Feb 13, 2017 - mechanism and the free energy profile associated to the full ... Sodium-calcium exchanger (NCX) transporters play a critical role in a variety.
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Monday, February 13, 2017
Transporters and Exchangers II 1339-Pos Board B407 Molecular Rationale Behind the Differential Substrate Specificity of RND Transporters AcrB and AcrD Venkata Krishnan Ramaswamy, Giuliano Malloci, Attilio Vittorio Vargiu, Paolo Ruggerone. Department of Physics, University of Cagliari, Monserrato, Italy. Resistance-Nodulation-Division (RND) transporters AcrB and AcrD in E. coli, recognize and expel into the medium a wide range of substrates ranging from lipophilic to amphiphilic molecules, contributing to the onset of bacterial multidrug resistance (MDR). Despite sharing an overall sequence identity of nearly 66% (similarity of nearly 80%), these Acr pumps feature distinct substrate specificity patterns whose underlying basis still remains elusive. In an attempt to understand the molecular basis responsible for this behavior, we performed a comparative analysis of multi-copy ms-long MD simulations of the apo-forms of the two transporters AcrB and AcrD, focusing on substrate binding and transport pathways. Our results would be informative to new drug design attempts to correlate the different specificity patterns of these two transporters to the physicochemical as well as topographical properties calculated on (or projected onto) the molecular surface of their multifunctional recognition sites. 1340-Pos Board B408 Molecular Insights into Compound-Transporter Interactions: The Case of Inhibitors of Gram-Negative Bacteria Efflux Pumps Hanno Sjuts1, Attilio V. Vargiu2, Steven M. Kwasny3, Son T. Nguyen3, Hong-Suk Kim4, Xiaoyuan Ding3, Alina R. Ornik1, Paolo Ruggerone2, Terry L. Bowlin5, Hiroshi Nikaido6, Klaas M. Pos1, Timothy J. Opperman3. 1 Institute of Biochemistry, Goethe-University, Frankfurt, Germany, 2Physics, Universita’ di Cagliari, Monserrato (CA), Italy, 3Department of Molecular and Cellular Biology, Microbiotix Inc., Worcester, MA, USA, 4University of California, Berkeley, MA, USA, 5Institute of Biochemistry, Microbiotix Inc., Worcester, MA, USA, 6Physics, University of California, Berkeley, CA, USA. The Escherichia coli AcrAB-TolC efflux pump is the archetype of the Resistance-Nodulation-cell Division (RND) exporters from Gram-negative bacteria. Overexpression of RND-type efflux pumps is a major factor in multidrug resistance (MDR), which makes these polyspecific pumps important antibacterial drug discovery targets. However, the development of potent efflux pump inhibitors has been hindered by the lack of structural information for rational drug design. Here, we describe the molecular basis for pyranopyridine-based inhibition of AcrB, which is responsible for the recognition and the initial extrusion of substrates, using a combination of cellular, X-ray crystallographic, and molecular dynamics simulations studies. We found that the pyranopyridines bind within a phenylalanine-rich cage that branches from the deep binding pocket of AcrB, where they form extensive hydrophobic interactions. Moreover, the increasing potency of improved inhibitors correlates with the formation of a delicate protein- and water-mediated hydrogen bond network. These detailed insights favour a deep understanding of the molecular interactions of compounds with AcrB and provide a molecular platform for the development of novel combinational therapies using efflux pump inhibitors for combating multidrug resistant Gram-negative pathogens. 1341-Pos Board B409 Computational Study of the Interaction between Antimicrobial Compounds and Efflux Systems of Gram-Negative Bacteria Alessio Atzori, Attilio Vittorio Vargiu, Giuliano Malloci, Paolo Ruggerone. Department of Physics, Universita´ degli Studi di Cagliari, Cagliari, Italy. Nowadays, multidrug resistance in Gram-negative bacteria is a major issue for public health. The efflux pumps of the resistance nodulation division (RND) family contribute largely to this phenomenon. A well-known example is represented by the AcrAB-TolC efflux system of E. coli, in which the inner membrane translocase AcrB is the main responsible for the uptake and extrusion of different substrates. Due to the limited availability of crystallographic structures of AcrB-substrate complexes, computational methods represent an alternative approach to elucidate the nature of interactions between diverse antimicrobial compounds and the efflux protein. Here, two antibiotics known for their different affinity for the efflux pump in P. aeruginosa, namely meropenem and imipenem (respectively, strongly and poorly affected by MexB, the homologous of AcrB in P. aeruginosa), were chosen for this study. A similar behaviour for the two antibiotics has not been yet reported in the literature for the AcrB of E. coli. In this study we first performed a preliminary docking investigation using ensemble of conformations extracted from molecular dynamics simulations
of both ligands and AcrB structures. The top poses of ligands were selected as initial conformations for molecular dynamics simulations. Results from MD simulations evidence a larger propensity of meropenem than imipenem to bind to the putative affinity sites of AcrB, in analogy to what observed for MexB. Moreover, a detailed analysis of the molecular interactions of the two compounds with AcrB allowed identifying the key residues involved in the extrusion/retention processes. Our results provide information that could help in designing new antibiotics less likely to be extruded by RND efflux pumps, as well as inhibitors more effective in blocking the whole efflux process. 1342-Pos Board B410 Transport Mechanism in the RND Transporter AcrD of E. coli Attilio V. Vargiu, Venkata R. Krishnan, Giuliano Malloci, Paolo Ruggerone. Physics, University of Cagliari, Monserrato, Italy. The RND efflux pump AcrAB-TolC is involved in antibiotic resistance in E. coli [1-2]. The core of this machinery is the proton-gradient-driven antiporter AcrB, a homotrimeric protein with an unreached ability to recognize antibiotics belonging to many different families. On the basis of available experimental data, a functional rotation mechanism has been hypothesized for this transporter, in which recognition and expulsion of substrates are coupled to concerted conformational changes occurring in each monomer of AcrB. While several computational studies have been performed aiming to confirm and detail this mechanism [3-5], no study addressed so far the energetics associated to the whole translocation process. In this work, we propose a new computational protocol to smoothly couple large-scale conformational changes of AcrB to full translocation of the substrate doxorubicin from the distal pocket of monomer B to the funnel domain. Our method allows characterizing the mechanism and the free energy profile associated to the full translocation of doxorubicin within AcrB. We show that translocation occurs via a full and cost-free rotation of the drug before it enters the putative Gate to the proximal region of AcrB. Moreover, the functional rotation mechanism crucially lowers the main free energy barrier associated with extrusion with respect to the same process with the protein in equilibrium. The analysis of different drug-protein interactions and of waters in assisting the process is detailed. [1] X-Z. Li et al., Clin. Microbiol. Rev., 2015, 28, 337. [2] P. Ruggerone et al., Curr. Top. Med. Chem., 2013, 13, 3079. [3] A.V. Vargiu et al., J. Am. Chem. Soc., 2011, 133, 10704. [4] R. Schulz et al., PLoS Comput. Biol., 2010, 6(6): e1000806. [5] Z. Zuo et al.,J. Phys. Chem. B, 2016, 120, 2145. 1343-Pos Board B411 Membrane Transport of Guanidium Ion Ali A. Kermani, Randy Stockbridge. Biophysics, University of Michigan, Ann Arbor, MI, USA. Guanidine, the functional group of the arginine sidechain, is a metabolic byproduct that is toxic to bacteria if it accumulates in the cytoplasm. We identified a new class of bacterial membrane proteins that export guanidium ion. These proteins are selective for guanidium over other compounds with guanidinyl groups. Using radioactive uptake assay, tryptophan fluorescence binding assays, and ion selective electrodes, we characterize guanidinium selectivity, co-transported ions, and basic transport characteristics. 1344-Pos Board B412 Characterization of Sodium-Calcium Exchanger NCX_Mj using Fluorescent Indicators Irina Shlosman1, Jose´ Faraldo-Go´mez2, Joseph Mindell1. 1 NIH/NINDS, Bethesda, MD, USA, 2NIH/NHLBI, Bethesda, MD, USA. What the flux? Sodium-calcium exchanger (NCX) transporters play a critical role in a variety of physiological processes involved in calcium signaling. In heart muscle, sodium calcium exchangers regulate contractility by extruding calcium during ventricular myocyte depolarization. Determination of key thermodynamic and kinetic parameters for these transporters would offer invaluable insight into their mechanism of action as well as provide tools for therapy. Here we present a methodology to study sodium-calcium exchangers in an in vitro reconstituted system with the use of fluorescent calcium indicators. This versatile system offers several advantages over the traditional method of employing radiolabeled substrate. Complete control over the experimental set-up is achieved through bidirectional flux, high sensitivity of the dyes and real-time tracking of changes in calcium concentration. We validate and test this approach by characterizing one of the members of the NCX family, an archaeal transporter from Methanocaldoccus jannaschii (NCX_Mj). NCX_Mj, for which structural data exist, can thus serve as a model system for characterization of its mammalian analogs, providing insight into the interplay between transporter structure and mechanism.
