Adequate membrane fluidity is required for a variety of key cellular processes and in particular for proper function of membrane proteins. In most eukaryotic cells, membrane fluidity is known to be ...regulated by fatty acid desaturation and cholesterol, although some cells, such as insect cells, are almost devoid of sterol synthesis. We show here that insect and mammalian cells present similar microviscosity at their respective physiological temperature. To investigate how both sterols and phospholipids control fluidity homeostasis, we quantified the lipidic composition of insect SF9 and mammalian HEK 293T cells under normal or sterol-modified condition. As expected, insect cells show minimal sterols compared with mammalian cells. A major difference is also observed in phospholipid content as the ratio of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) is inverted (4 times higher in SF9 cells). In vitro studies in liposomes confirm that both cholesterol and PE can increase rigidity of the bilayer, suggesting that both can be used by cells to maintain membrane fluidity. We then show that exogenously increasing the cholesterol amount in SF9 membranes leads to a significant decrease in PE:PC ratio whereas decreasing cholesterol in HEK 293T cells using statin treatment leads to an increase in the PE:PC ratio. In all cases, the membrane fluidity is maintained, indicating that both cell types combine regulation by sterols and phospholipids to control proper membrane fluidity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
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 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.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Multidrug efflux pumps present a challenge to the treatment of bacterial infections, making it vitally important to understand their mechanism of action. Here, we investigate the nature of substrate ...binding within Lactococcus lactis LmrP, a prototypical multidrug transporter of the major facilitator superfamily. We determined the crystal structure of LmrP in a ligand-bound outward-open state and observed an embedded lipid in the binding cavity of LmrP, an observation supported by native mass spectrometry analyses. Molecular dynamics simulations suggest that the anionic lipid stabilizes the observed ligand-bound structure. Mutants engineered to disrupt binding of the embedded lipid display reduced transport of some, but not all, antibiotic substrates. Our results suggest that a lipid within the binding cavity could provide a malleable hydrophobic component that allows adaptation to the presence of different substrates, helping to explain the broad specificity of this protein and possibly other multidrug transporters.
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IJS, NUK, SBMB, UL, UM, UPUK
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.
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IJS, NUK, SBMB, UL, UM, UPUK
In the past few years there has been a growth in the use of nanoparticles for stabilizing lipid membranes that contain embedded proteins. These bionanoparticles provide a solution to the challenging ...problem of membrane protein isolation by maintaining a lipid bilayer essential to protein integrity and activity. We have previously described the use of an amphipathic polymer （poly（styrene-co-maleic add）, SMA） to produce discoidal nanoparticles with a lipid bilayer core containing the embedded protein. However the structure of the nanoparticle itself has not yet been determined. This leaves a major gap in understanding how the SMA stabilizes the encapsulated bilayer and how the bilayer relates physically and structurally to an unencapsulated lipid bilayer. In this paper we address this issue by describing the structure of the SMA lipid particle （SMALP） using data from small angle neutron scattering （SANS）, electron microscopy （EM）, attenuated total reflection Fourier transform infrared spectroscopy （ATR-FTIR）, differential scanning calorimetry （DSC） and nuclear magnetic resonance spectroscopy （NMR）. We show that the particle is disc shaped containing a polymer ＂bracelet＂ encircling the lipid bilayer. The structure and orientation of the individual components within the bilayer and polymer are determined showing that styrene moieties within SMA intercalate between the lipid acyl chains. The dimensions of the encapsulated bilayer are also determined and match those measured for a natural membrane. Taken together, the description of the structure of the SMALP forms the foundation for future development and applications of SMALPs in membrane protein production and analysis.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
CCR5 was first characterized as a receptor for MIP-1, MIP-1β, and RANTES, and was rapidly shown to be the main coreceptor for M-tropic human immunodeficiency virus (HIV)-1 strains and simian ...immunodeficiency virus (SIV). Chemokines constitute a rapidly growing family of proteins and receptor-chemokine interactions are known to be promiscuous and redundant. We have therefore tested whether other CC-chemokines could bind to and activate CCR5. All CC-chemokines currently available were tested for their ability to compete with 125I-MIP-1β binding on a stable cell line expressing recombinant CCR5, and/or to induce a functional response in these cells. We found that in addition to MIP-1β, MIP-1, and RANTES, five other CC-chemokines could compete for 125I-MIP-1β binding: MCP-2, MCP-3, MCP-4, MCP-1, and eotaxin binding was characterized by IC50 values of 0.22, 2.14, 5.89, 29.9, and 21.7 nmol/L, respectively. Among these ligands, MCP-3 had the remarkable property of binding CCR5 with high affinity without eliciting a functional response, MCP-3 could also inhibit the activation of CCR5 by MIP-1β and may therefore be considered as a natural antagonist for CCR5. It was unable to induce significant endocytosis of the receptor. Chemokines that could compete with high affinity for MIP-1β binding could also compete for monomeric gp120 binding, although with variable potencies; maximal gp120 binding inhibition was 80% for MCP-2, but only 30% for MIP-1β. MCP-3 could compete efficiently for gp120 binding but was, however, found to be a weak inhibitor of HIV infection, probably as a consequence of its inability to downregulate the receptor.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Studies using low-resolution fiber diffraction, electron microscopy, and atomic force microscopy on various amyloid fibrils indicate that the misfolded conformers must be modular, compact, and adopt ...a cross-β structure. In an earlier study, we used electron crystallography to delineate molecular models of the N-terminally truncated, disease-causing isoform ( PrPSc) of the prion protein, designated PrP 27-30, which polymerizes into amyloid fibrils, but we were unable to choose between a trimeric or hexameric arrangement of right- or left-handed β-helical models. From a study of 119 all-β folds observed in globular proteins, we have now determined that, if PrPScfollows a known protein fold, it adopts either a β-sandwich or parallel β-helical architecture. With increasing evidence arguing for parallel β-sheet organization in amyloids, we contend that the sequence of PrP is compatible with a parallel left-handed β-helical fold. Left-handed β-helices readily form trimers, providing a natural template for a trimeric model of PrPSc. This trimeric model accommodates the PrP sequence from residues 89-175 in a β-helical conformation with the C terminus (residues 176-227), retaining the disulfide-linked α-helical conformation observed in the normal cellular isoform. In addition, the proposed model matches the structural constraints of the PrP 27-30 crystals, positioning residues 141-176 and the N-linked sugars appropriately. Our parallel left-handed β-helical model provides a coherent framework that is consistent with many structural, biochemical, immunological, and propagation features of prions. Moreover, the parallel left-handed β-helical model for PrPScmay provide important clues to the structure of filaments found in some other neurodegenerative diseases.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is essential to maintain fluid homeostasis in key organs. Functional impairment of CFTR due to mutations in the cftr gene ...leads to cystic fibrosis. Here, we show that the first nucleotide-binding domain (NBD1) of CFTR can spontaneously adopt an alternate conformation that departs from the canonical NBD fold previously observed. Crystallography reveals that this conformation involves a topological reorganization of NBD1. Single-molecule fluorescence resonance energy transfer microscopy shows that the equilibrium between the conformations is regulated by adenosine triphosphate binding. However, under destabilizing conditions, such as the disease-causing mutation F508del, this conformational flexibility enables unfolding of the β-subdomain. Our data indicate that, in wild-type CFTR, this conformational transition of NBD1 regulates channel function, but, in the presence of the F508del mutation, it allows domain misfolding and subsequent protein degradation. Our work provides a framework to design conformation-specific therapeutics to prevent noxious transitions.The cystic fibrosis transmembrane conductance regulator anion channel can adopt an alternate conformation of its nucleotide-binding domain, which affects channel activity and, under certain conditions, leads to unfolding and protein degradation.
Clones encoding a new human P2Y receptor, provisionally called P2Y11, have been isolated from human placenta complementary DNA and genomic DNA libraries. The 1113-base pair open reading frame is ...interrupted by one intron. The P2Y11 receptor is characterized by considerably larger second and third extracellular loops than the subtypes described so far. The deduced amino acid sequence exhibits 33% amino acid identity with the P2Y1 receptor, its closest homolog. Northern blot analysis detected human P2Y11 receptor messenger RNA in spleen and HL-60 cells. The level of P2Y11 transcripts was strongly increased in these cells after granulocyte differentiation induced by retinoic acid or dimethyl sulfoxide. The new receptor was stably expressed in 1321N1 astrocytoma and CHO-K1 cells, where it couples to the stimulation of both the phosphoinositide and adenylyl cyclase pathways, a unique feature among the P2Y family. The rank order of agonists potency was: ATP > 2-methylthio-ATP >>> ADP, whereas UTP and UDP were inactive, indicating that it behaves as a selective purinoceptor.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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