Understanding the Binding Mechanism of Ryanodine to the Open- and ...

Tuesday, February 14, 2017 concentration-dependent manner, with significant effects at 10 and 30 mM. The voltammetry data suggest that 5-MAPB reduces the rate of dopamine reuptake; while the peak dopamine efflux was not increased, the area under the curve was augmented. 5-MAPB can also cause reverse dopamine transport consistent with stimulant properties, more similar to amphetamine than cocaine. Molecular modelling and docking studies compared the binding site of DAT in complex with 5-MAPB to dopamine, amphetamine, 5-APB, MDMA, cocaine and RTI121. This structural comparison reveals a binding mode for 5-MAPB found in the primary binding (S1) site, central to transmembrane domains 1, 3, 6 and 8, which overlaps with the binding modes of dopamine, cocaine and its analogues. Atomistic molecular dynamics simulations further show that, when in complex with 5-MAPB, DAT can exhibit conformational transitions that spontaneously isomerize the transporter into inward-facing state, similarly to that observed in dopamine-bound DAT. These novel insights, offered by the combination of computational methods of biophysics with neurobiological procedures, provide structural context for NPS at DAT and relate them with their functional properties at DAT as the molecular target of stimulants. 1660-Plat Weighted Ensemble of Pathways for Ligand Unbinding from T4 Lysozyme Ariane Nunes-Alves1, Daniel M. Zuckerman2, Guilherme M. Arantes1. 1 Department of Biochemistry, Institute of Chemistry, University of Sa˜o Paulo, Sa˜o Paulo, Brazil, 2Department of Computational and Systems Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA. T4 lysozyme L99A mutant is a protein often used as a model to study small molecule binding to macromolecules. Experimental affinities and crystal structures are available for its complex with several ligands. But pathways for ligand entry and exit from the deeply buried and solvent inaccessible binding site have not been fully resolved. Protein conformational changes necessary to allow ligand excursion to the binding site have also been debated. Here, we present molecular dynamics (MD) simulations that sample the pathways for benzene exit from the T4 lysozyme binding site and the associated conformational changes. The weighted ensemble (WE) approach was used to search for possible unbinding pathways and to overcome the limitations of MD simulations for sampling low probability regions in pre-defined progress coordinates. Interaction energies were obtained using the CHARMM36 force field in combination with implicit solvation. Independent WE simulations employing different progress coordinates revealed four possible pathways for benzene exit. Displacements of alpha-helices backbone and amino acid side chains on the order of 0.4 nm or less were enough to allow ligand exit. The side chains of Tyr88 and Leu133 were identified as the main gates for two of the pathways sampled. These results complement previous proposals which identified only one or two transit pathways. 1661-Plat Exploring New Pharmacological Perspectives of Fusicoccin, A Stabilizer of 14-3-3 - Target Protein Complex Andrea Saponaro1, Alessandro Porro1, Antonio C. Sanjuan1, Chiara Donadoni1, Marco Nardini1, Gerhard Thiel2, Anna Moroni1. 1 Dept. Biosciences, University of Milan, Milan, Italy, 2Dept. Biology, TU-Darmstadt, Darmstadt, Germany. The development of small-molecules regulating protein - protein interaction has emerged as a powerful field in pharmacology. In this scenario, 14-3-3 proteins represent a promising target, as they are an important class of adapter proteins that regulate several hundred partners, many of them described as disease-relevant proteins such as p53, Raf1, a-Synuclein and HERG channel. The fungal phytoxin Fusicoccin (FC) has shown to possess stabilizing properties on the complex formed by 14-3-3 proteins with some of their targets, including ion channels and pumps. Thus, FC is a promising tool to control cellular processes regulated by 14-3-3 proteins. To date FC action was thought to be limited to a specific subset of targets displaying the 14-3-3 binding site at their C-terminus (MODE III). The structure of the ternary 14-3-3 - target - FC complex shows that FC displays its stabilizing effect by developing an hydrophobic interaction with the C-terminal non-polar residue of the target protein. In this work, by taking advantage of our structural and functional study on the interaction between 14-3-3 and the voltage-gated inward rectifier potassium channel KAT1 of Arabidopsis thaliana, we expanded the palette of pharmacological targets of FC to a new kind of internal and C-terminal binding motifs lacking the hydrophobic residue. By solving the structure of the ternary complex, we discovered a new orientation of FC within the 14-3-3 - KAT1 complex, that accounts for the unexpected stabilizing effect of the molecule. Thus, our structural and functional work opens new perspectives in the employment of FC as a stabilizing molecule for 14-3-3 - target complexes.

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1662-Plat Developing a Novel Class of CLC Chloride-Channel Inhibitors Anna K. Koster1, Chase Wood2, Rhiannon Thomas-Tran1, Tanmay S. Chavan2, Jonas Almqvist3, Kee-Hyun Choi2,4, Justin Du Bois1, Merritt Maduke2. 1 Chemistry, Stanford University, Stanford, CA, USA, 2Molecular & Cellular Physiology, Stanford University, Stanford, CA, USA, 3Uppsala University, Uppsala, Sweden, 4Korea Institute of Science and Technology, Seoul, Korea, Republic of. The chloride channel (CLC) family is a class of membrane proteins that regulates the flux of chloride ions across cell membranes. Two of the nine mammalian CLC isoforms, CLC-Ka and CLC-Kb, reside in the kidney where they are critical for maintaining proper water and salt balance and therefore are therapeutic targets for treating hypertension and hyponatremia. CLCs are traditionally difficult drug targets, as demonstrated by the low potency and general lack of isoform selectivity among existing classes of CLC inhibitors. In this work, we describe a novel small-molecule inhibitor that shows an unprecedented selectivity for CLC-Ka over CLC-Kb despite their 90% sequence similarity. Through homology modeling, site-directed mutagenesis, and structureactivity relationship studies, we have identified and validated the inhibitor binding site, as well as key molecular features that are stepping stones for designing CLC inhibitors with enhanced potency and isoform selectivity. 1663-Plat Mapping Cholesterol Binding Sites on the Human Dopamine Transporter Talia Zeppelin, Xavier Periole, Birgit Schiøtt. Chemistry, Aarhus University, Aarhus C, Denmark. The human dopamine transporter (hDAT) is essential for regulating dopaminergic neurotransmission by transporting dopamine from the synaptic cleft back into the presynaptic neuron. Dysregulation of hDAT is involved in several debilitating diseases such as Parkinson‘s disease, attention deficit hyperactive disease (ADHD) and Tourette‘s syndrome, among others. DAT is also the target of many illicit drugs and has been presumed to be involved in the development of addiction. It is therefore essential to further the understanding of the transporters architecture, transport mechanism and means of regulation to aid in drug development. Recently, a range of DAT structures has been published from the species Drosophilia Melanogaster (dDAT). All of which contain at least one conserved co-crystallized cholesterol molecule. It is believed that cholesterol acts in regulating hDAT activity, but it is unclear whether the two cholesterol sites found on dDAT also exist on hDAT. Within this contribution we use coarse-grained molecular dynamics simulations of a hDAT homology model embedded in a mixed POPC and cholesterol membrane. We will present the results, and furthermore compare any identified cholesterol binding sites to other membrane protein known to bind cholesterol. 1664-Plat Understanding the Binding Mechanism of Ryanodine to the Open- and Closed States of the Ryanodine Receptor Pore Williams E. Miranda1, Van A. Ngo1, Laura L. Perissinotti1, S.R. Wayne Chen2, Sergei Y. Noskov1. 1 Biological Sciences, University of Calgary, Calgary, AB, Canada, 2Libin Cardiovascular Institute, University of Calgary, Calgary, AB, Canada. Ryanodine (Ryd) is a poisonous plant alkaloid that specifically binds ryanodine receptors (RyRs) with distinguishable affinities. Some of its binding modes can sharply enhance the open probabilities, thus helping to demonstrate the vital roles of RyRs as a family of intracellular calcium release channels in skeletal, cardiac and neuronal cells. Although electrophysiology and mutagenesis experiments have shed some light on the binding mechanism of Ryd to RyRs, a full atomistic insight of the receptor-ligand recognition process is still lacking. We used all-atom molecular dynamics (MD) simulations-based approaches to study the binding of Ryd to the skeletal muscle isoform RyR1, ˚ resolution for a closed which has been recently solved by Cryo-EM at 3.8-A ˚ for an open structure. We applied enhanced and bistructure and 4.1-A directional sampling methodologies to study not only energetics and thermodynamics, but also geometrical binding modes of single and multiple Ryds along the pathways from the cytosol to cavity and passing the selectivity filter to the lumen. Using the closed structure, our preliminary results suggest that the pyrrolic ring of Ryd statistically favors R4892AGGG-F4921 residues of RyR1‘s cavity, which explain the effects of the corresponding mutations in RyR2 (cardiac isoform) in experiments. This will be compared with the binding modes and thermodynamics of Ryds in the open structure of RyR1, thus providing some atomistic insights into how Ryd interacts with major residues in the open and closed RyR1 that give rise to the enhancement of the open probabilities.