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Principles Of Fluorescence Spectroscopy

Infections arising from multidrug-resistant pathogenic bacteria are spreading rapidly throughout the world and threaten to become untreatable. The origins of resistance are numerous and complex, but one underlying factor is the capacity of bacteria to rapidly export drugs through the intrinsic activity of efflux pumps. In this Review, we describe recent advances that have increased our understanding of the structures and molecular mechanisms of multidrug efflux pumps in bacteria. Clinical and laboratory data indicate that efflux pumps function not only in the drug extrusion process but also in virulence and the adaptive responses that contribute to antimicrobial resistance during infection. The emerging picture of the structure, function and regulation of efflux pumps suggests opportunities for countering their activities.

/pdf/multidrug-efflux-pumps-structure-function-and-regulation-3y0335ts48.pdf

Multidrug efflux pumps: structure, function and regulation

Membrane proteins play a most important part in metabolism, signaling, cell motility, transport, development, and many other biochemical and biophysical processes which constitute fundamentals of life on the molecular level. Detailed understanding of these processes is necessary for the progress of life sciences and biomedical applications. Nanodiscs provide a new and powerful tool for a broad spectrum of biochemical and biophysical studies of membrane proteins and are commonly acknowledged as an optimal membrane mimetic system that provides control over size, composition, and specific functional modifications on the nanometer scale. In this review we attempted to combine a comprehensive list of various applications of nanodisc technology with systematic analysis of the most attractive features of this system and advantages provided by nanodiscs for structural and mechanistic studies of membrane proteins.

/pdf/nanodiscs-in-membrane-biochemistry-and-biophysics-7gg437700d.pdf

Nanodiscs in Membrane Biochemistry and Biophysics

The major facilitator superfamily (MFS) is the largest known superfamily of secondary active transporters. MFS transporters are responsible for transporting a broad spectrum of substrates, either down their concentration gradient or uphill using the energy stored in the electrochemical gradients. Over the last 10 years, more than a hundred different MFS transporter structures covering close to 40 members have provided an atomic framework for piecing together the molecular basis of their transport cycles. Here, we summarize the remarkable promiscuity of MFS members in terms of substrate recognition and proton coupling as well as the intricate gating mechanisms undergone in achieving substrate translocation. We outline studies that show how residues far from the substrate binding site can be just as important for fine-tuning substrate recognition and specificity as those residues directly coordinating the substrate, and how a number of MFS transporters have evolved to form unique complexes with chaperone and signaling functions. Through a deeper mechanistic description of glucose (GLUT) transporters and multidrug resistance (MDR) antiporters, we outline novel refinements to the rocker-switch alternating-access model, such as a latch mechanism for proton-coupled monosaccharide transport. We emphasize that a full understanding of transport requires an elucidation of MFS transporter dynamics, energy landscapes, and the determination of how rate transitions are modulated by lipids.

Structures and General Transport Mechanisms by the Major Facilitator Superfamily (MFS).

Secondary transporters undergo structural rearrangements to catalyze substrate translocation across the cell membrane - yet how such conformational changes happen within a lipid environment remains poorly understood. Here, we combine hydrogen-deuterium exchange mass spectrometry (HDX-MS) with molecular dynamics (MD) simulations to understand how lipids regulate the conformational dynamics of secondary transporters at the molecular level. Using the homologous transporters XylE, LacY and GlpT from Escherichia coli as model systems, we discover that conserved networks of charged residues act as molecular switches that drive the conformational transition between different states. We reveal that these molecular switches are regulated by interactions with surrounding phospholipids and show that phosphatidylethanolamine interferes with the formation of the conserved networks and favors an inward-facing state. Overall, this work provides insights into the importance of lipids in shaping the conformational landscape of an important class of transporters.

/pdf/direct-protein-lipid-interactions-shape-the-conformational-3c3b1zy5k8.pdf

Direct protein-lipid interactions shape the conformational landscape of secondary transporters

Direct interactions with lipids have emerged as key determinants of the folding, structure and function of membrane proteins, but an understanding of how lipids modulate protein dynamics is still lacking. Here, we systematically explored the effects of lipids on the conformational dynamics of the proton-powered multidrug transporter LmrP from Lactococcus lactis, using the pattern of distances between spin-label pairs previously shown to report on alternating access of the protein. We uncovered, at the molecular level, how the lipid headgroups shape the conformational-energy landscape of the transporter. The model emerging from our data suggests a direct interaction between lipid headgroups and a conserved motif of charged residues that control the conformational equilibrium through an interplay of electrostatic interactions within the protein. Together, our data lay the foundation for a comprehensive model of secondary multidrug transport in lipid bilayers.

/pdf/lipids-modulate-the-conformational-dynamics-of-a-secondary-2nyqnj81mk.pdf

Lipids modulate the conformational dynamics of a secondary multidrug transporter

LmrP from Lactoccoccus lactis: a tractable model to understand secondary multidrug transport in MFS.

The Major Facilitator Superfamily (MFS) is the largest family of secondary active membrane transporters and is found in all domains of Life. Their transport cycle mechanism involves conformational changes between outward- and inward-open states. Yet, the kinetic details of MFS proteins have been largely missing. To investigate MFS transporters in real-time, we studied the multidrug efflux pump LmrP as a model system for antibiotic resistance development in bacteria. While it is well-known that the conformational cycle of LmrP is triggered by the protonation of a network of specific amino acids, the role of the transported substrate in these transitions is still puzzling. For a robust readout of the real-time structure of LmrP, we performed solution-based single-molecule Foerster resonance energy transfer (smFRET) using a confocal microscope with direct alternating donor/acceptor excitation and multiparameter (intensity, lifetime, anisotropy) detection. Lowering pH from 8 to 5 triggered an overall conformational transition, corroborating that detergent solubilization allows studying the LmrP transport cycle using smFRET. Using a newly developed linear 3-state photon distribution analysis (PDA) model, we show that the apo protein interconverted between two structures at low rate (>>10 ms dwell time) at the cytosolic side while it interconverts dynamically between the 3 states (

https://www.biorxiv.org/content/biorxiv/early/2020/04/11/2020.04.09.034439.full.pdf

Aurélie Roth

Papers

Lipids modulate the conformational dynamics of a secondary multidrug transporter

LmrP from Lactoccoccus lactis: a tractable model to understand secondary multidrug transport in MFS.

Substrate binding modulates the conformational kinetics of the secondary multidrug transporter LmrP