13 research outputs found
Antiparallel Dimers of the Small Multidrug Resistance Protein EmrE Are More Stable Than Parallel Dimers
The bacterial multidrug transporter EmrE is a dual-topology membrane protein and as such is able to insert into the membrane in two opposite orientations. The functional form of EmrE is a homodimer; however, the relative orientation of the subunits in the dimer is under debate. Using EmrE variants with fixed, opposite orientations in the membrane, we now show that, although the proteins are able to form parallel dimers, an antiparallel organization of the subunits in the dimer is preferred. Blue-native PAGE analyses of intact oligomers and disulfide cross-linking demonstrate that in membranes, the proteins form parallel dimers only if no oppositely orientated partner is present. Co-expression of oppositely orientated proteins almost exclusively yields antiparallel dimers. Finally, parallel dimers can be disrupted and converted into antiparallel dimers by heating of detergent-solubilized protein. Importantly, in vivo function is correlated clearly to the presence of antiparallel dimers. Our results suggest that an antiparallel arrangement of the subunits in the dimer is more stable than a parallel organization and likely corresponds to the functional form of the protein
DirectMX – One-Step Reconstitution of Membrane Proteins From Crude Cell Membranes Into Salipro Nanoparticles
Integral membrane proteins (IMPs) are central to many physiological processes and represent ∼60% of current drug targets. An intricate interplay with the lipid molecules in the cell membrane is known to influence the stability, structure and function of IMPs. Detergents are commonly used to solubilize and extract IMPs from cell membranes. However, due to the loss of the lipid environment, IMPs usually tend to be unstable and lose function in the continuous presence of detergent. To overcome this problem, various technologies have been developed, including protein engineering by mutagenesis to improve IMP stability, as well as methods to reconstitute IMPs into detergent-free entities, such as nanodiscs based on apolipoprotein A or its membrane scaffold protein (MSP) derivatives, amphipols, and styrene-maleic acid copolymer-lipid particles (SMALPs). Although significant progress has been made in this field, working with inherently unstable human IMP targets (e.g., GPCRs, ion channels and transporters) remains a challenging task. Here, we present a novel methodology, termed DirectMX (for direct membrane extraction), taking advantage of the saposin-lipoprotein (Salipro) nanoparticle technology to reconstitute fragile IMPs directly from human crude cell membranes. We demonstrate the applicability of the DirectMX methodology by the reconstitution of a human solute carrier transporter and a wild-type GPCR belonging to the human chemokine receptor (CKR) family. We envision that DirectMX bears the potential to enable studies of IMPs that so far remained inaccessible to other solubilization, stabilization or reconstitution methods.QC 20211008</p
In Vivo Trp Scanning of the Small Multidrug Resistance Protein EmrE Confirms 3D Structure Models
The quaternary structure of the homodimeric small multidrug resistance protein EmrE has been studied intensely over the past decade. Structural models derived from both two- and three-dimensional crystals show EmrE as an anti-parallel homodimer. However, the resolution of the structures is rather low and their relevance for the in vivo situation has been questioned. Here, we have challenged the available structural models by a comprehensive in vivo Trp scanning of all four transmembrane helices in EmrE. The results are in close agreement with the degree of lipid exposure of individual residues predicted from coarse-grained molecular dynamics simulations of the anti-parallel dimeric structure obtained by X-ray crystallography, strongly suggesting that the X-ray structure provides a good representation of the active in vivo form of EmrE
Host cell-derived lactate functions as an effector molecule in <i>Neisseria meningitidis</i> microcolony dispersal
<div><p>The development of meningococcal disease, caused by the human pathogen <i>Neisseria meningitidis</i>, is preceded by the colonization of the epithelial layer in the nasopharynx. After initial adhesion to host cells meningococci form aggregates, through pilus-pilus interactions, termed microcolonies from which the bacteria later detach. Dispersal from microcolonies enables access to new colonization sites and facilitates the crossing of the cell barrier; however, this process is poorly understood. In this study, we used live-cell imaging to investigate the process of <i>N</i>. <i>meningitidis</i> microcolony dispersal. We show that direct contact with host cells is not required for microcolony dispersal, instead accumulation of a host-derived effector molecule induces microcolony dispersal. By using a host-cell free approach, we demonstrated that lactate, secreted from host cells, initiate rapid dispersal of microcolonies. Interestingly, metabolic utilization of lactate by the bacteria was not required for induction of dispersal, suggesting that lactate plays a role as a signaling molecule. Furthermore, <i>Neisseria gonorrhoeae</i> microcolony dispersal could also be induced by lactate. These findings reveal a role of host-secreted lactate in microcolony dispersal and virulence of pathogenic <i>Neisseria</i>.</p></div
A heat-stable, low-molecular weight compound present in CM can induce microcolony dispersal.
<p>The CM was pretreated with 95°C heat, a protease inhibitor cocktail, proteinase K, trypsin, chymotrypsin, DNase I, RNase A, EDTA, or EGTA (A) or passaged through a 3 kDa cut-off filter (B) prior to induction assay. Microcolony dispersal was examined in live-cell time-lapse microscopy after addition of treated CM. A black horizontal line in panels represents the 3 h time point when treated CM was added to preformed microcolonies. Data represent the mean ± SD of three independent experiments. *p < 0.05.</p
Cell-conditioned medium (CM) induces the dispersal of microcolonies.
<p>(A) Schematic drawing of the experimental set-up. Confluent FaDu cells were washed and incubated with DMEM. After 5 h, the CM was collected, sterile filtered, and added at a 1:1 volume ratio to bacterial meningococcal suspensions where microcolonies had been allowed to form for 3 h (initial bacterial concentration of 10<sup>7</sup> CFU/ml). A black horizontal line in panels B and D represents the 3 h time point. Bacteria were observed by live-cell time-lapse microscopy. DMEM was used as a control. (B) Microcolony dispersal upon addition of CM from infected (+) or uninfected (-) cells to FAM20 bacteria in liquid. (C) Microcolony dispersal was examined by live-cell time-lapse microscopy after addition of CM. Representative images are shown 10, 20 and 30 min after induction. Scale bar, 10 μm. (D) Microcolony dispersal upon addition of CM collected from FaDu cells after 5, 15, and 30 min and 1 h and 5 h. (E) Microcolony dispersal of FAM20 resuspended (10<sup>7</sup> CFU/ml) in either CM or DMEM (control) when the incubation was initiated. Data represent the mean ± SD of three independent experiments. <sup>*</sup>p < 0.05. ns, non-significant.</p
Lactate induces microcolony dispersal in <i>N</i>. <i>meningitidis</i> strain JB515 and <i>N</i>. <i>gonorrhoeae</i> strain MS11.
<p>(A) Bacteria (10<sup>7</sup> CFU/ml) were allowed to form microcolonies for 3 h (black horizontal line) before addition of DMEM supplemented with lactate at final concentrations ranging from 0.5–10 mM. Microcolony dispersal was examined by live-cell microscopy for 8 h. Data represent the mean ± SD of three independent experiments. <sup>*</sup>p < 0.05. (B) Representative images showing dispersal 40 min, 60 min and 80 min after addition of lactate (1 mM) to JB515 and MS11 microcolonies. Scale bar, 20 μm.</p
Environmental concentration of lactate influences microcolony dispersal in pathogenic <i>Neisseria</i>.
<p>Proposed model of lactate-induced microcolony dispersal in pathogenic <i>Neisseria</i>. (A) In presence of high lactate concentration the microcolony dispersal of pathogenic <i>Neisseria</i> is rapid regardless of whether microcolonies are in direct contact with host cells or not. (B) In low lactate concentrations, pathogenic <i>Neisseria</i> remain in microcolonies.</p
Lactate-induced microcolony dispersal is not dependent on metabolic utilization by <i>N</i>. <i>meningitidis</i>.
<p>Growth of FAM20 wild-type and its isogenic mutants Δ<i>lctP</i>, Δ<i>ldhA</i> Δ<i>ldhD</i>, Δ<i>lldA</i> in the presence of glucose (A), L-lactate (B) or D-lactate (C) as the major carbon source. (D-G) The timing of microcolony dispersal was examined after addition of DMEM (control), CM, L-lactate (10 mM) or D-lactate (10 mM) to preformed Δ<i>lctP</i> (D), Δ<i>ldhD</i> (E), Δ<i>ldhA</i> (F) or Δ<i>lldA</i> (G) microcolonies. Induction is represented by a black horizontal line. For panel A-C, one representative growth curve out of two is shown. For panel D-G, data represent the mean ± SD of three independent experiments. <sup>*</sup>p < 0.05. ns, non-significant.</p
Host cells accelerate microcolony dispersal independent of direct contact.
<p>Microcolony formation and dispersal of <i>N</i>. <i>meningitidis</i> FAM20 (2 × 10<sup>6</sup> CFU/ml) were monitored for 8 h by live-cell time-lapse microscopy after infection of FaDu cells. (A) FaDu cells at confluences of 100%, 80%, 50%, 20% and 0% were infected with FAM20. (B) The dispersal of FAM20 microcolonies on FaDu cells at 50% confluence was observed with live-cell time-lapse microscopy. Representative images are shown. Scale bar, 10 μm. (C) FaDu cells were infected with FAM20 or the Δ<i>pilC1</i> mutant. (D) FAM20 infecting fixed (3.7% paraformaldehyde) or live FaDu cells. Data represent the mean ± SD for at least three individual experiments. <sup>*</sup>p < 0.05. ns, non-significant.</p