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

Complete topology inversion can be part of normal membrane protein biogenesis

The topology of helical membrane proteins is generally defined during insertion of the transmembrane helices, yet it is now clear that it is possible for topology to change under unusual circumstances. It remains unclear, however, if topology reorientation is part of normal biogenesis. For dual topology dimer proteins such as the multidrug transporter EmrE, there may be evolutionary pressure to allow topology flipping so that the populations of both orientations can be equalized. We previously demonstrated that when EmrE is forced to insert in a distorted topology, topology flipping of the first transmembrane helix can occur during translation. Here, we show that topological malleability also extends to the C‐terminal helix and that even complete topology inversion of the entire EmrE protein can occur after the full protein is translated and inserted. Thus, topology rearrangements are possible during normal biogenesis. Wholesale topology flipping is remarkable given the physical constraints of the membrane and expands the range of possible membrane protein folding pathways, both productive and detrimental

Dual-topology insertion of a dual-topology membrane protein

Some membrane transporters are dual-topology dimers in which the subunits have inverted transmembrane topology. How a cell manages to generate equal populations of two opposite topologies from the same polypeptide chain remains unclear. For the dual-topology transporter EmrE, the evidence to date remains consistent with two extreme models. A post-translational model posits that topology remains malleable after synthesis and becomes fixed once the dimer forms. A second, co-translational model, posits that the protein inserts in both topologies in equal proportions. Here we show that while there is at least some limited topological malleability, the co-translational model likely dominates under normal circumstances

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

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 10⁷ 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 (10⁷ CFU/ml) in either CM or DMEM (control) when the incubation was initiated. Data represent the mean ± SD of three independent experiments. ^*p < 0.05. ns, non-significant.</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

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