Membrane transporters studied by EPR spectroscopy: structure determination and elucidation of functional dynamics

During their mechanistic cycles membrane transporters often undergo extensive conformational changes, sampling a range of orientations, in order to complete their function. Such membrane transporters present somewhat of a challenge to conventional structural studies; indeed, crystallization of membrane-associated proteins sometimes require conditions that vary vastly from their native environments. Moreover, this technique currently only allows for visualization of single selected conformations during any one experiment. EPR spectroscopy is a magnetic resonance technique that offers a unique opportunity to study structural, environmental and dynamic properties of such proteins in their native membrane environments, as well as readily sampling their substrate-binding-induced dynamic conformational changes especially through complementary computational analyses. Here we present a review of recent studies that utilize a variety of EPR techniques in order to investigate both the structure and dynamics of a range of membrane transporters and associated proteins, focusing on both primary (ABC-type transporters) and secondary active transporters which were key interest areas of the late Professor Stephen Baldwin to whom this review is dedicated

Lipids modulate the conformational dynamics of a secondary multidrug transporter

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

Common activation mechanism of class A GPCRs.

Funder: Young Talent Program of ShanghaiClass A G-protein-coupled receptors (GPCRs) influence virtually every aspect of human physiology. Understanding receptor activation mechanism is critical for discovering novel therapeutics since about one-third of all marketed drugs target members of this family. GPCR activation is an allosteric process that couples agonist binding to G-protein recruitment, with the hallmark outward movement of transmembrane helix 6 (TM6). However, what leads to TM6 movement and the key residue level changes of this movement remain less well understood. Here, we report a framework to quantify conformational changes. By analyzing the conformational changes in 234 structures from 45 class A GPCRs, we discovered a common GPCR activation pathway comprising of 34 residue pairs and 35 residues. The pathway unifies previous findings into a common activation mechanism and strings together the scattered key motifs such as CWxP, DRY, Na+ pocket, NPxxY and PIF, thereby directly linking the bottom of ligand-binding pocket with G-protein coupling region. Site-directed mutagenesis experiments support this proposition and reveal that rational mutations of residues in this pathway can be used to obtain receptors that are constitutively active or inactive. The common activation pathway provides the mechanistic interpretation of constitutively activating, inactivating and disease mutations. As a module responsible for activation, the common pathway allows for decoupling of the evolution of the ligand binding site and G-protein-binding region. Such an architecture might have facilitated GPCRs to emerge as a highly successful family of proteins for signal transduction in nature

Multidrug efflux pumps:structure, function and regulation

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

Molecular basis of secondary multidrug transport

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 Sciencesinfo:eu-repo/semantics/nonPublishe

Allosteric regulation of G protein-coupled receptor activity by phospholipids.

Lipids are emerging as key regulators of membrane protein structure and activity. These effects can be attributed either to the modification of bilayer properties (thickness, curvature and surface tension) or to the binding of specific lipids to the protein surface. For G protein-coupled receptors (GPCRs), the effects of phospholipids on receptor structure and activity remain poorly understood. Here we reconstituted purified β2-adrenergic receptor (β2R) in high-density lipoparticles to systematically characterize the effect of biologically relevant phospholipids on receptor activity. We observed that the lipid headgroup type affected ligand binding (agonist and antagonist) and receptor activation. Specifically, phosphatidylgycerol markedly favored agonist binding and facilitated receptor activation, whereas phosphatidylethanolamine favored antagonist binding and stabilized the inactive state of the receptor. We then showed that these effects could be recapitulated with detergent-solubilized lipids, demonstrating that the functional modulation occurred in the absence of a bilayer. Our data suggest that phospholipids act as direct allosteric modulators of GPCR activity.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

AlphaFold2 predicts the inward‐facing conformation of the multidrug transporter LmrP

As part of the CASP competition, the protein structure prediction algorithm AlphaFold2 generated multiple models of the proton/drug antiporter LmrP. Previous distance restraints from double electron-electron resonance (DEER) spectroscopy, a technique which reports distance distributions between spin labels attached to proteins, suggest that one of the lower-ranked models may have captured a conformation that has so far eluded experimental structure determination