Structure and Dynamics of a Small Multidrug Resistance Transporter, EmrE

EmrE is a small multidrug resistance transporter in E. coli. It effluxes a wide range of antibiotics, thus contributing to the evolving epidemic of drug resistance. Despite its small size, EmrE is a fully functional transporter making it an ideal model system for a comprehensive study of the multidrug transport mechanism. In the transport cycle, EmrE must alternate between outward- and inward-facing conformations upon substrate binding to translocate substrates across the membrane. High-resolution structures of EmrE in complex with substrates facing different sides of the membrane will shed light on the coupling mechanism between substrate binding and transport. However, the conformational plasticity that enables EmrE to transport diverse drugs also makes it a very challenging system for high-resolution structural studies. The conformational dynamics inherent in the transport process require experimental measures of structural transitions to provide the link between static structures and functional transport. This thesis aims to characterize the structure and dynamics of EmrE in atomic detail using NMR, a well-established technique to study structure and dynamics of biomolecules simultaneously under a variety of conditions. In the case of EmrE, NMR spectroscopy is the best approach for high-resolution structures because the dynamic nature and small size of EmrE hamper X-ray crystallography and cryoEM approaches. I have made significant progress towards a better structure of EmrE using a slow-dynamics mutant and have achieved a near complete backbone and side chain ILV methyl assignment for this highly challenging helical membrane protein system. I have also collected a large data set of distance and orientational restraints. I have also used NMR and functional assays to characterize a series of mutants located near the transmembrane helix 3 (TM3) kink and have demonstrated the important role of TM3 kink formation for the global conformational interconversion required for alternating-access. My NMR data also suggest that hydration within the transport pore may be an important property fine-tuning the rates of conformational interconversion. My NMR pH titrations show that the slow-dynamics mutant also has elevated pKa values for E14, the critical residue for proton-coupling in EmrE. This provides the first experimental evidence of the physicochemical link between proton and substrate binding and alternating-access necessary for achieving coupled transport. By correlating high-resolution structural and dynamic data with functional transport assays, this thesis provides key insights into the multidrug transport mechanism of EmrE. The principles learned for EmrE set the stage for understanding even more challenging transporters

Structural Basis for Potentiation by Alcohols and Anaesthetics in a Ligand-gated Ion Channel

Ethanol alters nerve signalling by interacting with proteins in the central nervous system, particularly pentameric ligand-gated ion channels. A recent series of mutagenesis experiments on Gloeobacter violaceus ligand-gated ion channel, a prokaryotic member of this family, identified a single-site variant that is potentiated by pharmacologically relevant concentrations of ethanol. Here we determine crystal structures of the ethanol-sensitized variant in the absence and presence of ethanol and related modulators, which bind in a transmembrane cavity between channel subunits and may stabilize the open form of the channel. Structural and mutagenesis studies defined overlapping mechanisms of potentiation by alcohols and anaesthetics via the inter-subunit cavity. Furthermore, homology modelling show this cavity to be conserved in human ethanol-sensitive glycine and GABA(A) receptors, and to involve residues previously shown to influence alcohol and anaesthetic action on these proteins. These results suggest a common structural basis for ethanol potentiation of an important class of targets for neurological actions of ethanol

Target highlights in CASP14 : Analysis of models by structure providers

Abstract The biological and functional significance of selected CASP14 targets are described by the authors of the structures. The authors highlight the most relevant features of the target proteins and discuss how well these features were reproduced in the respective submitted predictions. The overall ability to predict three-dimensional structures of proteins has improved remarkably in CASP14, and many difficult targets were modelled with impressive accuracy. For the first time in the history of CASP, the experimentalists not only highlighted that computational models can accurately reproduce the most critical structural features observed in their targets, but also envisaged that models could serve as a guidance for further studies of biologically-relevant properties of proteins. This article is protected by copyright. All rights reserved.Peer reviewe

Spectroscopic tools for the study of voltage gated sodium channel structure, function and ligand binding

As the proteins responsible for the initiation and propagation of the action potential in excitable cells, voltage-gated sodium channels (NaVs) are crucial drug targets. Antiarrhythmics, anaesthetics, and antiepileptic drugs all target NaVs. Yet despite the difference in NaV subtypes affecting these diseases, the drugs used to treat them are able to act while showing little subtype specificity. This is explained by drugs displaying state-dependant binding and kinetics. Hence an understanding of the structural changes in different gating states of NaVs is vital for structure-based drug design, of more effective and selective compounds. To date there is no atomic-resolution structure of a functional eukaryotic NaV, although the structures of several prokaryotic NaVs which share pharmacological characteristics with their eukaryotic counterparts have been determined. However these structures are in variety of conformations and the relationships between them remain unclear. This study presents biophysical techniques complementary to structural ones to investigate the gating of NaVs, using the channel from Magnetococcus Marinus (NaVMs) as a case study. Oriented circular dichroism (oCD) spectroscopy is used to report on the orientation of the pore-lining α-helices within the membrane, based on quantitative analysis of oCD data. Single molecule FRET spectroscopy is able to measure interatomic distances across biological molecules, and here is used to investigate the changes in pore diameter at the gate of NaVMs. Also presented is a new web-based application which uses protein structure files to define the orientation of α-helices that comprise the channel

MED15 prion-like domain forms a coiled-coil responsible for its amyloid conversion and propagation

Altres ajuts: "la Caixa" Foundation i ICREA-Academia 2016A disordered to β-sheet transition was thought to drive the functional switch of Q/N-rich prions, similar to pathogenic amyloids. However, recent evidence indicates a critical role for coiled-coil (CC) regions within yeast prion domains in amyloid formation. We show that many human prion-like domains (PrLDs) contain CC regions that overlap with polyQ tracts. Most of the proteins bearing these domains are transcriptional coactivators, including the Mediator complex subunit 15 (MED15) involved in bridging enhancers and promoters. We demonstrate that the human MED15-PrLD forms homodimers in solution sustained by CC interactions and that it is this CC fold that mediates the transition towards a β-sheet amyloid state, its chemical or genetic disruption abolishing aggregation. As in functional yeast prions, a GFP globular domain adjacent to MED15-PrLD retains its structural integrity in the amyloid state. Expression of MED15-PrLD in human cells promotes the formation of cytoplasmic and perinuclear inclusions, kidnapping endogenous full-length MED15 to these aggregates in a prion-like manner. The prion-like properties of MED15 are conserved, suggesting novel mechanisms for the function and malfunction of this transcription coactivator