Secondary multidrug transporters use the energy stored in transmembrane ion gradients to bind and extrude a variety of weakly related chemical structures. These polyspecific antiporters challenge the notions of high-affinity conformation and strict ion-substrate coupling, inherent to the alternating-access model of transport. In order to investigate the mechanism of secondary multidrug transport at a molecular level, we study LmrP, a Major Facilitator Superfamily (MFS) multidrug transporter from Lactococcus lactis, which relies on the proton-motive force to achieve the transport of its diverse substrates. We carried out Double Electron Electron (DEER) distance measurements to elucidate the conformational dynamics underlying the transport cycle. We monitored the conformational response of a library of labeled double cysteine mutants to the presence of ligand(s) and proton(s). We investigated the role of the lipid environment by performing the measurements on mutants reconstituted in nanoscale soluble lipid bilayers (nanodiscs). During this work, we have demonstrated that the transporter oscillates between two main conformations, the outward-open and the inward-open. We have shown that the protonation of conserved acidic residues is the driving force of the conformational transition. The lipid bilayer modulates the equilibrium and allows the transition to occur at higher and more physiological pH values. By using specific lipid compositions, we observe that the lipid headgroup is crucial in the regulation of the conformational equilibrium. Based on our data, we propose a model of secondary multidrug transport wherein substrate binding initiates the transport cycle by catalysing proton entrance from the extracellular side. Subsequent protonation of membrane-embedded acidic residues triggers a cascade of conformational changes that results in substrate extrusion to the extracellular side and proton release in the cytosol. We suggest the opening and closing of the extracellular site is tightly regulated while the cytoplasmic side is more flexible. To our knowledge, this work provides the first direct structural evidence of the role of the lipids in the regulation of the conformational dynamics of a membrane transporter. / La surexpression de transporteurs capables d’expulser des molécules cytotoxiques est un mécanisme connu de résistance aux antibiotiques de la cellule bactérienne. Certains transporteurs ont développé la capacité de reconnaitre et d’expulser des substrats de structures diverses, donnant lieu à une résistance multidrogue de la part de leur hôte. Ces transporteurs multidrogues sont présents dans une variété de classes de protéines, distribués dans tous les règnes du vivant. Parmi celles - ci, la famille MFS (Major Facilitator Superfamily) comprend la majorité des transporteurs multidrogues activé par une source d’énergie secondaire, et jouent un rôle crucial dans la propagation de maladies nosocomiales d’origine bactérienne. Une meilleure compréhension des mécanismes fondamentaux du transport multidrogue secondaire est le prérequis indispensable à l’élaboration de thérapies adaptées. En particulier, une description détaillée des changements conformationnels impliqués dans le transport, et une identification des mécanismes moléculaires qui permettent de lier la source d’énergie au transport fait actuellement défaut. Afin de pallier ce manque, ce travail vise à étudier LmrP (Lactococcus lactis multidrug resistance Protein) un transporteur MFS qui confère à son hôte Lactococcus lactis la résistance à divers antibiotiques et agents cytotoxiques de structure et de charge variable. Cette extrusion active est alimentée par un cotransport énergétiquement favorable de protons. Nous avons étudié le mécanisme de transport de LmrP à l’échelle moléculaire en utilisant la technique spectroscopique Double Electron Electron Resonance (DEER), qui permet de mesurer des variations de distances à l’échelle nanométrique, idéale pour observer les mouvements intramoléculaires d’un transporteur MFS. Différents aspects moléculaires susceptibles de réguler le cycle de transport sont étudiés de façon indépendante et couplée :le rôle des protons, des différents substrats, et de l’environnement lipidique. Sur base de cette cartographie conformationnelle, un mécanisme de transport couplant tous les acteurs moléculaires est proposé :la liaison du proton à un motif d’acides aminés conservé constitue la base de la transition conformationnelle, les divers substrats ayant pour rôle de permettre aux protons d’accéder à ce motif. La compétition substrat-proton est la base du transport couplé. Notre travail a mis en évidence le rôle fondamental de l’environnement lipidique, qui module l’équilibre conformationnel du transporteur en interagissant avec un ou plusieurs motif(s) conservé(s). Par ailleurs, notre étude questionne le paradigme actuel de transport au sein de la famille MFS car elle démontre que les changements conformationnels globaux passent par des réarrangements locaux et coordonnés. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

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Yes / Protein quality control mechanisms are essential for cell health and involve delivery of proteins to specific cellular compartments for recycling or degradation. In particular, stray hydrophobic proteins are captured in the aqueous cytosol by a co-chaperone, the small glutamine-rich, tetratricopeptide repeat-containing protein alpha (SGTA), which facilitates the correct targeting of tail-anchored membrane proteins, as well as the sorting of membrane and secretory proteins that mislocalize to the cytosol and endoplasmic reticulum-associated degradation. Full-length SGTA has an unusual elongated dimeric structure that has, until now, evaded detailed structural analysis. The Cterminal region of SGTA plays a key role in binding a broad range of hydrophobic substrates, yet in contrast to the well-characterized N-terminal and TPR domains, there is a lack of structural information on the C-terminal domain. In this study, we present new insights into the conformation and organization of distinct domains of SGTA and show that the C-terminal domain possesses a conserved region essential for substrate processing in vivo. We show that the C-terminal domain region is characterized by α-helical propensity and an intrinsic ability to dimerize independently of the N-terminal domain. Based on the properties of different regions of SGTA that are revealed using cell biology, NMR, SAXS, Native MS, and EPR, we observe that its C-terminal domain can dimerize in the full-length protein and propose that this reflects a closed conformation of the substrate-binding domain. Our results provide novel insights into the structural complexity of SGTA and provide a new basis for mechanistic studies of substrate binding and release at the C-terminal region. / MRC New Investigator Research Grant: G0900936; BBSRC grants: BB/L006952/1 and BB/L006510/1; BBSRC grant: BB/N006267/1; Wellcome Trust Investigator Award in Science: 204957/Z/16/Z; BBSRC grant: BB/J014567/1

The Major Facilitator Superfamily groups a vast number of secondary transporters that import or export distinct substrates. Among these, multidrug antiporters constitute a peculiar class of transporters, both because of their multispecificity, recognizing structurally very diverse substrates, and because of their transport mechanism, that relies on bilayer-mediated extrusion of cytotoxic compounds. An accurate and detailed description of the conformational changes that underlie the transport cycle is still lacking and the structural basis for energetic coupling in these transporters has not been elucidated, with so far only limited crystallographic evidence available. We investigate the molecular basis of secondary multidrug transport with biochemical and biophysical studies on LmrP, a Major Facilitator Superfamily multidrug transporter from Lactococcus lactis. We used extensive continuous-wave electron paramagnetic resonance and double electron-electron resonance measurements on a library of spin-labeled LmrP mutants to uncover the conformational states involved in transport and to investigate how protons and ligands shift the equilibrium between conformers to enable transport. We find that the transporter switches between outward-open and outward-closed conformations depending on the protonation states of specific acidic residues forming a transmembrane protonation relay. We observe that substrate binding restricts the conformational freedom of LmrP and induces localized conformational changes. Our data allows to build a model of secondary multidrug transport wherein substrate binding initiates the transport cycle by opening the extracellular side to protons. Subsequent protonation of membrane-embedded acidic residues induces substrate release to the extracellular side and triggers a cascade of conformational changes that culminates in a proton release to the intracellular side. Parallel to this, we have optimized our purification and expression protocol in order to set up crystallization trials on LmrP. Through extensive screening and optimization of the lipidation state of LmrP, using ad hoc methods for sample preparation, we were able to obtain low-resolution diffracting crystals. By improving our lipidation technique and modifying the lipid composition we further improved crystal quality. Other factors such as ligand addition, the presence of secondary detergent and additives for controlling phase separation and nucleation were tested, paving the way to high resolution structure determination of LmrP. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

The human α-lactalbumin (α-LA) molten globule formed at low pH is a model for the study of protein folding intermediates. The molten globule lacks native-like side-chain interactions, resulting in a fluctuating ensemble of tertiary structures, characterisation of which has been precluded by severe line-broadening in NMR spectra and a lack of long-range NOEs. Paramagnetic relaxation enhancements (PREs) have been measured in a variant of α-LA in which all native cysteines have been mutated to alanine (all-Ala α-LA). Cysteine residues have been mutated into regions of interest and spin labelled with MTSL. These measurements have confirmed that all-Ala α-LA forms a compact molten globule. Transient, long-range interactions that are stabilising the compact fold have also been identified using PREs measured in urea-denatured states. This has identified several interactions formed by hydrophobic residues from both the α- and β-domain, which could be important for initiating and driving folding. The molten globule’s 3D topology has been probed by measuring long-range distances between MTSL pairs using Double Electron-Electron Resonance (DEER). Broad distance distributions have been identified between elements of secondary structure, indicative of a fluctuating but compact fold. By contrast, a narrower distance distribution has been measured within one of the major helices, indicative of native-like secondary structure. The surface accessibility of all-Ala α-LA and that of two other variants ([28-111] α-LA and 4SS α-LA) has been probed using solvent PREs obtained using TEMPOL, a paramagnetic co-solute. This has revealed differences in the solvent-exposure of hydrophobic residues due to the removal of disulphide bonds. This method has also identified buried hydrophobic residues that contribute to forming the molten globule’s stable, native-like core.

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