, the number of subunits per ring, and the shape of the distribution of subunits per ring are tightly controlled by the angle and rigidity of the V building blocks. The rings may be hierarchically assembled into giant, giga-dalton scale tubes. We also describe progress on the selfassembly of 3D polyhedral cages such as cubes and dodecaheder from appropriately shaped building blocks. (1) T. Gerling, K. Wagenbauer, A. Neuner, and H. Dietz ‘Dynamic DNA devices and assemblies formed by shape-complementary, nonbasepairing 3D components’. Science, vol 347 (2015), p1446-1452. 2337-Plat The Energy Landscape of Human Chromosomes Michele Di Pierro. Center for Theoretical Biological Physics, Rice University, Houston, TX, USA. In vivo, the human genome folds into a characteristic ensemble of threedimensional structures. The mechanism driving the folding process remains unknown. We report a theoretical model for chromatin (Minimal Chromatin Model) that explains the folding of interphase chromosomes and generates chromosome conformations consistent with experimental data. The energy landscape of the model was derived by using the maximum entropy principle and relies on two inputs: a classification of loci into chromatin types and a catalog of the positions of CTCF-mediated chromatin loops. Chromatin types, which are distinct from DNA sequence, are partially epigenetically controlled and change during cell differentiation, thus constituting a link between epigenetics, chromosomal organization, and cell development. We trained our energy function using the Hi-C contact map of chromosome 10 from human GM12878 lymphoblastoid cells. Then, we used the model to perform molecular dynamics simulations producing an ensemble of 3D structures for all GM12878 autosomes. We used these 3D structures to generate contact maps. We found that simulated contact maps closely agree with experimental results for all GM12878 autosomes. The ensemble of structures resulting from these simulations exhibited unknotted chromosomes, phase separation of chromatin types, and a tendency for open chromatin to lie at the periphery of chromosome territories. Finally, we analyzed the dynamics generated by MiChroM. We found our model to be in excellent agreement with experimental observations reporting anomalous diffusion and spatial coherence in chromatin in vivo.
Symposium: Channel Gating Mechanisms 2338-Symp Dynamic Coupling between the Gates in the NaK Channel Katherine Henzler-Wildman. Biochemistry, University of Wisconsin, Madison, WI, USA. Flux-dependent inactivation that arises from functional coupling between the inner gate and the selectivity filter is widespread in ion channels. The structural basis of this coupling has only been well characterized in KcsA. Here we present NMR data demonstrating structural and dynamic coupling between the selectivity filter and intracellular constriction point in the bacterial non-selective cation channel, NaK. High-resolution crystal structures are available for a number of NaK mutants with different numbers of ion binding sites in the selectivity filter and different ion selectivity properties. NMR spectra of non-selective and potassium-selective NaK mutants indicate that the equilibrium distribution of structural states for the entire channel varies with the ion-selectivity of the channel. Our results highlight the tight structural and dynamic coupling between the selectivity filter, the surrounding scaffold, and the inner gate. NaK offers a distinct and valuable model to study this physiologically essential connection between ion conduction and channel gating. 2339-Symp Structure of the TPC1 Channel from Arabidopsis Thaliana Jiangtao Guo1, Weizhong Zeng1, Youxing Jiang1,2. 1 University of Texas Southwestern Medical Center, Dallas, TX, USA, 2 Howard Hughes Medical Institute, Dallas, TX, USA. Two-pore channels (TPCs) belong to the family of voltage-gated tetrameric cation channels and are ubiquitously expressed in organelles of animals and plants. Each TPC subunit contains 12 transmembrane segments that can be
475a
divided into two homologous copies of an S1-S6 Shaker-like 6-TM domain. A functional TPC channel assembles as a dimer - the equivalent of a voltage-gated tetrameric cation channel. Plant TPC channel (TPC1) is localized to the vacuole membrane and is responsible for generating the slow vacuolar (SV) current observed long before its molecular identification; therefore, plant TPC1 is also called SV channel. Plant TPC1 is a non-selective cation channel, permeable to various monovalent cations as well as Ca2þ and likely plays an important role in regulating cytosolic ion concentrations. The channel is voltage-gated and its voltage-dependent activation can be modulated by both cytosolic and vacuolar Ca2þ. Recently, we determined the first crystal structure of a plant vacuolar two-pore channel from Arabidopsis thaliana, AtTPC1, and were also able to functionally characterize the channel activity using patch clamp recording. Our structural and functional studies demonstrate that Ca2þ activates the channel by triggering conformational changes at the EF-hand domain that are coupled to the pair of pore-lining inner helices from the first 6-TM domains, whereas membrane potential only activates the second voltage-sensing domain (VSD2), whose conformational changes may only be coupled to the other pair of inner helices from the second 6-TM domains. More importantly, the current AtTPC1 structure is in a closed state and, for the first time, provides a long-awaited structural view of a channel voltage sensor in the resting state. This major breakthrough on AtTPC1 study provides us with an excellent model system to further investigate the structural mechanisms of channel gating and ion selectivity in the TPC channel family. 2340-Symp Gating Pathways for a Pentameric Ligand-Gated Ion Channel Solved by Atomistic String Method Simulations Bogdan Lev1, Samuel Murail2, Fre´de´ric Poitevin3, Brett A. Cromer1, Marc Baaden2, Marc Delarue4, Toby W. Allen1,5. 1 Science, RMIT University, Melbourne, Australia, 2Laboratoire de Biochimie The´orique, CNRS, UPR9080, Univ Paris Diderot, Sorbonne, Paris, France, 3Structural Biology, Stanford University, Stanford, CA, USA, 4 Structural Biology and Chemistry, Institut Pasteur and UMR 3528 du CNRS, Paris, France, 5Chemistry, University of California, Davis, CA, USA. Pentameric ligand-gated ion channels control synaptic neurotransmission via an allosteric mechanism, where agonist binding induces global protein conformational changes that open an ion-conducting pore. We use an atomistic molecular dynamics string method to solve for the minimum free energy gating pathways for the proton-activated GLIC channel. We describe stable open and closed states, and uncover conformational changes associated with communication between agonist-binding extracellular and ion-conducting trans-membrane domains. Transition analysis is used to compute free energy surfaces that suggest pathways, allosteric stabilization with pH, and intermediate states that facilitate channel closing in the presence of agonist. We describe a switching mechanism that senses proton binding to induce asynchronous pore-lining M2 helix movements. These results provide new insight into the allosteric mechanisms for the super-family of pentameric ligand-gated channels, with potential applications in improved anesthetics, neuromodulatory drugs, antiparasitics and pesticides. 2341-Symp Fine Tuning HCN Channel Activity Anna Moroni. Biosciences, University of Milan, Milan, Italy. HCN (hyperopolarization-activated cyclic-nucleotide gated) channels are activated by membrane hyperopolarization and further modulated by binding of cAMP to the CNBD (cyclic nucleotide binding domain). Understanding the molecular mechanisms of regulation of this family of ion channels is critical as it pertains to the physiological processes as well as to diseases associated dysfunctions in the cardiac and neuronal If/Ih currents. In the brain, cAMP modulation of HCN isoforms 1 and 2 is controlled and fine tuned by the auxillary protein TRIP8b while, in the heart, cAMP regulation of the cardiac isoform HCN4 is prevented by cyclic dinucleotides, an emerging class of second messengers in mammals. Starting from a structural and functional description of the molecular determinants of the aforementioned regulatory systems, we are developing new tools (peptides, chimeric channels and optogenetic approaches) to control and analyse cAMP action on HCN channels. Our goals are: i) to fully describe the cAMP-induced conformational changes which affectpore gating; ii) to understand the physiological role of TRIP8b modulation of HCN channel gating in vivo; iii) to identify terapeutical approaches based on the control of cAMP modulation. Our results on the regulatory network of HCN activity highlight an efficient mechanism for preventing b-adrenergic stimulation on If and identify potential drug targets in HCN channels.
Symposium: Channel Gating Mechanisms 2338-Symp Dynamic Coupling between the Gates in the NaK Channel Katherine Henzler-Wildman. Biochemistry, University of Wisconsin, Madison, WI, USA. Flux-dependent inactivation that arises from functional coupling between the inner gate and the selectivity filter is widespread in ion channels. The structural basis of this coupling has only been well characterized in KcsA. Here we present NMR data demonstrating structural and dynamic coupling between the selectivity filter and intracellular constriction point in the bacterial non-selective cation channel, NaK. High-resolution crystal structures are available for a number of NaK mutants with different numbers of ion binding sites in the selectivity filter and different ion selectivity properties. NMR spectra of non-selective and potassium-selective NaK mutants indicate that the equilibrium distribution of structural states for the entire channel varies with the ion-selectivity of the channel. Our results highlight the tight structural and dynamic coupling between the selectivity filter, the surrounding scaffold, and the inner gate. NaK offers a distinct and valuable model to study this physiologically essential connection between ion conduction and channel gating. 2339-Symp Structure of the TPC1 Channel from Arabidopsis Thaliana Jiangtao Guo1, Weizhong Zeng1, Youxing Jiang1,2. 1 University of Texas Southwestern Medical Center, Dallas, TX, USA, 2 Howard Hughes Medical Institute, Dallas, TX, USA. Two-pore channels (TPCs) belong to the family of voltage-gated tetrameric cation channels and are ubiquitously expressed in organelles of animals and plants. Each TPC subunit contains 12 transmembrane segments that can be
475a
divided into two homologous copies of an S1-S6 Shaker-like 6-TM domain. A functional TPC channel assembles as a dimer - the equivalent of a voltage-gated tetrameric cation channel. Plant TPC channel (TPC1) is localized to the vacuole membrane and is responsible for generating the slow vacuolar (SV) current observed long before its molecular identification; therefore, plant TPC1 is also called SV channel. Plant TPC1 is a non-selective cation channel, permeable to various monovalent cations as well as Ca2þ and likely plays an important role in regulating cytosolic ion concentrations. The channel is voltage-gated and its voltage-dependent activation can be modulated by both cytosolic and vacuolar Ca2þ. Recently, we determined the first crystal structure of a plant vacuolar two-pore channel from Arabidopsis thaliana, AtTPC1, and were also able to functionally characterize the channel activity using patch clamp recording. Our structural and functional studies demonstrate that Ca2þ activates the channel by triggering conformational changes at the EF-hand domain that are coupled to the pair of pore-lining inner helices from the first 6-TM domains, whereas membrane potential only activates the second voltage-sensing domain (VSD2), whose conformational changes may only be coupled to the other pair of inner helices from the second 6-TM domains. More importantly, the current AtTPC1 structure is in a closed state and, for the first time, provides a long-awaited structural view of a channel voltage sensor in the resting state. This major breakthrough on AtTPC1 study provides us with an excellent model system to further investigate the structural mechanisms of channel gating and ion selectivity in the TPC channel family. 2340-Symp Gating Pathways for a Pentameric Ligand-Gated Ion Channel Solved by Atomistic String Method Simulations Bogdan Lev1, Samuel Murail2, Fre´de´ric Poitevin3, Brett A. Cromer1, Marc Baaden2, Marc Delarue4, Toby W. Allen1,5. 1 Science, RMIT University, Melbourne, Australia, 2Laboratoire de Biochimie The´orique, CNRS, UPR9080, Univ Paris Diderot, Sorbonne, Paris, France, 3Structural Biology, Stanford University, Stanford, CA, USA, 4 Structural Biology and Chemistry, Institut Pasteur and UMR 3528 du CNRS, Paris, France, 5Chemistry, University of California, Davis, CA, USA. Pentameric ligand-gated ion channels control synaptic neurotransmission via an allosteric mechanism, where agonist binding induces global protein conformational changes that open an ion-conducting pore. We use an atomistic molecular dynamics string method to solve for the minimum free energy gating pathways for the proton-activated GLIC channel. We describe stable open and closed states, and uncover conformational changes associated with communication between agonist-binding extracellular and ion-conducting trans-membrane domains. Transition analysis is used to compute free energy surfaces that suggest pathways, allosteric stabilization with pH, and intermediate states that facilitate channel closing in the presence of agonist. We describe a switching mechanism that senses proton binding to induce asynchronous pore-lining M2 helix movements. These results provide new insight into the allosteric mechanisms for the super-family of pentameric ligand-gated channels, with potential applications in improved anesthetics, neuromodulatory drugs, antiparasitics and pesticides. 2341-Symp Fine Tuning HCN Channel Activity Anna Moroni. Biosciences, University of Milan, Milan, Italy. HCN (hyperopolarization-activated cyclic-nucleotide gated) channels are activated by membrane hyperopolarization and further modulated by binding of cAMP to the CNBD (cyclic nucleotide binding domain). Understanding the molecular mechanisms of regulation of this family of ion channels is critical as it pertains to the physiological processes as well as to diseases associated dysfunctions in the cardiac and neuronal If/Ih currents. In the brain, cAMP modulation of HCN isoforms 1 and 2 is controlled and fine tuned by the auxillary protein TRIP8b while, in the heart, cAMP regulation of the cardiac isoform HCN4 is prevented by cyclic dinucleotides, an emerging class of second messengers in mammals. Starting from a structural and functional description of the molecular determinants of the aforementioned regulatory systems, we are developing new tools (peptides, chimeric channels and optogenetic approaches) to control and analyse cAMP action on HCN channels. Our goals are: i) to fully describe the cAMP-induced conformational changes which affectpore gating; ii) to understand the physiological role of TRIP8b modulation of HCN channel gating in vivo; iii) to identify terapeutical approaches based on the control of cAMP modulation. Our results on the regulatory network of HCN activity highlight an efficient mechanism for preventing b-adrenergic stimulation on If and identify potential drug targets in HCN channels.