632-Pos Board B397 Computational Investigation of the Serotonin Transporter Conformation and Reset Mechanism Emily M. Benner, Jeffry D. Madura. Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA, USA. The aid of monoamine transporter (MAT) proteins in terminating the effect of neurotransmitters in the synaptic cleft is crucial for the maintenance of proper neurotransmitter concentrations. Termination of effect is achieved via the process of reuptake, where the proteins bind neurotransmitters to a central substrate binding site and transport the substrate to the other side of the membrane through a permeation pathway. The MAT family of pro- teins includes the serotonin transporter (SERT), which is responsible for the reuptake of serotonin from the synaptic cleft. SERT, as well as the other MAT proteins are implicated in several psychological disorders, including depression, anxiety, and addiction. Treatments for these disorders are focused on the inhibition of the MAT system, especially SERT. These drugs are known as selective serotonin reuptake inhibitors, which act by binding to the substrate site of SERT, preventing the reuptake of seroto- nin. Recent work in our lab has focused on identifying the complete transport mechanism of SERT computationally, employing both a single bilayer and dual bilayer system to investigate this phenomenon. The work presented here represents the data collected from our most recent dual bilayer system. Herein we utilized the newly crystallized human SERT pro- tein (PDB 5I6X), embedded in POPE lipid membranes. The dual bilayer setup allows the system to maintain ion concentrations on either side of the membrane when periodic boundary conditions are implemented. Ions were added to either side of the membrane at physiological concentra- tions, keeping the membrane potential at around À70mV, representative of resting potential. Understanding more fully the complete transport process, with specific attention paid to the reset mechanism, can aid in drug discov- ery and design for treatments that better alleviate symptoms of depression and related disorders. Presented here are the molecular dynamics and conformational data from our ongoing simulations of the dual bilayer SERT system. 633-Pos Board B398 Characterizing Outward- to Inward-Facing Transition Pathway of Dopamine Transporter Zhiyu Zhao 1 , Emad Tajkhorshid 2 . 1 Center for Biophysics and Quantitative Biology, UIUC, Champaign, IL, USA, 2 UIUC, Champaign, IL, USA. The dopamine transporter (DAT) belongs to the family of neurotransmitter sodium symporters (NSSs), which harness transmembrane electrochemical ionic gradients to transport neurotransmitters against their chemical gradi- ents. Dysregulation of DAT is associated with serious neurological disor- ders, such as Parkinson’s disease, depression, anxiety and epilepsy. As with all transporters, substrate translocation through DAT follows the alternating-access mechanism in which the protein swiches between outward-facing (OF) and inward-facing (IF) states. The details of these structural changes and their coupling to chemical events such as substrate and ion binding events remain elusive. In the present study, we charactrized large-scale transition from the OF state to IF state using all-atom molecular dynamics simulations of membrane-bound models of DAT. As all crystal structures of DAT are in the OF state, the initial phase of the study included modeling of a stable IF structure of DAT in the context of a membrane using the bacterial sodium-coupled leucine symporter (LeuT) as a template. Furthermore, equilibrium simulations performed in this phase revealed a novel sodium binding site located between TM3 and TM8 helices, which are elements involved in coupling of protein structural changes to substrate binding and translocation. Using the orientations of helics TM1e/TM8e and TM1i/TM8i as collective variables, and employing two dimensional bias- exchange umbrella sampling and string method with swarms of trajectories, we characerize a structural transition pathway between the OF and IF states of DAT. The results of this study provide a deeper understanding of the functional mechanism of DAT, wiht implications to all members of the NSS family. 634-Pos Board B399 Structural Insights into Sodium-Dependent Sugar Transporters and their Inhibition Mechanism Paola Bisignano 1 , Chakrapani Kalyanaraman 2 , Chiara Ghezzi 3 , Ernest M. Wright 3 , Jeff Abramson 3 , Aviv Paz 3 , Matthew P. Jacobson 2 , Rosmarie Friemann 4,5 , Michael Grabe 1 . 1 Cardiovascular Research Institute and Department of Pharmaceutical Chemistry, UCSF, San Francisco, CA, USA, 2 Department of Pharmaceutical Chemistry, UCSF, San Francisco, CA, USA, 3 Department of Physiology, UCLA, Los Angeles, CA, USA, 4 Department of Chemistry and Molecular Biology, University of Gothenburg, Go ¨teborg, Sweden, 5 Department of Structural Biology, Stanford, Stanford, CA, USA. Sodium-dependent glucose transporters (SGLTs) are members of the large solute carrier (SLC) family of proteins that exploit the sodium ion concentration gradient to transport a myriad of small molecules across the plasma membrane. In humans, there are six SGLT subtypes labeled 1-6 that are expressed widely in the small intestine, kidney, lung, muscle, and brain. Due to their role in sugar reabsorption, SGLTs are currently exploited as drug targets for the treatment of type 2 diabetes, especially hSGLT2, which is responsible for 98% of glucose reabsorption in the kidneys. Current inhibitors are chemical derivatives of the naturally occurring small molecule phlorizin, which is expressed in the bark of fruit trees, such as apple and pear. The structural basis of binding is not known, in part, because high-resolution structures of mammalian SGLTs do not exist. However, the inward-facing structure of the bacterial homologue from vibrio parahaemolyticus (vSGLT) has been solved both in apo and in complex with galactose, and our collaborators recently solved the outward-facing structure of a closely related homologue (unpub- lished). Here we combine homology modeling, virtual screening techniques and molecular dynamics simulations to achieve two goals: 1) model the outward-facing state of SGLTs, and 2) predict the binding mode of phlorizin and its derivatives hSGLT1 and 2. As a result, the spectroscopic data (double electron-electron resonance) probing outward facing state of SGLTs validate our homology model and mutagenesis studies testing binding to hSGLT1 and 2 are in agreement with our predicted binding modes. 635-Pos Board B400 Uptake Dynamics in the LacY Membrane Protein Transporter Dari Kimanius 1 , Stephen White 2 , Erik Lindahl 3 , Ronald Kaback 4 , Magnus Andersson 5 . 1 Stockholm University, Stockholm, Sweden, 2 University of California Irvine, Irvine, CA, USA, 3 KTH Royal Institute of Technology/Stockholm University, Stockholm, Sweden, 4 University of California Los Angeles, Los Angeles, CA, USA, 5 KTH Royal Institute of Technology, Stockholm, Sweden. Membrane protein transporters govern important cellular processes and are therefore central to human health. To accomplish transport, these proteins rearrange their structures to alternatively expose an internal binding site to either side of the membrane. Recent advances in protein structural determination methods have resulted in a steadily increasing number of high-resolution structures of membrane transporters trapped in different intermediate states. However, to understand the under- lying transport mechanism, the molecular details of uptake and release need to be determined. We have used specialized simulation hardware to simulate uptake of galactoside sugar into the Lactose permease (LacY) of Escherichia coli. The extended brute-force simulation revealed large-scale structural rearrangements, lipid and amino acid interactions, and hydration associated with sugar uptake. The free energy landscape of sugar entry was determined by parallel bias-exchange metadynamics simulations and identified a global free energy minimum that coincided with the crystallo- graphic binding site and also a local free energy minimum in the larger periplasmic cavity of LacY. Together, our observations show a putative molecular mechanism for sugar uptake in this prototype membrane transporter. 636-Pos Board B401 Elevator-Like Mechanism of Transport in the EIIC Glucose Superfamily of Transporters Zhenning Ren 1 , Yin Nian 1 , Jumin Lee 2 , Jason McCoy 3 , Wonpil Im 2 , Ming Zhou 1 . 1 Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA, 2 University of Kansas, Lawrence, KS, USA, 3 Broad Institute, Cambridge, MA, USA. The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is crucial for sugar uptake in bacteria. It has a membrane embedded compo- nent, EIIC, that translocates a sugar from the extracellular to the intracel- lular side of the cell. Before the sugar is released into the cytosol, a 128a Sunday, February 12, 2017