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20240603_membrane digest


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A molecular switch controls assembly of bacterial focal adhesions.

Attia B, My L, Castaing JP, Dinet C, Le Guenno H, Schmidt V, Espinosa L, Anantharaman V, Aravind L, Sebban-Kreuzer C, Nouailler M, Bornet O, Viollier P, Elantak L, Mignot T.

Sci Adv. 2024 May 31;10(22):eadn2789.

doi: 10.1126/sciadv.adn2789. Epub 2024 May 29.

PMID: 38809974.

Cell motility universally relies on spatial regulation of focal adhesion complexes (FAs) connecting the substrate to cellular motors. In bacterial FAs, the Adventurous gliding motility machinery (Agl-Glt) assembles at the leading cell pole following a Mutual gliding-motility protein (MglA)-guanosine 5′-triphosphate (GTP) gradient along the cell axis. Here, we show that GltJ, a machinery membrane protein, contains cytosolic motifs binding MglA-GTP and AglZ and recruiting the MreB cytoskeleton to initiate movement toward the lagging cell pole. In addition, MglA-GTP binding triggers a conformational shift in an adjacent GltJ zinc-finger domain, facilitating MglB recruitment near the lagging pole. This prompts GTP hydrolysis by MglA, leading to complex disassembly. The GltJ switch thus serves as a sensor for the MglA-GTP gradient, controlling FA activity spatially.


Maintenance of bacterial outer membrane lipid asymmetry: insight into MlaA.

Kaur M, Mingeot-Leclercq MP.

BMC Microbiol. 2024 May 28;24(1):186.

doi: 10.1186/s12866-023-03138-8. PMID: 38802775.

The outer membrane (OM) of Gram-negative bacteria acts as an effective barrier to protect against toxic compounds. By nature, the OM is asymmetric with the highly packed lipopolysaccharide (LPS) at the outer leaflet and glycerophospholipids at the inner leaflet. OM asymmetry is maintained by the Mla system, in which is responsible for the retrograde transport of glycerophospholipids from the OM to the inner membrane. This system is comprised of six Mla proteins, including MlaA, an OM lipoprotein involved in the removal of glycerophospholipids that are mis-localized at the outer leaflet of the OM. Interestingly, MlaA was initially identified – and called VacJ – based on its role in the intracellular spreading of Shigella flexneri.Many open questions remain with respect to the Mla system and the mechanism involved in the translocation of mislocated glycerophospholipids at the outer leaflet of the OM, by MlaA. After summarizing the current knowledge on MlaA, we focus on the impact of mlaA deletion on OM lipid composition and biophysical properties of the OM. How changes in OM lipid composition and biophysical properties can impact the generation of membrane vesicles and membrane permeability is discussed. Finally, we explore whether and how MlaA might be a candidate for improving the activity of antibiotics and as a vaccine candidate.Efforts dedicated to understanding the relationship between the OM lipid composition and the mechanical strength of the bacterial envelope and, in turn, how such properties act against external stress, are needed for the design of new targets or drugs for Gram-negative infections.


Coupling enzymatic activity and gating in an ancient TRPM chanzyme and its molecular evolution.

Huang Y, Kumar S, Lee J, Lü W, Du J.

Nat Struct Mol Biol. 2024 May 21.

doi: 10.1038/s41594-024-01316-4. Epub ahead of print.

PMID: 38773335.

Channel enzymes represent a class of ion channels with enzymatic activity directly or indirectly linked to their channel function. We investigated a TRPM2 chanzyme from choanoflagellates that integrates two seemingly incompatible functions into a single peptide: a channel module activated by ADP-ribose with high open probability and an enzyme module (NUDT9-H domain) consuming ADP-ribose at a remarkably slow rate. Using time-resolved cryogenic-electron microscopy, we captured a complete series of structural snapshots of gating and catalytic cycles, revealing the coupling mechanism between channel gating and enzymatic activity. The slow kinetics of the NUDT9-H enzyme module confers a self-regulatory mechanism: ADPR binding triggers NUDT9-H tetramerization, promoting channel opening, while subsequent hydrolysis reduces local ADPR, inducing channel closure. We further demonstrated how the NUDT9-H domain has evolved from a structurally semi-independent ADP-ribose hydrolase module in early species to a fully integrated component of a gating ring essential for channel activation in advanced species.


Oligomerization of drug transporters: Forms, functions, and mechanisms.

Ni C, Hong M.

Acta Pharm Sin B. 2024 May;14(5):1924-1938.

doi: 10.1016/j.apsb.2024.01.007. Epub 2024 Jan 20.

PMID: 38799641.

Drug transporters are essential players in the transmembrane transport of a wide variety of clinical drugs. The broad substrate spectra and versatile distribution pattern of these membrane proteins infer their pharmacological and clinical significance. With our accumulating knowledge on the three-dimensional structure of drug transporters, their oligomerization status has become a topic of intense study due to the possible functional roles carried out by such kind of post-translational modification (PTM). In-depth studies of oligomeric complexes formed among drug transporters as well as their interactions with other regulatory proteins can help us better understand the regulatory mechanisms of these membrane proteins, provide clues for the development of novel drugs, and improve the therapeutic efficacy. In this review, we describe different oligomerization forms as well as their structural basis of major drug transporters in the ATP-binding cassette and solute carrier superfamilies, summarize our current knowledge on the influence of oligomerization for protein expression level and transport function of these membrane proteins, and discuss the regulatory mechanisms of oligomerization. Finally, we highlight the challenges associated with the current oligomerization studies and propose some thoughts on the pharmaceutical application of this important drug transporter PTM.


SecYEG-mediated translocation in a model synthetic cell.

Schoenmakers LLJ, den Uijl MJ, Postma JL, van den Akker TAP, Huck WTS, Driessen AJM.

Synth Biol (Oxf). 2024 May 10;9(1):ysae007.

doi: 10.1093/synbio/ysae007.

PMID: 38807757.

Giant unilamellar vesicles (GUVs) provide a powerful model compartment for synthetic cells. However, a key challenge is the incorporation of membrane proteins that allow for transport, energy transduction, compartment growth and division. Here, we have successfully incorporated the membrane protein complex SecYEG-the key bacterial translocase that is essential for the incorporation of newly synthesized membrane proteins-in GUVs. Our method consists of fusion of small unilamellar vesicles containing reconstituted SecYEG into GUVs, thereby forming SecGUVs. These are suitable for large-scale experiments while maintaining a high protein:lipid ratio. We demonstrate that incorporation of SecYEG into GUVs does not inhibit its translocation efficiency. Robust membrane protein functionalized proteo-GUVs are promising and flexible compartments for use in the formation and growth of synthetic cells.


Single transmembrane GPCR modulating proteins: neither single nor simple.

Meng Wang, Jianjun Lyu, Chao Zhang,

Protein & Cell, Volume 15, Issue 6, June 2024, Pages 395–402,

The discovery of G-protein coupled receptor (GPCR) accessory proteins has fundamentally redefined the pharmacological concept of GPCR signaling, demonstrating a more complex molecular basis for receptor specificity on the plasma membrane and impressionable downstream intracellular cascades. GPCR accessory proteins not only contribute to the proper folding and trafficking of receptors but also exhibit selectable receptor preferences. The melanocortin receptor accessory proteins (MRAP1 and MRAP2) as well as receptor activity-modifying proteins (RAMPs) are two well-known single transmembrane partners for the regulation of the melanocortin receptors (MC1R–MC5R) and the glucagon receptor (GCGR), respectively. Especially, the MRAP family participates in the pathological control of multiple endocrine disorders and RAMPs contribute to the endogenous regulation of glucose homeostasis. However, the precise mechanisms by which the MRAP and RAMP proteins regulate receptor signaling at atomic resolution remain unknown. Recent progress made in the determination of RAMP2-bound GCGR complexes published on Cell unraveled the importance of RAMP2 for the promotion of extracellular receptor dynamics leading to cytoplasmic surface inactivation. Moreover, the new findings on Cell Research of the adrenocorticotropic hormone (ACTH)-bound MC2R–Gs–MRAP1 complex disclosed the essential role of MRAP1 for MC2R activation and specificity of ligand recognition. In this article, we reviewed a series of key findings of MRAP proteins in the last decade, the recent structural investigation of the MRAP–MC2R and RAMP–GCGR functional complex, and the expanded identification of new GPCR partners of MRAP proteins. An in-depth understanding of GPCR modulation by single transmembrane accessory proteins will provide valuable insights for the therapeutic drug development to treat multiple GPCR-associated human disorders.


A unifying model for membrane protein biogenesis.

Hegde RS, Keenan RJ.

Nat Struct Mol Biol. 2024 May 29.

doi: 10.1038/s41594-024-01296-5. Epub ahead of print.

PMID: 38811793.

α-Helical integral membrane proteins comprise approximately 25% of the proteome in all organisms. The membrane proteome is highly diverse, varying in the number, topology, spacing and properties of transmembrane domains. This diversity imposes different constraints on the insertion of different regions of a membrane protein into the lipid bilayer. Here, we present a cohesive framework to explain membrane protein biogenesis, in which different parts of a nascent substrate are triaged between Oxa1 and SecY family members for insertion. In this model, Oxa1 family proteins insert transmembrane domains flanked by short translocated segments, whereas the SecY channel is required for insertion of transmembrane domains flanked by long translocated segments. Our unifying model rationalizes evolutionary, genetic, biochemical and structural data across organisms and provides a foundation for future mechanistic studies of membrane protein biogenesis.


Affinity-directed substrate/H+-antiport by a MATE transporter.

Takeuchi K, Ueda T, Imai M, Fujisaki M, Shimura M, Tokunaga Y, Kofuku Y, Shimada I.

Structure. 2024 May 18:S0969-2126(24)00181-3.

doi: 10.1016/j.str.2024.05.004. Epub ahead of print.

PMID: 38815577.

Multidrug and toxin extrusion (MATE) family transporters excrete toxic compounds coupled to Na+/H+ influx. Although structures of MATE transporters are available, the mechanism by which substrate export is coupled to ion influx remains unknown. To address this issue, we conducted a structural analysis of Pyrococcus furiosus MATE (PfMATE) using solution nuclear magnetic resonance (NMR). The NMR analysis, along with thorough substitutions of all non-exposed acidic residues, confirmed that PfMATE is under an equilibrium between inward-facing (IF) and outward-facing (OF) conformations, dictated by the Glu163 protonation. Importantly, we found that only the IF conformation exhibits a mid-μM affinity for substrate recognition. In contrast, the OF conformation exhibited only weak mM substrate affinity, suitable for releasing substrate to the extracellular side. These results indicate that PfMATE is an affinity-directed H+ antiporter where substrates selectively bind to the protonated IF conformation in the equilibrium, and subsequent proton release mechanistically ensures H+-coupled substrate excretion by the transporter.


Dynamics underlie the drug recognition mechanism by the efflux transporter EmrE.

Li J, Her AS, Besch A, Ramirez-Cordero B, Crames M, Banigan JR, Mueller C, Marsiglia WM, Zhang Y, Traaseth NJ.

Nat Commun. 2024 May 28;15(1):4537.

doi: 10.1038/s41467-024-48803-2.

PMID: 38806470.

The multidrug efflux transporter EmrE from Escherichia coli requires anionic residues in the substrate binding pocket for coupling drug transport with the proton motive force. Here, we show how protonation of a single membrane embedded glutamate residue (Glu14) within the homodimer of EmrE modulates the structure and dynamics in an allosteric manner using NMR spectroscopy. The structure of EmrE in the Glu14 protonated state displays a partially occluded conformation that is inaccessible for drug binding by the presence of aromatic residues in the binding pocket. Deprotonation of a single Glu14 residue in one monomer induces an equilibrium shift toward the open state by altering its side chain position and that of a nearby tryptophan residue. This structural change promotes an open conformation that facilitates drug binding through a conformational selection mechanism and increases the binding affinity by approximately 2000-fold. The prevalence of proton-coupled exchange in efflux systems suggests a mechanism that may be shared in other antiporters where acid/base chemistry modulates access of drugs to the substrate binding pocket.



Thicket and Mesh: How the Outer Membrane Can Resist Tension Imposed by the Cell Wall.

Ryoo D, Hwang H, Gumbart JC.

J Phys Chem B. 2024 May 24.

doi: 10.1021/acs.jpcb.3c08510. Epub ahead of print.

PMID: 38787347.

The cell envelope of Gram-negative bacteria is composed of an outer membrane (OM) and an inner membrane (IM) and a peptidoglycan cell wall (CW) between them. Combined with Braun’s lipoprotein (Lpp), which connects the OM and the CW, and numerous membrane proteins that exist in both OM and IM, the cell envelope creates a mechanically stable environment that resists various physical and chemical perturbations to the cell, including turgor pressure caused by the solute concentration difference between the cytoplasm of the cell and the extracellular environment. Previous computational studies have explored how individual components (OM, IM, and CW) can resist turgor pressure although combinations of them have been less well studied. To that end, we constructed multiple OM-CW systems, including the Lpp connections with the CW under increasing degrees of strain. The results show that the OM can effectively resist the tension imposed by the CW, shrinking by only 3-5% in area even when the CW is stretched to 2.5× its relaxed area. The area expansion modulus of the system increases with increasing CW strain, although the OM remains a significant contributor to the envelope’s mechanical stability. Additionally, we find that when the protein TolC is embedded in the OM, its stiffness increases.


Phospholipid Membrane Interactions of Model Ac-WL-X-LL-OH Peptides Investigated by Solid-State Nuclear Magnetic Resonance.

Alsaker NE, Halskau Ø, Haug BE, Reuter N, Nerdal W.

Membranes (Basel). 2024 May 1;14(5):105.

doi: 10.3390/membranes14050105. PMID: 38786939.

The role of aromatic amino acids in peripheral protein membrane binding has been reported to involve cation-π interactions with choline lipids. In this study, we have investigated the interactions of the model pentapeptide Ac-WL-X-LL-OH (where X = L, Y, F, or W) with the phospholipid membrane using solid-state NMR. The effect of guest residue X on the peptide-lipid interactome was complementary to the seminal report on the interfacial hydrophobicity scale by Wimley and White. We found that the phospholipids retained a lamellar phase in the presence of each of the peptides with an aromatic X residue, whereas the Leu peptide perturbed the bilayer to an extent where an additional isotropic phase was observed. The solid-state NMR 13C and 31P data provide additional information on the influence of these short peptides on the membrane that has not been previously reported. The magnitude of membrane perturbation was in the order of guest residue X = L > Y~F > W, which is consistent with the relative amino acid interfacial affinity reported by Wimley and White. Further work is, however, required to uncover the behavior of the peptide and localization in the membrane domain due to ambiguity of the 13C NMR data. We have launched efforts in this regard for the objective of better understanding the role of aromatic amino acids in peripheral membrane protein binding.


A unifying mechanism for seipin-mediated lipid droplet formation.

Klug YA, Ferreira JV, Carvalho P.

FEBS Lett. 2024 May;598(10):1116-1126.

doi: 10.1002/1873-3468.14825. Epub 2024 Feb 13.

PMID: 38785192.

Lipid droplets (LDs) are dynamic organelles essential for cellular lipid homeostasis. Assembly of LDs occurs in the endoplasmic reticulum (ER), and the conserved ER membrane protein seipin emerged as a key player in this process. Here, we review recent advances provided by structural, biochemical, and in silico analysis that revealed mechanistic insights into the molecular role of the seipin complexes and led to an updated model for LD biogenesis. We further discuss how other ER components cooperate with seipin during LD biogenesis. Understanding the molecular mechanisms underlying seipin-mediated LD assembly is important to uncover the fundamental aspects of lipid homeostasis and organelle biogenesis and to provide hints on the pathogenesis of lipid storage disorders.


Influence of antimicrobial peptides on the bacterial membrane curvature and vice versa.

Cardoso MH, de la Fuente-Nunez C, Santos NC, Zasloff MA, Franco OL.

Trends Microbiol. 2024 May 21:S0966-842X(24)00113-6.

doi: 10.1016/j.tim.2024.04.012. Epub ahead of print.

PMID: 38777700.

Many factors contribute to bacterial membrane stabilization, including steric effects between lipids, membrane spontaneous curvature, and the difference in the number of neighboring molecules. This forum provides an overview of the physicochemical properties associated with membrane curvature and how this parameter can be tuned to design more effective antimicrobial peptides.


Different lateral packing stress in acyl chains alters KcsA orientation and structure in lipid membranes.

Hayakawa ESH, Ueki M, Alhatmi E, Oiki S, Tokumasu F, Mitchell DC, Iwamoto M.

Biochim Biophys Acta Biomembr. 2024 May 18;1866(6):184338.

doi: 10.1016/j.bbamem.2024.184338. Epub ahead of print.

PMID: 38763269.

The molecular structures of the various intrinsic lipids in membranes regulate lipid-protein interactions. These different lipid structures with unique volumes produce different lipid molecular packing stresses/lateral stresses in lipid membranes. Most studies examining lipid packing effects have used phosphatidylcholine and phosphatidylethanolamine (PE), which are the main phospholipids of eukaryotic cell membranes. In contrast, Gram-negative or Gram-positive bacterial membranes are composed primarily of phosphatidylglycerol (PG) and PE, and the physical and thermodynamic properties of each acyl chain in PG at the molecular level remain unresolved. In this study, we used 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG, 16:0-18:1 PG) and 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (PAPG, 16:0-20:4 PG) to prepare lipid bilayers (liposome) with the rod-type fluorescence probe DPH. We measured the lipid packing conditions by determining the rotational freedom of DPH in POPG or PAPG bilayers. Furthermore, we investigated the effect of different monoacyl chains on a K+ channel (KcsA) structure when embedded in POPG or PAPG membranes. The results revealed that differences in the number of double bonds and carbon chain length in the monoacyl chain at sn-2 affected the physicochemical properties of the membrane and the structure and orientation of KcsA.


Membrane specificity of the human cholesterol transfer protein STARD4.

Talandashti R, van Ek L, Gehin C, Xue D, Moqadam M, Gavin AC, Reuter N.

J Mol Biol. 2024 Jun 1;436(11):168572.

doi: 10.1016/j.jmb.2024.168572. Epub 2024 Apr 12.

PMID: 38615744.

STARD4 regulates cholesterol homeostasis by transferring cholesterol between the plasma membrane and endoplasmic reticulum. The STARD4 structure features a helix-grip fold surrounding a large hydrophobic cavity holding the sterol. Its access is controlled by a gate formed by the Ω1 and Ω4 loops and the C-terminal α-helix. Little is known about the mechanisms by which STARD4 binds to membranes and extracts/releases cholesterol. All available structures of STARD4 are without a bound sterol and display the same closed conformation of the gate. The cholesterol transfer activity of the mouse STARD4 is enhanced in the presence of anionic lipids, and in particular of phosphatidylinositol biphosphates (PIP2) for which two binding sites were proposed on the mouse STARD4 surface. Yet only one of these sites is conserved in human STARD4. We here report the results of a liposome microarray-based assay and microseconds-long molecular dynamics simulations of human STARD4 with complex lipid bilayers mimicking the composition of the donor and acceptor membranes. We show that the binding of apo form of human STARD4 is sensitive to the presence of PIP2 through two specific binding sites, one of which was not identified on mouse STARD4. We report two novel conformations of the gate in holo-STARD4: a yet-unobserved close conformation and an open conformation of Ω4 shedding light on the opening/closure mechanism needed for cholesterol uptake/release. Overall, the modulation of human STARD4 membrane-binding by lipid composition, and by the presence of the cargo supports the capacity of human STARD4 to achieve directed transfer between specific organelle membranes.


There and back again: bridging meso- and nanoscales to understand lipid vesicle patterning.

Julie Cornet, Nelly Coulonges, Weria Pezeshkian, Mael Penissat-Mahaut, Hermes Desgrez-Dautet, Siewert J. Marrink, Nicolas Destainville, Matthieu Chavent and Manoel Manghi.

Soft Matter


We describe a complete methodology to bridge the scales between nanoscale Molecular Dynamics and (micrometer) mesoscale Monte Carlo simulations in lipid membranes and vesicles undergoing phase separation, in which curving molecular species are furthermore embedded. To go from the molecular to the mesoscale, we notably appeal to physical renormalization arguments enabling us to rigorously infer the mesoscale interaction parameters from its molecular counterpart. We also explain how to deal with the physical timescales at stake at the mesoscale. Simulating the so-obtained mesoscale system enables us to equilibrate the long wavelengths of the vesicles of interest, up to the vesicle size. Conversely, we then backmap from the meso- to the nano- scale, which enables us to equilibrate in turn the short wavelengths down to the molecular length-scales. By applying our approach to the specific situation of the patterning of a vesicle membrane, we show that macroscopic membranes can thus be equilibrated at all length-scales in achievable computational time offering an original strategy to address the fundamental challenge of time scale in simulations of large bio-membrane systems.



Flow-based bioconjugation of coumarin phosphatidylethanolamine probes: Optimised synthesis and membrane molecular dynamics studies.

Varandas PAMM, Belinha R, Cobb AJA, Prates Ramalho JP, Segundo MA, Loura LMS, Silva EMP.

Biochim Biophys Acta Biomembr. 2024 May 17:184335.

doi: 10.1016/j.bbamem.2024.184335. Epub ahead of print.

PMID: 38763271.

A series of phosphatidylethanolamine fluorescent probes head-labelled with 3-carboxycoumarin was prepared by an improved bioconjugation approach through continuous flow synthesis. The established procedure, supported by a design of experiment (DoE) set-up, resulted in a significant reduction in the reaction time compared to the conventional batch method, in addition to a minor yield increase. The characterization of these probes was enhanced by an in-depth molecular dynamics (MD) study of the behaviour of a representative probe of this family, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine labelled with 3-carboxycoumarin (POPE-COUM), in bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (SLPC) 2:1, mimicking the composition of the egg yolk lecithin membranes recently used experimentally by our group to study POPE-COUM as a biomarker of the oxidation state and integrity of large unilamellar vesicles (LUVs). The MD simulations revealed that the coumarin group is oriented towards the bilayer interior, leading to a relatively internal location, in agreement with what is observed in the nitrobenzoxadiazole fluorophore of commercial head-labelled NBD-PE probes. This behaviour is consistent with the previously stated hypothesis that POPE-COUM is entirely located within the LUVs structure. Hence, the delay on the oxidation of the probe in the oxygen radical absorbance capacity (ORAC) assays performed is related with the inaccessibility of the probe until alteration of the LUV structure occurs. Furthermore, our simulations show that POPE-COUM exerts very little global and local perturbation on the host bilayer, as evaluated by key properties of the unlabelled lipids. Together, our findings establish PE-COUM as suitable fluorescent lipid analogue probes.



Efficient Segmental Isotope Labeling of Integral Membrane Proteins for High-Resolution NMR Studies.

Daniilidis M, Sperl LE, Müller BS, Babl A, Hagn F.

J Am Chem Soc. 2024 May 24.

doi: 10.1021/jacs.4c03294. Epub ahead of print. PMID: 38787792.

High-resolution structural NMR analyses of membrane proteins are challenging due to their large size, resulting in broad resonances and strong signal overlap. Among the isotope labeling methods that can remedy this situation, segmental isotope labeling is a suitable strategy to simplify NMR spectra and retain high-resolution structural information. However, protein ligation within integral membrane proteins is complicated since the hydrophobic protein fragments are insoluble, and the removal of ligation side-products is elaborate. Here, we show that a stabilized split-intein system can be used for rapid and high-yield protein trans-splicing of integral membrane proteins under denaturing conditions. This setup enables segmental isotope labeling experiments within folded protein domains for NMR studies. We show that high-quality NMR spectra of markedly reduced complexity can be obtained in detergent micelles and lipid nanodiscs. Of note, the nanodisc insertion step specifically selects for the ligated and correctly folded membrane protein and simultaneously removes ligation byproducts. Using this tailored workflow, we show that high-resolution NMR structure determination is strongly facilitated with just two segmentally isotope-labeled membrane protein samples. The presented method will be broadly applicable to structural and dynamical investigations of (membrane-) proteins and their complexes by solution and solid-state NMR but also other structural methods where segmental labeling is beneficial.


Optimizing Coarse-Grained Models for Large-Scale Membrane Protein Simulation.

Wen CY, Luo YL, Madsen JJ.

bioRxiv [Preprint]. 2024 May 15:2024.05.13.594009.

doi: 10.1101/2024.05.13.594009. PMID: 38798639.

Coarse-grained (CG) models have been developed for studying membrane proteins at physiologically relevant scales. Such methods, including popular CG lipid models, exhibit stability and efficiency at moderate scales, but they can become impractical or even unusable beyond a critical size due to various technical issues. Here, we report that these scale-dependent issues can arise from progressively slower relaxation dynamics and become confounded by unforeseen instabilities observed only at larger scales. To address these issues, we systemically optimized a 4-site solvent-free CG lipid model that is suitable for conducting micron-scale molecular dynamics simulations of membrane proteins under various membrane properties. We applied this lipid model to explore the long-range membrane deformation induced by a large mechanosensitive ion channel, PIEZO. We show that the optimized CG models are powerful in elucidating the structural and dynamic interplay between PIEZO and the membrane. Furthermore, we anticipate that our methodological insights can prove useful for resolving issues stemming from scale-dependent limitations of similar CG methodologies.


Nanodiscs for the study of membrane proteins.

Denisov IG, Sligar SG.

Curr Opin Struct Biol. 2024 May 24;87:102844.

doi: 10.1016/ Epub ahead of print.

PMID: 38795563.

Nanodiscs represent a versatile tool for studies of membrane proteins and protein-membrane interactions under native-like conditions. Multiple variations of the Nanodisc platform, as well as new experimental methods, have been recently developed to understand various aspects of structure, dynamics and functional properties of systems involved in signaling, transport, blood coagulation and many other critically important processes. In this mini-review, we focus on some of these exciting recent developments that utilize the Nanodisc platform.


Transporter function characterization via continuous-exchange cell-free synthesis and solid supported membrane-based electrophysiology.

Dong F, Lojko P, Bazzone A, Bernhard F, Borodina I.

Bioelectrochemistry. 2024 May 23;159:108732.

doi: 10.1016/j.bioelechem.2024.108732. Epub ahead of print.

PMID: 38810322.

Functional characterization of transporters is impeded by the high cost and technical challenges of current transporter assays. Thus, in this work, we developed a new characterization workflow that combines cell-free protein synthesis (CFPS) and solid supported membrane-based electrophysiology (SSME). For this, membrane protein synthesis was accomplished in a continuous exchange cell-free system (CECF) in the presence of nanodiscs. The resulting transporters expressed in nanodiscs were incorporated into proteoliposomes and assayed in the presence of different substrates using the surface electrogenic event reader. As a proof of concept, we validated this workflow to express and characterize five diverse transporters: the drug/H+-coupled antiporters EmrE and SugE, the lactose permease LacY, the Na+/H+ antiporter NhaA from Escherichia coli, and the mitochondrial carrier AAC2 from Saccharomyces cerevisiae. For all transporters kinetic parameters, such as KM, IMAX, and pH dependency, were evaluated. This robust and expedite workflow (e.g., can be executed within only five workdays) offers a convenient direct functional assessment of transporter protein activity and has the ability to facilitate applications of transporters in medical and biotechnological research.



RND Efflux Pump Induction: A Crucial Network Unveiling Adaptive Antibiotic Resistance Mechanisms of Gram-Negative Bacteria.

Novelli, M.; Bolla, J.-M.

Antibiotics 2024, 13, 501.

The rise of multi-drug-resistant (MDR) pathogenic bacteria presents a grave challenge to global public health, with antimicrobial resistance ranking as the third leading cause of mortality worldwide. Understanding the mechanisms underlying antibiotic resistance is crucial for developing effective treatments. Efflux pumps, particularly those of the resistance-nodulation-cell division (RND) superfamily, play a significant role in expelling molecules from bacterial cells, contributing to the emergence of multi-drug resistance. These are transmembrane transporters naturally produced by Gram-negative bacteria. This review provides comprehensive insights into the modulation of RND efflux pump expression in bacterial pathogens by numerous and common molecules (bile, biocides, pharmaceuticals, additives, plant extracts, etc.). The interplay between these molecules and efflux pump regulators underscores the complexity of antibiotic resistance mechanisms. The clinical implications of efflux pump induction by non-antibiotic compounds highlight the challenges posed to public health and the urgent need for further investigation. By addressing antibiotic resistance from multiple angles, we can mitigate its impact and preserve the efficacy of antimicrobial therapies.



Antibiotic influx and efflux in Pseudomonas aeruginosa: Regulation and therapeutic implications.

Wu W, Huang J, Xu Z.

Microb Biotechnol. 2024 May;17(5):e14487.

doi: 10.1111/1751-7915.14487. PMID: 38801351.

Pseudomonas aeruginosa is a notorious multidrug-resistant pathogen that poses a serious and growing threat to the worldwide public health. The expression of resistance determinants is exquisitely modulated by the abundant regulatory proteins and the intricate signal sensing and transduction systems in this pathogen. Downregulation of antibiotic influx porin proteins and upregulation of antibiotic efflux pump systems owing to mutational changes in their regulators or the presence of distinct inducing molecular signals represent two of the most efficient mechanisms that restrict intracellular antibiotic accumulation and enable P. aeruginosa to resist multiple antibiotics. Treatment of P. aeruginosa infections is extremely challenging due to the highly inducible mechanism of antibiotic resistance. This review comprehensively summarizes the regulatory networks of the major porin proteins (OprD and OprH) and efflux pumps (MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY) that play critical roles in antibiotic influx and efflux in P. aeruginosa. It also discusses promising therapeutic approaches using safe and efficient adjuvants to enhance the efficacy of conventional antibiotics to combat multidrug-resistant P. aeruginosa by controlling the expression levels of porins and efflux pumps. This review not only highlights the complexity of the regulatory network that induces antibiotic resistance in P. aeruginosa but also provides important therapeutic implications in targeting the inducible mechanism of resistance.


AdeIJK Pump-Specific Inhibitors Effective against Multidrug Resistant Acinetobacter baumannii.

Tambat R, Kinthada RK, Saral Sariyer A, Leus IV, Sariyer E, D’Cunha N, Zhou H, Leask M, Walker JK, Zgurskaya HI.

ACS Infect Dis. 2024 May 24.

doi: 10.1021/acsinfecdis.4c00190. Epub ahead of print.

PMID: 38787939.

Multidrug-resistant Acinetobacter baumannii is a serious threat pathogen rapidly spreading in clinics and causing a range of complicated human infections. The major contributor to A. baumannii antibiotic resistance is the overproduction of AdeIJK and AdeABC multidrug efflux pumps of the resistance-nodulation-division (RND) superfamily of proteins. The dominant role of efflux in antibiotic resistance and the relatively high permeability of the A. baumannii outer membrane to amphiphilic compounds make this pathogen a promising target for the discovery of clinically relevant efflux pump inhibitors. In this study, we identified 4,6-diaminoquoniline analogs with inhibitory activities against A. baumannii AdeIJK efflux pump and followed up on these compounds with a focused synthetic program to improve the target specificity and to reduce cytotoxicity. We identified several candidates that potentiate antibacterial activities of antibiotics erythromycin, tetracycline, and novobiocin not only in the laboratory antibiotic susceptible strain A. baumannii ATCC17978 but also in multidrug-resistant clinical isolates AB5075 and AYE. The best analogs potentiated the activities of antibiotics in low micromolar concentrations, did not have antibacterial activities on their own, inhibited AdeIJK-mediated efflux of its fluorescent substrate ethidium ion, and had low cytotoxicity in A549 human lung epithelial cells.


Effects of pleiotropic ompR and envZ alleles of Escherichia coli on envelope stress and antibiotic sensitivity.

Gerken H, Shetty D, Kern B, Kenney LJ, Misra R.

J Bacteriol. 2024 May 29:e0017224.

doi: 10.1128/jb.00172-24. Epub ahead of print.

PMID: 38809006.

The EnvZ-OmpR two-component system of Escherichia coli regulates the expression of the ompF and ompC porin genes in response to medium osmolarity. However, certain mutations in envZ confer pleiotropy by affecting the expression of genes of the iron and maltose regulons not normally controlled by EnvZ-OmpR. In this study, we obtained two novel envZ and ompR pleiotropic alleles, envZT15P and ompRL19Q, among revertants of a mutant with heightened envelope stress and an outer membrane (OM) permeability defect. Unlike envZ, pleiotropic mutations in ompR have not been described previously. The mutant alleles reduced the expression of several outer membrane proteins (OMPs), overcame the temperature-sensitive growth defect of a protease-deficient (ΔdegP) strain, and lowered envelope stress and OM permeability defects in a background lacking the BamB protein of an essential β-barrel assembly machinery complex. Biochemical analysis showed OmpRL19Q, like wild-type OmpR, is readily phosphorylated by EnvZ, but the EnvZ-dependent dephosphorylation of OmpRL19Q~P was drastically impaired compared to wild-type OmpR. This defect would lead to a prolonged half-life for OmpRL19Q~P, an outcome remarkably similar to what we had previously described for EnvZR397L, resulting in pleiotropy. By employing null alleles of the OMP genes, it was determined that the three pleiotropic alleles lowered envelope stress by reducing OmpF and LamB levels. The absence of LamB was principally responsible for lowering the OM permeability defect, as assessed by the reduced sensitivity of a ΔbamB mutant to vancomycin and rifampin. Possible mechanisms by which novel EnvZ and OmpR mutants influence EnvZ-OmpR interactions and activities are discussed.IMPORTANCEMaintenance of the outer membrane (OM) integrity is critical for the survival of Gram-negative bacteria. Several envelope homeostasis systems are activated when OM integrity is perturbed. Through the isolation and characterization of novel pleiotropic ompR/envZ alleles, this study highlights the involvement of the EnvZ-OmpR two-component system in lowering envelope stress and the OM permeability defect caused by the loss of proteins that are involved in OM biogenesis, envelope homeostasis, and structural integrity.


Structure and activity of the septal peptidoglycan hydrolysis machinery crucial for bacterial cell division.

Chen Y, Gu J, Yang B, Yang L, Pang J, Luo Q, Li Y, Li D, Deng Z, Dong C, Dong H, Zhang Z.

PLoS Biol. 2024 May 30;22(5):e3002628.

doi: 10.1371/journal.pbio.3002628.

PMID: 38814940.

The peptidoglycan (PG) layer is a critical component of the bacterial cell wall and serves as an important target for antibiotics in both gram-negative and gram-positive bacteria. The hydrolysis of septal PG (sPG) is a crucial step of bacterial cell division, facilitated by FtsEX through an amidase activation system. In this study, we present the cryo-EM structures of Escherichia coli FtsEX and FtsEX-EnvC in the ATP-bound state at resolutions of 3.05 Å and 3.11 Å, respectively. Our PG degradation assays in E. coli reveal that the ATP-bound conformation of FtsEX activates sPG hydrolysis of EnvC-AmiB, whereas EnvC-AmiB alone exhibits autoinhibition. Structural analyses indicate that ATP binding induces conformational changes in FtsEX-EnvC, leading to significant differences from the apo state. Furthermore, PG degradation assays of AmiB mutants confirm that the regulation of AmiB by FtsEX-EnvC is achieved through the interaction between EnvC-AmiB. These findings not only provide structural insight into the mechanism of sPG hydrolysis and bacterial cell division, but also have implications for the development of novel therapeutics targeting drug-resistant bacteria.


‘Smart’ antibiotic can kill deadly bacteria while sparing the microbiome.

Schwaller F.

Nature. 2024 May 29.

doi: 10.1038/d41586-024-01566-8. Epub ahead of print.

PMID: 38811787.

An antibiotic called lolamicin targets disease-causing Gram-negative bacteria without disturbing healthy gut bacteria. Broad-spectrum antibiotics against these pathogens wreak havoc on the gut microbiome and can allow potentially deadly Clostridioides difficile to take over. Mice infected with antibiotic-resistant Gram-negative bacteria survived after being given lolamicin whereas almost 90% of those that didnt receive the drug died within three days. Lolamicin did not seem to disrupt the gut microbiome and spared mice from C. difficile infections.



The AI revolution is coming to robots: how will it change them?

Gibney E.

Nature. 2024 Jun;630(8015):22-24.

doi: 10.1038/d41586-024-01442-5.

PMID: 38822186.

Artificial intelligence (AI) algorithms such as those that power chatbots could imbue robots with the common-sense knowledge they need to cook dinner or run errands. I wouldnt be surprised if we are the last generation for which those sci-fi scenes are not a reality” says robotics researcher Alexander Khazatsky. There are already some impressive demonstrations of AI-powered robots but the dearth of movement data for robots to learn from coupled with temperamental hardware means that it could be a while until we can rely on droid helpers. And there are concerns that AI systemsbiases and mistakes could cause physical harm. Until we have confidence in robots we will need a lot of human supervision” says AI researcher Keerthana Gopalakrishnan.


Does sleep really clean the brain? Maybe not new paper argues

A mouse study challenges popular glymphatic” theory but its methodology is drawing criticism.

A study seems to cast doubt on the theory that we need sleep to clean waste products from the brain. In mice sleep apparently slows down rather than speeds up their brains ability to remove a dye. Some researchers see this as a blow to the sleep clearance theory. Critics say that the studys method for measuring dye removal could have damaged the brains waste-clearance system and is too different from previous studies to credibly challenge them. The brain could even have different ways to clear small compounds such as a dye and large ones such as the beta-amyloid protein linked to Alzheimers disease says brain circulation specialist Erik Bakker.

Brain clearance is reduced during sleep and anesthesia.

Miao A, Luo T, Hsieh B, Edge CJ, Gridley M, Wong RTC, Constandinou TG, Wisden W, Franks NP.

Nat Neurosci. 2024 May 13.

doi: 10.1038/s41593-024-01638-y. Epub ahead of print.

PMID: 38741022.


Why do we clap?

Behavioural scientist Richard Mann investigated how applause spreads — a useful perspective when watching audiences applaud for up to 22 minutes on the trot at the Cannes Film Festival.

You have this social pressure to start (clapping) but once youve started theres an equally strong social pressure not to stop.”