Unique Insights into the Structural and Functional Biology of ...

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Feb 19, 2018 - USA, 3Institute of Molecular Biophysics, Florida State University,. Tallahassee, FL, USA, 4City University of New York, New York, NY, USA,.
Monday, February 19, 2018 drug efflux as a homodimer by coupling transport with the electrochemical potential across the inner membrane of E. coli. While it is known that a conserved glutamate residue (Glu-14) within the first transmembrane domain of EmrE plays a central role in transport, the mechanistic details concerning how this anionic residue couples efflux with the pH gradient are not fully understood due to the lack of a high-resolution view of the substrate binding pocket. Our experiments use a combination of solution and solid-state NMR spectroscopy to uncover the conformational dynamics and allosteric communication within EmrE that underlie the ion-coupled mechanism. In addition, we offer highresolution structural insight of EmrE’s binding pocket in lipid bilayers with the goal of defining the multidrug recognition mechanism. 1043-Symp Unique Insights into the Structural and Functional Biology of Membrane Proteins from Solid State NMR Spectroscopy Timothy Cross1, Joana Paulino1, Huajun Qin2, Yiseul Shin2, Cristian Escobar3, Rongfu Zhang2, Joshua Taylor2, Yimin Miao4, Riqiang Fu1, Eduard Chekmenev5, Ivan Hung1, Zhehong Gan1, Petr Gor’kov1. 1 Natl High Mag Field Lab, Florida State University, Tallahassee, FL, USA, 2 Chemistry and Biochemistry, Florida State University, Tallahassee, FL, USA, 3Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, USA, 4City University of New York, New York, NY, USA, 5 Vanderbilt University, Nashville, TN, USA. The Influenza A M2 protein is an extensively studied tetrameric membrane protein whose structure in liquid crystalline lipid bilayers from solid state NMR (ssNMR) has been the basis for detailing the proton conductance mechanism, and has illuminated the role of M2 in the viral budding process, where it takes advantage of cholesterol. Specific proton histidyl proton exchange rates with water have been measured distinguishing futile exchange in which the proton binds and dissociates from the same site versus protonation of one site and deprotonation of another that could lead to proton conductance. CrgA is a protein associated with the cell division apparatus of Mycobacterium tuberculosis. The two helix transmembrane (TM) structure and orientation in liquid crystalline lipid bilayers has been characterized by ssNMR. The N-terminus is largely disordered except for a short sequence of b-strand close to the initiation of the first TM helix. CrgA represents the first example of a bstrand stabilized helical membrane protein dimer. At very high field ssNMR of 17O has greatly enhanced sensitivity and resolution. While the high resolution structure of gramicidin A (gA) was the first peptide or protein characterized in a liquid crystalline lipid bilayer 25 years ago, the dimer was shown to ‘perfectly’ symmetric. Not so, now that we have high resolution 17O data obtained 35.2 T (1500 MHz for 1H) that can characterize the sites water and cation binding. Single carbonyl 17O labeled gA associated with the cation binding site clearly shows two resonances (one from each monomer even in the absence of cations suggesting that the water interactions at one binding site influences the water interactions in the other monomer binding site ˚ away. 20A

Platform: Molecular Dynamics I 1044-Plat Mechanism of Substrate Translocation in an Alternating Access Transporter Naomi R. Latorraca, Nathan M. Fastman, Liang Feng, Ron O. Dror. Stanford University, Stanford, CA, USA. Transporters shuttle molecules across cell membranes by alternating among distinct conformational states. Despite a wealth of structural data supporting the existence of such states, a critical challenge in the field is to capture transitions from one conformational state to another, providing insights into how structural rearrangements regulate substrate translocation. Molecular dynamics simulations represent a powerful means for observing such transitions. However, many commonly studied transporters, including members of the GLUT and SGLT families, are large (> 50 kDa) and may transport more than one substrate, making their spontaneous conformational changes difficult to observe with unguided simulation. Here, we performed over 175 ms of unguided molecular dynamics simulations of SemiSWEET, a minimal, model transporter, to capture the substrate translocation process, providing an atomic-level description of alternating access transport. Simulations of this transporter initiated from an outward-open, glucose-bound structure spontaneously adopt occluded and inward-open conformations (Latorraca, Fastman et al., Cell 2017). Strikingly, both of these simulated conformations match existing crystal structures, including a new inward-open structure. Our results reveal that state transitions are driven by favorable interactions formed upon closure of extracellular and intracellular ‘‘gates’’ and by an unfavorable configuration of transmembrane

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helices when both gates are closed simultaneously. This mechanism leads to tight allosteric coupling between gates, preventing them from opening simultaneously. Interestingly, the substrate appears to take a ‘‘free ride’’ across the membrane without causing major structural rearrangements in the transporter. 1045-Plat Neurotransmitter Transporter Conformational Dynamics using HDX-MS and Molecular Dynamics Simulation Richard T. Bradshaw1, Anu Nagarajan1, Suraj Adhikary2, Daniel J. Deredge3, Patrick L. Wintrode3, Satinder K. Singh2, Lucy R. Forrest1. 1 NINDS, National Institutes of Health, Bethesda, MD, USA, 2Department of Cellular and Molecular Physiology, School of Medicine, Yale University, New Haven, CT, USA, 3Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD, USA. Experimental observations of protein dynamics frequently make use of protein variants with suitably located covalent labels. For mammalian neurotransmitter transporters, however, the difficulty in constructing and functionally verifying these mutant systems has hampered such investigations. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) is an attractive method for investigating dynamics of fully active transporters, reconstituted into a realistic membrane lipid environment, without such modifications. An atomistic rationale for HDX-MS observations can be provided by complementary computational predictions of deuteration rates. Nevertheless, although successfully applied previously to globular proteins in aqueous solvent, it remains unclear whether these computational approaches are transferable to large transporter proteins in the diverse membrane environment. Here we have used HDX-MS to measure backbone deuteration of the bacterial amino acid transporter LeuT as a model membrane protein system. HDX-MS experiments and molecular dynamics (MD) simulations compared LeuT in two distinct conformational ensembles, representing extracellular- or intracellular-open states in the transport cycle. Simulations, analyzed with an empirical model for estimating deuteration rates from the accessibility of backbone amide groups, have shown good agreement with the experimental deuteration data, and highlight areas of divergence between the outward and inward-facing ensembles (Adhikary et al., PNAS, 2017, 114, E1786). Despite the overall good agreement, some discrepancies were observed between predicted & experimental deuteration rates for the TM1a and EL4 protein segments. These were investigated in detail to identify deficiencies in either the (empirically parameterized) deuteration rate model, or the sampling accessible during our MD timescales. Our results emphasize the potential of HDX-MS complemented with simulations as a strategy for probing conformational dynamics of membrane proteins and highlight improved strategies for applying this approach to clinically relevant mammalian neurotransmitter transporters, such as SERT, DAT or NET. 1046-Plat Transport Pathways in Membrane Transporters Sayane Shome1, Edward Yu2, Robert Jernigan3. 1 Bioinformatics and Computational Biology, Iowa State University, Ames, IA, USA, 2Department of Pharmacology, Case Western Reserve University, Cleveland, OH, USA, 3Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA, USA. Substrate transport through membrane transporters is critical for many biological processes. One of the most interesting questions is how to understand the substrate specificity of transporters. Due to the limitations of experimental methods, computational approaches can be applied advantageously to screen a large number of possible transported molecules. The experimental determination of the mechanistic details of transport is difficult. We have employed steered molecular dynamics simulations to determine the critical factors responsible for the transport and how they interact with protein components along the pathway. Systems that we have investigated include the transport of: 1) sulfonamide drugs by the AbgT transporter YdaH protein, 2) inorganic carbon (CO2 and bicarbonate ion) by the Low CO2 inducible protein Lci1 and 3) long-chain cyclic lipids (Hopanoid and Steroid) by the RND-like HpnN protein. VMD software has been used. Protein-embedded lipid systems were minimized for 250,000 steps, followed by equilibration until the system temperature reaches 310 K. Steered Molecular dynamics simulations at constant velocity (cv-SMD) were carried out on the equilibrated systems via NAMD, with the direction and magnitude of the pulling being adjusted for the system of interest. Based on the simulation trajectories, we have determined the transport pathway through the protein for the ligand transport in these three cases, as well as the roles of functionally important residues along the pathway. These pathways have been confirmed by experimental findings.