MP

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.

Distinct roles of the major binding residues in the cation-binding pocket of the melibiose transporter MelB.

Hariharan P, Bakhtiiari A, Liang R, Guan L.

J Biol Chem. 2024 May 31;300(7):107427.

doi: 10.1016/j.jbc.2024.107427. Epub ahead of print.

PMID: 38823641.

Salmonella enterica serovar Typhimurium melibiose permease (MelBSt) is a prototype of the major facilitator superfamily (MFS) transporters, which play important roles in human health and diseases. MelBSt catalyzed the symport of galactosides with Na+, Li+, or H+ but prefers the coupling with Na+. Previously, we determined the structures of the inward- and outward-facing conformation of MelBSt and the molecular recognition for galactoside and Na+. However, the molecular mechanisms for H+– and Na+-coupled symport remain poorly understood. In this study, we solved two x-ray crystal structures of MelBSt, the cation-binding site mutants D59C at an unliganded apo-state and D55C at a ligand-bound state, and both structures display the outward-facing conformations virtually identical as published. We determined the energetic contributions of three major Na+-binding residues for the selection of Na+ and H+ by free energy simulations. Transport assays showed that the D55C mutant converted MelBSt to a solely H+-coupled symporter, and together with the free-energy perturbation calculation, Asp59 is affirmed to be the sole protonation site of MelBSt. Unexpectedly, the H+-coupled melibiose transport exhibited poor activities at greater bulky ΔpH and better activities at reversal ΔpH, supporting the novel theory of transmembrane-electrostatically localized protons and the associated membrane potential as the primary driving force for the H+-coupled symport mediated by MelBSt. This integrated study of crystal structure, bioenergetics, and free energy simulations, demonstrated the distinct roles of the major binding residues in the cation-binding pocket of MelBSt.

Structure-based drug design for TSPO: Challenges and opportunities.

Giladi M, Montgomery AP, Kassiou M, Danon JJ.

Biochimie. 2024 May 22:S0300-9084(24)00120-2.

doi: 10.1016/j.biochi.2024.05.018. Epub ahead of print.

PMID: 38782353.

The translocator protein 18 kDa (TSPO) is an evolutionarily conserved mitochondrial transmembrane protein implicated in various neuropathologies and inflammatory conditions, making it a longstanding diagnostic and therapeutic target of interest. Despite the development of various classes of TSPO ligand chemotypes, and the elucidation of bacterial and non-human mammalian experimental structures, many unknowns exist surrounding its differential structural and functional features in health and disease. There are several limitations associated with currently used computational methodologies for modelling the native structure and ligand-binding behaviour of this enigmatic protein. In this perspective, we provide a critical analysis of the developments in the uses of these methods, outlining their uses, inherent limitations, and continuing challenges. We offer suggestions of unexplored opportunities that exist in the use of computational methodologies which offer promise for enhancing our understanding of the TSPO.

Rational Design of Drugs Targeting G-Protein-Coupled Receptors: A Structural Biology Perspective.

Khorn PA, Luginina AP, Pospelov VA, Dashevsky DE, Khnykin AN, Moiseeva OV, Safronova NA, Belousov AS, Mishin AV, Borshchevsky VI.

Biochemistry (Mosc). 2024 Apr;89(4):747-764.

doi: 10.1134/S0006297924040138. PMID: 38831510.

G protein-coupled receptors (GPCRs) play a key role in the transduction of extracellular signals to cells and regulation of many biological processes, which makes these membrane proteins one of the most important targets for pharmacological agents. A significant increase in the number of resolved atomic structures of GPCRs has opened the possibility of developing pharmaceuticals targeting these receptors via structure-based drug design (SBDD). SBDD employs information on the structure of receptor-ligand complexes to search for selective ligands without the need for an extensive high-throughput experimental ligand screening and can significantly expand the chemical space for ligand search. In this review, we describe the process of deciphering GPCR structures using X-ray diffraction analysis and cryoelectron microscopy as an important stage in the rational design of drugs targeting this receptor class. Our main goal was to present modern developments and key features of experimental methods used in SBDD of GPCR-targeting agents to a wide range of specialists.

The Role of Cornichons in the Biogenesis and Functioning of Monovalent-Cation Transport Systems.

Papoušková K, Černá K, Radová V, Zimmermannová O.

Physiol Res. 2024 Jun 5. Epub ahead of print.

PMID: 38836370.

Monovalent-cation homeostasis, crucial for all living cells, is ensured by the activity of various types of ion transport systems located either in the plasma membrane or in the membranes of organelles. A key prerequisite for the functioning of ion-transporting proteins is their proper trafficking to the target membrane. The cornichon family of COPII cargo receptors is highly conserved in eukaryotic cells. By simultaneously binding their cargoes and a COPII-coat subunit, cornichons promote the incorporation of cargo proteins into the COPII vesicles and, consequently, the efficient trafficking of cargoes via the secretory pathway. In this review, we summarize current knowledge about cornichon proteins (CNIH/Erv14), with an emphasis on yeast and mammalian cornichons and their role in monovalent-cation homeostasis. Saccharomyces cerevisiae cornichon Erv14 serves as a cargo receptor of a large portion of plasma-membrane proteins, including several monovalent-cation transporters. By promoting the proper targeting of at least three housekeeping ion transport systems, Na+, K+/H+ antiporter Nha1, K+ importer Trk1 and K+ channel Tok1, Erv14 appears to play a complex role in the maintenance of alkali-metal-cation homeostasis. Despite their connection to serious human diseases, the repertoire of identified cargoes of mammalian cornichons is much more limited. The majority of current information is about the structure and functioning of CNIH2 and CNIH3 as auxiliary subunits of AMPAR multi-protein complexes. Based on their unique properties and easy genetic manipulation, we propose yeast cells to be a useful tool for uncovering a broader spectrum of human cornichons´ cargoes.

Exploring transmembrane allostery in the MexB: DB08385 variant as a promising inhibitor-like candidate against Pseudomonas aeruginosa antibiotic resistance: a computational study.

Bera A, Mukherjee S, Patra N.

Phys Chem Chem Phys. 2024 Jun 19;26(24):17011-17027.

doi: 10.1039/d4cp01620c. PMID: 38835320.

Pseudomonas aeruginosa, a formidable pathogen renowned for its antimicrobial resistance, poses a significant threat to immunocompromised individuals. In this regard, the MexAB-OprM efflux pump acts as a pivotal line of defense by extruding antimicrobials from bacterial cells. The inner membrane homotrimeric protein MexB captures antibiotics and translocates them into the outer membrane OprM channel protein connected through the MexA adaptor protein. Despite extensive efforts, competitive inhibitors targeting the tight (T) protomer of the MexB protein have not received FDA approval for medical use. Over the past few years, allosteric inhibitors have become popular as alternatives to the classical competitive inhibitor-based approach because of their higher specificity, lower dosage, and reduced toxicological effects. Hence, in this study, we unveiled the existence of a transmembrane allosteric binding pocket of MexB inspired by the recent discovery of an important allosteric inhibitor, BDM88855, for the homolog AcrB protein. While repurposing BDM88855 proved ineffective in controlling the MexB loose (L) protomer, our investigation identified a promising alternative: a chlorine-containing variant of DB08385 (2-Cl DB08385 or Variant 1). Molecular dynamics simulations, including binding free energy estimation coupled with heterogeneous dielectric implicit membrane model (implicit-membrane MM/PBSA), interaction entropy (IE) analysis and potential of mean force (PMF) calculation, demonstrated Variant 1’s superior binding affinity to the transmembrane pocket, displaying the highest energy barrier in the ligand unbinding process. To elucidate the allosteric crosstalk between the transmembrane and porter domain of MexB, we employed the ‘eigenvector centrality’ measure in the linear mutual information obtained from the protein correlation network. Notably, this study confirmed the presence of an allosteric transmembrane site in the MexB L protomer. In addition to this, Variant 1 emerged as a potent regulator of allosteric crosstalk, inducing an ‘O-L intermediate state’ in the MexB L protomer. This induced state might hold the potential to diminish substrate intake into the access pocket, leading to the ineffective efflux of antibiotics.

MFSD1 with its accessory subunit GLMP functions as a general dipeptide uniporter in lysosomes.

Jungnickel KEJ, Guelle O, Iguchi M, Dong W, Kotov V, Gabriel F, Debacker C, Dairou J, McCort-Tranchepain I, Laqtom NN, Chan SH, Ejima A, Sato K, Massa López D, Saftig P, Mehdipour AR, Abu-Remaileh M, Gasnier B, Löw C, Damme M.

Nat Cell Biol. 2024 Jun 5.

doi: 10.1038/s41556-024-01436-5. Epub ahead of print.

PMID: 38839979.

The lysosomal degradation of macromolecules produces diverse small metabolites exported by specific transporters for reuse in biosynthetic pathways. Here we deorphanized the major facilitator superfamily domain containing 1 (MFSD1) protein, which forms a tight complex with the glycosylated lysosomal membrane protein (GLMP) in the lysosomal membrane. Untargeted metabolomics analysis of MFSD1-deficient mouse lysosomes revealed an increase in cationic dipeptides. Purified MFSD1 selectively bound diverse dipeptides, while electrophysiological, isotope tracer and fluorescence-based studies in Xenopus oocytes and proteoliposomes showed that MFSD1-GLMP acts as a uniporter for cationic, neutral and anionic dipeptides. Cryoelectron microscopy structure of the dipeptide-bound MFSD1-GLMP complex in outward-open conformation characterized the heterodimer interface and, in combination with molecular dynamics simulations, provided a structural basis for its selectivity towards diverse dipeptides. Together, our data identify MFSD1 as a general lysosomal dipeptide uniporter, providing an alternative route to recycle lysosomal proteolysis products when lysosomal amino acid exporters are overloaded.

Dimerization and antidepressant recognition at noradrenaline transporter.

Zhang H, Yin YL, Dai A, Zhang T, Zhang C, Wu C, Hu W, He X, Pan B, Jin S, Yuan Q, Wang MW, Yang D, Xu HE, Jiang Y.

Nature. 2024 Jun;630(8015):247-254.

doi: 10.1038/s41586-024-07437-6. Epub 2024 May 15.

PMID: 38750358.

The noradrenaline transporter has a pivotal role in regulating neurotransmitter balance and is crucial for normal physiology and neurobiology1. Dysfunction of noradrenaline transporter has been implicated in numerous neuropsychiatric diseases, including depression and attention deficit hyperactivity disorder2. Here we report cryo-electron microscopy structures of noradrenaline transporter in apo and substrate-bound forms, and as complexes with six antidepressants. The structures reveal a noradrenaline transporter dimer interface that is mediated predominantly by cholesterol and lipid molecules. The substrate noradrenaline binds deep in the central binding pocket, and its amine group interacts with a conserved aspartate residue. Our structures also provide insight into antidepressant recognition and monoamine transporter selectivity. Together, these findings advance our understanding of noradrenaline transporter regulation and inhibition, and provide templates for designing improved antidepressants to treat neuropsychiatric disorders.

Re-evaluating TRP channel mechanosensitivity.

Cox CD, Poole K, Martinac B.

Trends Biochem Sci. 2024 Jun 7:S0968-0004(24)00114-2.

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

PMID: 38851904.

Transient receptor potential (TRP) channels are implicated in a wide array of mechanotransduction processes. However, a question remains whether TRP channels directly sense mechanical force, thus acting as primary mechanotransducers. We use several recent examples to demonstrate the difficulty in definitively ascribing mechanosensitivity to TRP channel subfamilies. Ultimately, despite being implicated in an ever-growing list of mechanosignalling events in most cases limited robust or reproducible evidence supports the contention that TRP channels act as primary transducers of mechanical forces. They either (i) possess unique and as yet unspecified structural or local requirements for mechanosensitivity; or (ii) act as mechanoamplifiers responding downstream of the activation of a primary mechanotransducer that could include Ca2+-permeable mechanosensitive (MS) channels or other potentially unidentified mechanosensors.

Design of a water-soluble transmembrane receptor kinase with intact molecular function by QTY code.

Li M, Tang H, Qing R, Wang Y, Liu J, Wang R, Lyu S, Ma L, Xu P, Zhang S, Tao F.

Nat Commun. 2024 Jun 10;15(1):4293.

doi: 10.1038/s41467-024-48513-9.

PMID: 38858360.

Membrane proteins are critical to biological processes and central to life sciences and modern medicine. However, membrane proteins are notoriously challenging to study, mainly owing to difficulties dictated by their highly hydrophobic nature. Previously, we reported QTY code, which is a simple method for designing water-soluble membrane proteins. Here, we apply QTY code to a transmembrane receptor, histidine kinase CpxA, to render it completely water-soluble. The designed CpxAQTY exhibits expected biophysical properties and highly preserved native molecular function, including the activities of (i) autokinase, (ii) phosphotransferase, (iii) phosphatase, and (iv) signaling receptor, involving a water-solubilized transmembrane domain. We probe the principles underlying the balance of structural stability and activity in the water-solubilized transmembrane domain. Computational approaches suggest that an extensive and dynamic hydrogen-bond network introduced by QTY code and its flexibility may play an important role. Our successful functional preservation further substantiates the robustness and comprehensiveness of QTY code.

Molecular mechanism of choline and ethanolamine transport in humans.

Ri K, Weng TH, Claveras Cabezudo A, Jösting W, Zhang Y, Bazzone A, Leong NCP, Welsch S, Doty RT, Gursu G, Lim TJY, Schmidt SL, Abkowitz JL, Hummer G, Wu D, Nguyen LN, Safarian S.

Nature. 2024 Jun;630(8016):501-508.

doi: 10.1038/s41586-024-07444-7. Epub 2024 May 22.

PMID: 38778100.

Human feline leukaemia virus subgroup C receptor-related proteins 1 and 2 (FLVCR1 and FLVCR2) are members of the major facilitator superfamily1. Their dysfunction is linked to several clinical disorders, including PCARP, HSAN and Fowler syndrome2-7. Earlier studies concluded that FLVCR1 may function as a haem exporter8-12, whereas FLVCR2 was suggested to act as a haem importer13, yet conclusive biochemical and detailed molecular evidence remained elusive for the function of both transporters14-16. Here, we show that FLVCR1 and FLVCR2 facilitate the transport of choline and ethanolamine across the plasma membrane, using a concentration-driven substrate translocation process. Through structural and computational analyses, we have identified distinct conformational states of FLVCRs and unravelled the coordination chemistry underlying their substrate interactions. Fully conserved tryptophan and tyrosine residues form the binding pocket of both transporters and confer selectivity for choline and ethanolamine through cation-π interactions. Our findings clarify the mechanisms of choline and ethanolamine transport by FLVCR1 and FLVCR2, enhance our comprehension of disease-associated mutations that interfere with these vital processes and shed light on the conformational dynamics of these major facilitator superfamily proteins during the transport cycle.

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 Jun 14;10(6):2239-2249.

doi: 10.1021/acsinfecdis.4c00190. Epub 2024 May 24.

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.

A novel function of the M2 muscarinic receptor.

Wess J, Liu L.

Trends Pharmacol Sci. 2024 Jun 8:S0165-6147(24)00116-0.

doi: 10.1016/j.tips.2024.05.010. Epub ahead of print.

PMID: 38853101.

The M2 muscarinic receptor (M2R) is a prototypic class A G protein-coupled receptor (GPCR). Interestingly, Fasciani et al. recently identified an internal translation start site within the M2 receptor mRNA, directing the expression of a C-terminal receptor fragment. Elevated during cellular stress, this polypeptide localizes to mitochondria where it inhibits oxidative phosphorylation.

Computational design of soluble and functional membrane protein analogues.

Goverde CA, Pacesa M, Goldbach N, Dornfeld LJ, Balbi PEM, Georgeon S, Rosset S, Kapoor S, Choudhury J, Dauparas J, Schellhaas C, Kozlov S, Baker D, Ovchinnikov S, Vecchio AJ, Correia BE.

Nature. 2024 Jun 19.

doi: 10.1038/s41586-024-07601-y. Epub ahead of print.

PMID: 38898281.

De novo design of complex protein folds using solely computational means remains a substantial challenge1. Here we use a robust deep learning pipeline to design complex folds and soluble analogues of integral membrane proteins. Unique membrane topologies, such as those from G-protein-coupled receptors2, are not found in the soluble proteome, and we demonstrate that their structural features can be recapitulated in solution. Biophysical analyses demonstrate the high thermal stability of the designs, and experimental structures show remarkable design accuracy. The soluble analogues were functionalized with native structural motifs, as a proof of concept for bringing membrane protein functions to the soluble proteome, potentially enabling new approaches in drug discovery. In summary, we have designed complex protein topologies and enriched them with functionalities from membrane proteins, with high experimental success rates, leading to a de facto expansion of the functional soluble fold space.

Structures reveal how SGLT inhibitors work.

Sun Z, Cui W, Chen L.

Trends Pharmacol Sci. 2024 Jun 18:S0165-6147(24)00101-9.

doi: 10.1016/j.tips.2024.05.009. Epub ahead of print.

PMID: 38897851.

Sodium glucose cotransporters (SGLTs) transport glucose against its concentration gradient by harnessing the electrochemical potential gradient of sodium ions. SGLT inhibitors are widely prescribed to treat diabetes and other conditions. Recent structural studies have uncovered how chemically diverse SGLT inhibitors bind and inhibit the transporter at the atomic level.

Nanobody-assisted cryoEM structural determination for challenging proteins.

Wu X.

Trends Biochem Sci. 2024 Jun 20:S0968-0004(24)00136-1.

TECHNOLOGY OF THE MONTH

doi: 10.1016/j.tibs.2024.06.002. Epub ahead of print.

PMID: 38906724.

Nanobodies (~15 kDa) are particularly useful for cryoEM study. Targets of interest are screened for specific nanobody binders that are then used to stabilize different conformations of targets. To improve the signal:noise ratio and to add more recognizable features, nanobodies can be further enlarged using methods such as Megabody, Legobody, and NabFab. Subunits of large complexes can also be screened for nanobodies. Nanobody-conjugated beads can capture and enrich the entire complex from endogenous tissues, especially in cases where genetic manipulation is challenging. The purified complex can then be subjected to cryoEM analysis.

Mg2+-dependent mechanism of environmental versatility in a multidrug efflux pump.

Lewis BR, Uddin MR, Kuo KM, Shah LMN, Harris NJ, Booth PJ, Hammerschmid D, Gumbart JC, Zgurskaya HI, Reading E.

bioRxiv [Preprint]. 2024 Jun 10:2024.06.10.597921.

doi: 10.1101/2024.06.10.597921.

PMID: 38915626.

Tripartite resistance nodulation and cell division multidrug efflux pumps span the periplasm and are a major driver of multidrug resistance among Gram-negative bacteria. The periplasm provides a distinct environment between the inner and outer membranes of Gram-negative bacteria. Cations, such as Mg2+, become concentrated within the periplasm and, in contrast to the cytoplasm, its pH is sensitive to conditions outside the cell. Here, we reveal an interplay between Mg2+ and pH in modulating the dynamics of the periplasmic adaptor protein, AcrA, and its function within the prototypical AcrAB-TolC multidrug efflux pump from Escherichia coli. In the absence of Mg2+, AcrA becomes increasingly plastic within acidic conditions, but when Mg2+ is bound this is ameliorated, resulting in domain specific organisation in neutral to weakly acidic regimes. We establish a unique histidine residue directs these structural dynamics and is essential for sustaining pump efflux activity across acidic, neutral, and alkaline conditions. Overall, we propose Mg2+ conserves the structural mobility of AcrA to ensure optimal AcrAB-TolC function within rapid changing environments commonly faced by the periplasm during bacterial infection and colonization. This work highlights that Mg2+ is an important mechanistic component in this pump class and possibly across other periplasmic lipoproteins.

Structural analysis of resistance-nodulation cell division transporters.

Klenotic PA, Yu EW.

Microbiol Mol Biol Rev. 2024 Jun 27;88(2):e0019823.

doi: 10.1128/mmbr.00198-23. Epub 2024 Mar 29.

PMID: 38551344.

SUMMARYInfectious bacteria have both intrinsic and acquired mechanisms to combat harmful biocides that enter the cell. Through adaptive pressures, many of these pathogens have become resistant to many, if not all, of the current antibiotics used today to treat these often deadly infections. One prominent mechanism is the upregulation of efflux systems, especially the resistance-nodulation-cell division class of exporters. These tripartite systems consist of an inner membrane transporter coupled with a periplasmic adaptor protein and an outer membrane channel to efficiently transport a diverse array of substrates from inside the cell to the extracellular space. Detailed mechanistic insight into how these inner membrane transporters recognize and shuttle their substrates can ultimately inform both new antibiotic and efflux pump inhibitor design. This review examines the structural basis of substrate recognition of these pumps and the molecular mechanisms underlying multidrug extrusion, which in turn mediate antimicrobial resistance in bacterial pathogens.

Dimeric transport mechanism of human vitamin C transporter SVCT1.

Kobayashi TA, Shimada H, Sano FK, Itoh Y, Enoki S, Okada Y, Kusakizako T, Nureki O.

Nat Commun. 2024 Jul 2;15(1):5569.

doi: 10.1038/s41467-024-49899-2.

PMID: 38956111.

Vitamin C plays important roles as a cofactor in many enzymatic reactions and as an antioxidant against oxidative stress. As some mammals including humans cannot synthesize vitamin C de novo from glucose, its uptake from dietary sources is essential, and is mediated by the sodium-dependent vitamin C transporter 1 (SVCT1). Despite its physiological significance in maintaining vitamin C homeostasis, the structural basis of the substrate transport mechanism remained unclear. Here, we report the cryo-EM structures of human SVCT1 in different states at 2.5-3.5 Å resolutions. The binding manner of vitamin C together with two sodium ions reveals the counter ion-dependent substrate recognition mechanism. Furthermore, comparisons of the inward-open and occluded structures support a transport mechanism combining elevator and distinct rotational motions. Our results demonstrate the molecular mechanism of vitamin C transport with its underlying conformational cycle, potentially leading to future industrial and medical applications.

Computational design of de novo bioenergetic membrane proteins.

Hardy BJ, Curnow P.

Biochem Soc Trans. 2024 Jul 3:BST20231347.

doi: 10.1042/BST20231347. Epub ahead of print.

PMID: 38958574.

The major energy-producing reactions of biochemistry occur at biological membranes. Computational protein design now provides the opportunity to elucidate the underlying principles of these processes and to construct bioenergetic pathways on our own terms. Here, we review recent achievements in this endeavour of ‘synthetic bioenergetics’, with a particular focus on new enabling tools that facilitate the computational design of biocompatible de novo integral membrane proteins. We use recent examples to showcase some of the key computational approaches in current use and highlight that the overall philosophy of ‘surface-swapping’ – the replacement of solvent-facing residues with amino acids bearing lipid-soluble hydrophobic sidechains – is a promising avenue in membrane protein design. We conclude by highlighting outstanding design challenges and the emerging role of AI in sequence design and structure ideation.

Interdomain communication in a homodimeric ABC transporter.

Lindt KA, Frühschulz S, Tampé R, Abele R.

J Biol Chem. 2024 Jun 5;300(7):107440.

doi: 10.1016/j.jbc.2024.107440. Epub ahead of print.

PMID: 38844133.

ABC transporters are found in all organisms and almost every cellular compartment. They mediate the transport of various solutes across membranes, energized by ATP binding and hydrolysis. Dysfunctions can result in severe diseases, such as cystic fibrosis or antibiotic resistance. In type IV ABC transporters, each of the two nucleotide-binding domains is connected to a transmembrane domain by two coupling helices, which are part of cytosolic loops. Although there are many structural snapshots of different conformations, the interdomain communication is still enigmatic. Therefore, we analyzed the function of three conserved charged residues in the intracytosolic loop 1 of the human homodimeric, lysosomal peptide transporter TAPL (transporter associated with antigen processing-like). Substitution of D278 in coupling helix 1 by alanine interrupted peptide transport by impeding ATP hydrolysis. Alanine substitution of R288 and D292, both localized next to the coupling helix 1 extending to transmembrane helix 3, reduced peptide transport but increased basal ATPase activity. Surprisingly, the ATPase activity of the R288A variant dropped in a peptide-dependent manner, whereas ATPase activity of wildtype and D292A was unaffected. Interestingly, R288A and D292A mutants did not differentiate between ATP and GTP in respect of hydrolysis. However, in contrast to wildtye TAPL, only ATP energized peptide transport. In sum, D278 seems to be involved in bidirectional interdomain communication mediated by network of polar interactions, whereas the two residues in the cytosolic extension of transmembrane helix 3 are involved in regulation of ATP hydrolysis, most likely by stabilization of the outward-facing conformation.

Discovery and Characterization of Two Folded Intermediates for Outer Membrane Protein TolC Biogenesis.

Ikujuni AP, Dhar R, Cordova A, Bowman AM, Noga S, Slusky JSG.

J Mol Biol. 2024 Jun 11;436(16):168652.

doi: 10.1016/j.jmb.2024.168652. Epub ahead of print.

PMID: 38871177.

TolC is the outer membrane protein responsible for antibiotic efflux in E. coli. Compared to other outer membrane proteins it has an unusual fold and has been shown to fold independently of commonly used periplasmic chaperones, SurA and Skp. Here we find that the assembly of TolC involves the formation of two folded intermediates using circular dichroism, gel electrophoresis, site-specific disulfide bond formation and radioactive labeling. First the TolC monomer folds, and then TolC assembles into a trimer both in detergent-free buffer and in the presence of detergent micelles. We find that a TolC trimer also forms in the periplasm and is present in the periplasm before it inserts in the outer membrane. The monomeric and trimeric folding intermediates may be used in the future to develop a new approach to antibiotic efflux pump inhibition by targeting the assembly pathway of TolC.

Cryo-EM structure of a conjugative type IV secretion system suggests a molecular switch regulating pilus biogenesis.

Macé K, Waksman G.

EMBO J. 2024 Jun 17. doi: 10.1038/s44318-024-00135-z.

Epub ahead of print. PMID: 38886579.

Conjugative type IV secretion systems (T4SS) mediate bacterial conjugation, a process that enables the unidirectional exchange of genetic materials between a donor and a recipient bacterial cell. Bacterial conjugation is the primary means by which antibiotic resistance genes spread among bacterial populations (Barlow 2009; Virolle et al, 2020). Conjugative T4SSs form pili: long extracellular filaments that connect with recipient cells. Previously, we solved the cryo-electron microscopy (cryo-EM) structure of a conjugative T4SS. In this article, based on additional data, we present a more complete T4SS cryo-EM structure than that published earlier. Novel structural features include details of the mismatch symmetry within the OMCC, the presence of a fourth VirB8 subunit in the asymmetric unit of both the arches and the inner membrane complex (IMC), and a hydrophobic VirB5 tip in the distal end of the stalk. Additionally, we provide previously undescribed structural insights into the protein VirB10 and identify a novel regulation mechanism of T4SS-mediated pilus biogenesis by this protein, that we believe is a key checkpoint for this process.

One-step drug transport across two membranes of Gram-negative bacteria

Ben Luisi, Zhaojun Zhong, Tuerxunjiang Maimaiti, Xueyan Gao, Rui Dong, Matthew Jackson, Wenyu Shang, Hongnian Jiang, Jinliang Guo, Shangrong Li, Huimin Zhao, Qing Ouyang, Huanjun Liu, Yanjie Chao, Dijun Du

https://www.researchsquare.com/article/rs-4468934/latest

preprint on Research Square

Transport of proteins and small molecules across the complex cell envelope of Gram-negative bacteria is crucial for their survival and interaction with their environment and is facilitated by specialized macromolecular machines that enable direct one-step or indirect two-step translocation of substrates. Major facilitator superfamily (MFS)-type tripartite efflux pumps and type I secretion systems likely employ a similar one-step mechanism for substrate transport across cell membranes, but the structural details remain elusive. A representative MFS-type tripartite efflux pump, EmrAB-TolC, mediates multidrug resistance through proton-coupled EmrB, a member of the DHA2 transporter family. Here, we demonstrate that the EmrAB-TolC pump confers resistance to clinical antibiotics, including polymyxin B and neomycin, and report the high-resolution (3.11 Å) structure of the pump, revealing a unique, asymmetric architecture emerging from the TolC:EmrA:EmrB ratio of 3:6:1. This structure identifies two critical subdomains, AssA and AssB, essential for pump assembly and key residues involved in pump assembly, drug recognition, proton translocation and coupling, which are corroborated by mutagenesis and antibiotic sensitivity assays. The delineation of the complete translocation pathway reveals the molecular mechanism for one-step drug transport process across the entire cell envelope.

Membrane

Functional diversity among cardiolipin binding sites on the mitochondrial ADP/ATP carrier.

Senoo N, Chinthapalli DK, Baile MG, Golla VK, Saha B, Oluwole AO, Ogunbona OB, Saba JA, Munteanu T, Valdez Y, Whited K, Sheridan MS, Chorev D, Alder NN, May ER, Robinson CV, Claypool SM.

EMBO J. 2024 Jun 5.

doi: 10.1038/s44318-024-00132-2. Epub ahead of print.

PMID: 38839991.

Lipid-protein interactions play a multitude of essential roles in membrane homeostasis. Mitochondrial membranes have a unique lipid-protein environment that ensures bioenergetic efficiency. Cardiolipin (CL), the signature mitochondrial lipid, plays multiple roles in promoting oxidative phosphorylation (OXPHOS). In the inner mitochondrial membrane, the ADP/ATP carrier (AAC in yeast; adenine nucleotide translocator, ANT in mammals) exchanges ADP and ATP, enabling OXPHOS. AAC/ANT contains three tightly bound CLs, and these interactions are evolutionarily conserved. Here, we investigated the role of these buried CLs in AAC/ANT using a combination of biochemical approaches, native mass spectrometry, and molecular dynamics simulations. We introduced negatively charged mutations into each CL-binding site of yeast Aac2 and established experimentally that the mutations disrupted the CL interactions. While all mutations destabilized Aac2 tertiary structure, transport activity was impaired in a binding site-specific manner. Additionally, we determined that a disease-associated missense mutation in one CL-binding site in human ANT1 compromised its structure and transport activity, resulting in OXPHOS defects. Our findings highlight the conserved significance of CL in AAC/ANT structure and function, directly tied to specific lipid-protein interactions.

SANS reveals lipid-dependent oligomerization of an intramembrane aspartyl protease from H. volcanii.

Thomas GM, Wu Y, Leite W, Pingali SV, Weiss KL, Grant AJ, Diggs MW, Schmidt-Krey I, Gutishvili G, Gumbart JC, Urban VS, Lieberman RL.

Biophys J. 2024 Jul 2;123(13):1846-1856.

doi: 10.1016/j.bpj.2024.05.029. Epub 2024 Jun 1.

PMID: 38824390.

Reactions that occur within the lipid membrane involve, at minimum, ternary complexes among the enzyme, substrate, and lipid. For many systems, the impact of the lipid in regulating activity or oligomerization state is poorly understood. Here, we used small-angle neutron scattering (SANS) to structurally characterize an intramembrane aspartyl protease (IAP), a class of membrane-bound enzymes that use membrane-embedded aspartate residues to hydrolyze transmembrane segments of biologically relevant substrates. We focused on an IAP ortholog from the halophilic archaeon Haloferax volcanii (HvoIAP). HvoIAP purified in n-dodecyl-β-D-maltoside (DDM) fractionates on size-exclusion chromatography (SEC) as two fractions. We show that, in DDM, the smaller SEC fraction is consistent with a compact HvoIAP monomer. Molecular dynamics flexible fitting conducted on an AlphaFold2-generated monomer produces a model in which loops are compact alongside the membrane-embedded helices. In contrast, SANS data collected on the second SEC fraction indicate an oligomer consistent with an elongated assembly of discrete HvoIAP monomers. Analysis of in-line SEC-SANS data of the HvoIAP oligomer, the first such experiment to be conducted on a membrane protein at Oak Ridge National Lab (ORNL), shows a diversity of elongated and spherical species, including one consistent with the tetrameric assembly reported for the Methanoculleus marisnigri JR1 IAP crystal structure not observed previously in solution. Reconstitution of monomeric HvoIAP into bicelles increases enzyme activity and results in the assembly of HvoIAP into a species with similar dimensions as the ensemble of oligomers isolated from DDM. Our study reveals lipid-mediated HvoIAP self-assembly and demonstrates the utility of in-line SEC-SANS in elucidating oligomerization states of small membrane proteins.

Lipid osmosis, membrane tension, and other mechanochemical driving forces of lipid flow.

Zhang Y, Lin C.

Curr Opin Cell Biol. 2024 Jun;88:102377.

doi: 10.1016/j.ceb.2024.102377. Epub 2024 May 31.

PMID: 38823338.

Nonvesicular lipid transport among different membranes or membrane domains plays crucial roles in lipid homeostasis and organelle biogenesis. However, the forces that drive such lipid transport are not well understood. We propose that lipids tend to flow towards the membrane area with a higher membrane protein density in a process termed lipid osmosis. This process lowers the membrane tension in the area, resulting in a membrane tension difference called osmotic membrane tension. We examine the thermodynamic basis and experimental evidence of lipid osmosis and osmotic membrane tension. We predict that lipid osmosis can drive bulk lipid flows between different membrane regions through lipid transfer proteins, scramblases, or similar barriers that selectively pass lipids but not membrane proteins. We also speculate on the biological functions of lipid osmosis. Finally, we explore other driving forces for lipid transfer and describe potential methods and systems to further test our theory.

TREK2 Lipid Binding Preferences Revealed by Native Mass Spectrometry.

Stover L, Zhu Y, Schrecke S, Laganowsky A.

J Am Soc Mass Spectrom. 2024 Jul 3;35(7):1516-1522.

doi: 10.1021/jasms.4c00112. Epub 2024 Jun 6.

PMID: 38843438.

TREK2, a two-pore domain potassium channel, is recognized for its regulation by various stimuli, including lipids. While previous members of the TREK subfamily, TREK1 and TRAAK, have been investigated to elucidate their lipid affinity and selectivity, TREK2 has not been similarly studied in this regard. Our findings indicate that while TRAAK and TREK2 exhibit similarities in terms of electrostatics and share an overall structural resemblance, there are notable distinctions in their interaction with lipids. Specifically, SAPI(4,5)P2,1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-(1′-myo-inositol-4′,5′-bisphosphate) exhibits a strong affinity for TREK2, surpassing that of dOPI(4,5)P2,1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol-4′,5′-bisphosphate), which differs in its acyl chains. TREK2 displays lipid binding preferences not only for the headgroup of lipids but also toward the acyl chains. Functional studies draw a correlation for lipid binding affinity and activity of the channel. These findings provide important insight into elucidating the molecular prerequisites for specific lipid binding to TREK2 important for function.

Computational analysis of the simultaneous application of ultrasound and electric fields in a lipid bilayer.

Müller WA, Sarkis JR, Marczak LDF, Muniz AR.

Biochim Biophys Acta Biomembr. 2024 Jun 18;1866(7):184364.

doi: 10.1016/j.bbamem.2024.184364. Epub ahead of print.

PMID: 38901662.

The combined application of electric fields and ultrasonic waves has shown promise in controlling cell membrane permeability, potentially resulting in synergistic effects that can be explored in the biotechnology industry. However, further clarification on how these processes interact is still needed. The objective of the present study was to investigate the atomic-scale effects of these processes on a DPPC lipid bilayer using molecular dynamics simulations. For higher electric fields, capable of independently forming pores, the application of an ultrasonic wave in the absence of cavitation yielded no additional effects on pore formation. However, for lower electric fields, the reduction in bilayer thickness induced by the shock wave catalyzed the electroporation process, effectively shortening the mean path that water molecules must traverse to form pores. When cavitation was considered, synergistic effects were evident only if the wave alone was able to generate pores through the formation of a water nanojet. In these cases, sonoporation acted as a mean to focus the electroporation effects on the initial pore formed by the nanojet. This study contributes to a better understanding of the synergy between electric fields and ultrasonic waves and to an optimal selection of processing parameters in practical applications of these processes.

Photobiomodulation of Na,K-ATPase in native membrane fraction and reconstituted in DPPC:DPPE-liposome.

Scanavachi G, Yoneda JS, Sebinelli HG, Barbosa LRS, Ciancaglini P, Itri R.

Photochem Photobiol. 2024 Jun 24. doi: 10.1111/php.13987.

Epub ahead of print.

PMID: 38922888.

Studies focusing on how photobiomodulation (PBM) can affect the structure and function of proteins are scarce in the literature. Few previous studies have shown that the enzymatic activity of Na,K-ATPAse (NKA) can be photo-modulated. However, the variability of sample preparation and light irradiation wavelengths have not allowed for an unequivocal conclusion about the PBM of NKA. Here, we investigate minimal membrane models containing NKA, namely, native membrane fraction and DPPC:DPPE proteoliposome upon laser irradiation at wavelengths 532, 650, and 780 nm. Interestingly, we show that the PBM on the NKA enzymatic activity has a bell-shaped profile with a stimulation peak (~15% increase) at around 20 J.cm-2 and 6 J.cm-2 for the membrane-bound and the proteoliposome samples, respectively, and are practically wavelength independent. Further, by normalizing the enzymatic activity by the NKA enzyme concentration, we show that the PBM response is related to the protein amount with small influence due to protein’s environment. The stimulation decays over time reaching the basal level around 6 h after the irradiation for the three lasers and both NKA samples. Our results demonstrate the potential of using low-level laser therapy to modulate NKA activity, which may have therapeutic implications and benefits.

Lipids and proteins: Insights into the dynamics of assembly, recognition, condensate formation. What is still missing?

Argudo PG.

Biointerphases. 2024 May 1;19(3):038501.

doi: 10.1116/6.0003662.

PMID: 38922634.

Lipid membranes and proteins, which are part of us throughout our lives, have been studied for decades. However, every year, new discoveries show how little we know about them. In a reader-friendly manner for people not involved in the field, this paper tries to serve as a bridge between physicists and biologists and new young researchers diving into the field to show its relevance, pointing out just some of the plethora of lines of research yet to be unraveled. It illustrates how new ways, from experimental to theoretical approaches, are needed in order to understand the structures and interactions that take place in a single lipid, protein, or multicomponent system, as we are still only scratching the surface.

Application of Generative Artificial Intelligence in Predicting Membrane Partitioning of Drugs: Combining Denoising Diffusion Probabilistic Models and MD Simulations Reduces the Computational Cost to One-Third.

Obi P, Gc JB, Mariasoosai C, Diyaolu A, Natesan S.

J Chem Theory Comput. 2024 Jun 28.

doi: 10.1021/acs.jctc.4c00315. Epub ahead of print.

PMID: 38942732.

The optimal interaction of drugs with plasma membranes and membranes of subcellular organelles is a prerequisite for desirable pharmacology. Importantly, for drugs targeting the transmembrane lipid-facing sites of integral membrane proteins, the relative affinity of a drug to the bilayer lipids compared to the surrounding aqueous phase affects the partitioning, access, and binding of the drug to the target site. Molecular dynamics (MD) simulations, including enhanced sampling techniques such as steered MD, umbrella sampling (US), and metadynamics, offer valuable insights into the interactions of drugs with the membrane lipids and water in atomistic detail. However, these methods are computationally prohibitive for the high-throughput screening of drug candidates. This study shows that applying denoising diffusion probabilistic models (DDPMs), a generative AI method, to US simulation data reduces the computational cost significantly. Specifically, the models used only partial (one-third) data from the US simulations and reproduced the complete potential of mean force (PMF) profiles for three FDA-approved drugs (β2-adrenergic agonists) and ∼20 biologically relevant chemicals with known experimentally characterized bilayer locations. Intriguingly, the model can predict the solvation-free energies for partitioning and crossing the bilayer, preferred bilayer locations (low-energy well), and orientations of the ligands with high accuracy. The results indicate that DDPMs can be used to characterize the complete membrane partitioning profile of drug molecules using fewer umbrella sampling simulations at select positions along the bilayer normal (z-axis), irrespective of their amphiphilic-lipophilic-cephalophilic characteristics.

Distance tuneable integral membrane protein containing floating bilayers via in situ directed self-assembly.

Hall SCL, Hardy DJ, Bragginton ÉC, Johnston H, Onose T, Holyfield R, Sridhar P, Knowles TJ, Clifton LA.

Nanoscale. 2024 Jun 28.

doi: 10.1039/d3nr04622b. Epub ahead of print.

PMID: 38940744.

Model membranes allow for structural and biophysical studies on membrane biochemistry at the molecular level, albeit on systems of reduced complexity which can limit biological accuracy. Floating supported bilayers offer a means of producing planar lipid membrane models not adhered to a surface, which allows for improved accuracy compared to other model membranes. Here we communicate the incorporation of an integral membrane protein complex, the multidomain β-barrel assembly machinery (Bam), into our recently developed in situ self-assembled floating supported bilayers. Using neutron reflectometry and quartz crystal microbalance measurements we show this sample system can be fabricated using a two-step self-assembly process. We then demonstrate the complexity of the model membrane and tuneability of the membrane-to-surface distance using changes in the salt concentration of the bulk solution. Results demonstrate an easily fabricated, biologically accurate and tuneable membrane assay system which can be utilized for studies on integral membrane proteins within their native lipid matrix.

Lipid Landscapes: Vibrational Spectroscopy for Decoding Membrane Complexity.

Chen X, Al-Mualem ZA, Baiz CR.

Annu Rev Phys Chem. 2024 Jun;75(1):283-305.

doi: 10.1146/annurev-physchem-090722-010230. Epub 2024 Jun 14.

PMID: 38382566.

Cell membranes are incredibly complex environments containing hundreds of components. Despite substantial advances in the past decade, fundamental questions related to lipid-lipid interactions and heterogeneity persist. This review explores the complexity of lipid membranes, showcasing recent advances in vibrational spectroscopy to characterize the structure, dynamics, and interactions at the membrane interface. We include an overview of modern techniques such as surface-enhanced infrared spectroscopy as a steady-state technique with single-bilayer sensitivity, two-dimensional sum-frequency generation spectroscopy, and two-dimensional infrared spectroscopy to measure time-evolving structures and dynamics with femtosecond time resolution. Furthermore, we discuss the potential of multiscale molecular dynamics (MD) simulations, focusing on recently developed simulation algorithms, which have emerged as a powerful approach to interpret complex spectra. We highlight the ongoing challenges in studying heterogeneous environments in multicomponent membranes via current vibrational spectroscopic techniques and MD simulations. Overall, this review provides an up-to-date comprehensive overview of the powerful combination of vibrational spectroscopy and simulations, which has great potential to illuminate lipid-lipid, lipid-protein, and lipid-water interactions in the intricate conformational landscape of cell membranes.

POTRA domains of the TamA insertase interact with the outer membrane and modulate membrane properties.

Mellouk A, Jaouen P, Ruel LJ, Lê M, Martini C, Moraes TF, El Bakkouri M, Lagüe P, Boisselier E, Calmettes C.

Proc Natl Acad Sci U S A. 2024 Jul 9;121(28):e2402543121.

doi: 10.1073/pnas.2402543121. Epub 2024 Jul 3.

PMID: 38959031.

The outer membrane (OM) of gram-negative bacteria serves as a vital organelle that is densely populated with OM proteins (OMPs) and plays pivotal roles in cellular functions and virulence. The assembly and insertion of these OMPs into the OM represent a fundamental process requiring specialized molecular chaperones. One example is the translocation and assembly module (TAM), which functions as a transenvelope chaperone promoting the folding of specific autotransporters, adhesins, and secretion systems. The catalytic unit of TAM, TamA, comprises a catalytic β-barrel domain anchored within the OM and three periplasmic polypeptide-transport-associated (POTRA) domains that recruit the TamB subunit. The latter acts as a periplasmic ladder that facilitates the transport of unfolded OMPs across the periplasm. In addition to their role in recruiting the auxiliary protein TamB, our data demonstrate that the POTRA domains mediate interactions with the inner surface of the OM, ultimately modulating the membrane properties. Through the integration of X-ray crystallography, molecular dynamic simulations, and biomolecular interaction methodologies, we located the membrane-binding site on the first and second POTRA domains. Our data highlight a binding preference for phosphatidylglycerol, a minor lipid constituent present in the OM, which has been previously reported to facilitate OMP assembly. In the context of the densely OMP-populated membrane, this association may serve as a mechanism to secure lipid accessibility for nascent OMPs through steric interactions with existing OMPs, in addition to creating favorable conditions for OMP biogenesis.

Cell-inspired, massive electromodulation of friction via transmembrane fields across lipid bilayers.

Zhang Y, Jin D, Tivony R, Kampf N, Klein J.

Nat Mater. 2024 Jun 24.

doi: 10.1038/s41563-024-01926-9. Epub ahead of print.

PMID: 38914644.

Transient electric fields across cell bilayer membranes can lead to electroporation and cell fusion, effects crucial to cell viability whose biological implications have been extensively studied. However, little is known about these behaviours in a materials context. Here we find that transmembrane electric fields can lead to a massive, reversible modulation of the sliding friction between surfaces coated with lipid-bilayer membranes-a 200-fold variation, up to two orders of magnitude greater than that achieved to date. Atomistic simulations reveal that the transverse fields, resembling those at cell membranes, lead to fully reversible electroporation of the confined bilayers and the formation of inter-bilayer bridges analogous to the stalks preceding intermembrane fusion. These increase the interfacial dissipation through reduced hydration at the slip plane, forcing it to revert in part from the low-dissipation, hydrated lipid-headgroup plane to the intra-bilayer, high-dissipation acyl tail interface. Our results demonstrate that lipid bilayers under transmembrane electric fields can have striking materials modification properties.

Molecules

Improved Highly Mobile Membrane Mimetic Model for Investigating Protein-Cholesterol Interactions.

Lihan M, Tajkhorshid E.

J Chem Inf Model. 2024 Jun 24;64(12):4822-4834.

doi: 10.1021/acs.jcim.4c00619. Epub 2024 Jun 6.

PMID: 38844760.

Cholesterol (CHL) plays an integral role in modulating the function and activity of various mammalian membrane proteins. Due to the slow dynamics of lipids, conventional computational studies of protein-CHL interactions rely on either long-time scale atomistic simulations or coarse-grained approximations to sample the process. A highly mobile membrane mimetic (HMMM) has been developed to enhance lipid diffusion and thus used to facilitate the investigation of lipid interactions with peripheral membrane proteins and, with customized in silico solvents to replace phospholipid tails, with integral membrane proteins. Here, we report an updated HMMM model that is able to include CHL, a nonphospholipid component of the membrane, henceforth called HMMM-CHL. To this end, we had to optimize the effect of the customized solvents on CHL behavior in the membrane. Furthermore, the new solvent is compatible with simulations using force-based switching protocols. In the HMMM-CHL, both improved CHL dynamics and accelerated lipid diffusion are integrated. To test the updated model, we have applied it to the characterization of protein-CHL interactions in two membrane protein systems, the human β2-adrenergic receptor (β2AR) and the mitochondrial voltage-dependent anion channel 1 (VDAC-1). Our HMMM-CHL simulations successfully identified CHL binding sites and captured detailed CHL interactions in excellent consistency with experimental data as well as other simulation results, indicating the utility of the improved model in applications where an enhanced sampling of protein-CHL interactions is desired.

Nanostructured lipopeptide-based membranomimetics for stabilizing bacteriorhodopsin.

Gurung AB, Chakraborty K, Ghosh S, Jan S, Gayen P, Biswas A, Mallick AM, Hembram M, Tripathi A, Mukherjee A, Mukherjee S, Mukherjee A, Bhattacharyya D, Sinha Roy R.

Biomater Sci. 2024 Jun 21.

doi: 10.1039/d4bm00250d. Epub ahead of print.

PMID: 38904161.

Nanostructured 7-9-residue cyclic and unstructured lipopeptide-based facial detergents have been engineered to stabilize the model integral membrane protein, bacteriorhodopsin. Formation of a cylindrical-type micelle assembly induced by facial amphipathic lipopeptides resembles a biological membrane more effectively than conventional micelles. The hydrophobic face of this cylindrical-type micelle provides extended stability to the membrane protein and the hydrophilic surface interacts with an aqueous environment. In our present study, we have demonstrated experimentally and computationally that lipopeptide-based facial detergents having an unstructured or β-turn conformation can stabilize membrane proteins. However, constrained peptide detergents can provide enhanced stability to bacteriorhodopsin. In this study, we have computationally examined the structural stability of bacteriorhodopsin in the presence of helical, beta-strand, and cyclic unstructured peptide detergents, and conventional detergent-like peptides. Our study demonstrates that optimal membranomimetics (detergents) for stabilizing a specific membrane protein can be screened based on the following criteria: (i) hydrodynamic radii of the self-assembled peptide detergents, (ii) stability assay of detergent-encased membrane proteins, (iii) percentage covered area of detergent-encased membrane proteins obtained computationally and (iv) protein-detergent interaction energy.

NDT-C11 as a viable novel detergent for single particle cryo-EM.

Jiko C, Li J, Moon Y, Tanaka Y, Gopalasingam CC, Shigematsu H, Chae PS, Kurisu G, Gerle C.

Chempluschem. 2024 Jun 17:e202400242.

doi: 10.1002/cplu.202400242. Epub ahead of print.

PMID: 38881532.

Single particle cryo electron microscopy (cryo-EM) is now the major method for the determination of integral membrane protein structure. For the success of a given project the type of membrane mimetic used for extraction from the native cell membrane, purification to homogeneity and finally cryo-grid vitrification is crucial. Although small molecule amphiphiles – detergents – are the most widely used membrane mimetic, specific tailoring of detergent structure for single particle cryo-EM is rare and the demand for effective detergents not satisfied. Here, we compare the popular detergent lauryl maltose-neopentyl glycol (LMNG) with the novel detergent neopentyl glycol-derived triglucoside-C11 (NDT-C11) in its behavior as free detergent and when bound to two types of multisubunit membrane protein complexes – cyanobacterial photosystem I (PSI) and mammalian F-ATP synthase. We conclude that NDT-C11 has high potential to become a very useful detergent for single particle cryo-EM of integral membrane proteins.

Nanodisc Technology: Direction toward Physicochemical Characterization of Chemosensory Membrane Proteins in Food Flavor Research.

Karanth S, Benthin J, Wiesenfarth M, Somoza V, Koehler M.

J Agric Food Chem. 2024 Jul 3;72(26):14521-14529.

doi: 10.1021/acs.jafc.4c01827. Epub 2024 Jun 21.

PMID: 38906535.

Chemosensory membrane proteins such as G-protein-coupled receptors (GPCRs) drive flavor perception of food formulations. To achieve this, a detailed understanding of the structure and function of these membrane proteins is needed, which is often limited by the extraction and purification methods involved. The proposed nanodisc methodology helps overcome some of these existing challenges such as protein stability and solubilization along with their reconstitution from a native cell-membrane environment. Being well-established in structural biology procedures, nanodiscs offer this elegant solution by using, e.g., a membrane scaffold protein (MSP) or styrene-maleic acid (SMA) polymer, which interacts directly with the cell membrane during protein reconstitution. Such derived proteins retain their biophysical properties without compromising the membrane architecture. Here, we seek to show that these lipidic systems can be explored for insights with a focus on chemosensory membrane protein morphology and structure, conformational dynamics of protein-ligand interactions, and binding kinetics to answer pending questions in flavor research. Additionally, the compatibility of nanodiscs across varied (labeled or label-free) techniques offers significant leverage, which has been highlighted here.

Methods

Identification of lipid-specific proteins with high-density lipid-immobilized beads.

Morito M, Yasuda H, Matsufuji T, Kinoshita M, Matsumori N.

Analyst. 2024 Jul 8;149(14):3747-3755.

doi: 10.1039/d4an00579a. PMID: 38829210.

In biological membranes, lipids often interact with membrane proteins (MPs), regulating the localization and activity of MPs in cells. Although elucidating lipid-MP interactions is critical to comprehend the physiological roles of lipids, a systematic and comprehensive identification of lipid-binding proteins has not been adequately established. Therefore, we report the development of lipid-immobilized beads where lipid molecules were covalently immobilized. Owing to the detergent tolerance, these beads enable screening of water-soluble proteins and MPs, the latter of which typically necessitate surfactants for solubilization. Herein, two sphingolipid species-ceramide and sphingomyelin-which are major constituents of lipid rafts, were immobilized on the beads. We first showed that the density of immobilized lipid molecules on the beads was as high as that of biological lipid membranes. Subsequently, we confirmed that these beads enabled the selective pulldown of known sphingomyelin- or ceramide-binding proteins (lysenin, p24, and CERT) from protein mixtures, including cell lysates. In contrast, commercial sphingomyelin beads, on which lipid molecules are sparsely immobilized through biotin-streptavidin linkage, failed to capture lysenin, a well-known protein that recognizes clustered sphingomyelin molecules. This clearly demonstrates the applicability of our beads for obtaining proteins that recognize not only a single lipid molecule but also lipid clusters or lipid membranes. Finally, we demonstrated the screening of lipid-binding proteins from Neuro2a cell lysates using these beads. This method is expected to significantly contribute to the understanding of interactions between lipids and proteins and to unravel the complexities of lipid diversity.

Membrane Proteins in Action Monitored by pH-Responsive Liquid Crystal Biosensors.

Bao P, Phillips K, Raval R.

ACS Appl Mater Interfaces. 2024 Jun 19;16(24):31843-31850.

doi: 10.1021/acsami.4c06614. Epub 2024 Jun 6.

PMID: 38841859.

Liquid crystal (LC) biosensors have received significant attention for their potential applications for point-of-care devices due to their sensitivity, low cost, and easy read-out. They have been employed to detect a wide range of important biological molecules. However, detecting the function of membrane proteins has been extremely challenging due to the difficulty of integrating membrane proteins, lipid membranes, and LCs into one system. In this study, we addressed this challenge by monitoring the proton-pumping function of bacteriorhodopsin (bR) using a pH-sensitive LC thin film biosensor. To achieve this, we deposited purple membranes (PMs) containing a 2D crystal form of bRs onto an LC-aqueous interface. Under light, the PM patches changed the local pH at the LC-aqueous interface, causing a color change in the LC thin film that is observable through a polarizing microscope with crossed polarizers. These findings open up new opportunities to study the biofunctions of membrane proteins and their induced local environmental changes in a solution using LC biosensors.

Simultaneous Native Mass Spectrometry Analysis of Single and Double Mutants To Probe Lipid Binding to Membrane Proteins.

Jayasekera HS, Mohona FA, Ewbank M, Marty MT.

Anal Chem. 2024 Jun 25;96(25):10426-10433.

doi: 10.1021/acs.analchem.4c01704. Epub 2024 Jun 10.

PMID: 38859611.

Lipids are critical modulators of membrane protein structure and function. However, it is challenging to investigate the thermodynamics of protein-lipid interactions because lipids can simultaneously bind membrane proteins at different sites with different specificities. Here, we developed a native mass spectrometry (MS) approach using single and double mutants to measure the relative energetic contributions of specific residues on Aquaporin Z (AqpZ) toward cardiolipin (CL) binding. We first mutated potential lipid-binding residues on AqpZ, and mixed mutant and wild-type proteins together with CL. By using native MS to simultaneously resolve lipid binding to the mutant and wild-type proteins in a single spectrum, we directly determined the relative affinities of CL binding, thereby revealing the relative Gibbs free energy change for lipid binding caused by the mutation. Comparing different mutants revealed that W14 contributes to the tightest CL binding site, with R224 contributing to a lower affinity site. Using double mutant cycling, we investigated the synergy between W14 and R224 sites on CL binding. Overall, this novel native MS approach provides unique insights into the binding of lipids to specific sites on membrane proteins.

Structures reveal how SGLT inhibitors work.

Sun Z, Cui W, Chen L.

Trends Pharmacol Sci. 2024 Jun 18:S0165-6147(24)00101-9.

doi: 10.1016/j.tips.2024.05.009. Epub ahead of print.

PMID: 38897851.

Sodium glucose cotransporters (SGLTs) transport glucose against its concentration gradient by harnessing the electrochemical potential gradient of sodium ions. SGLT inhibitors are widely prescribed to treat diabetes and other conditions. Recent structural studies have uncovered how chemically diverse SGLT inhibitors bind and inhibit the transporter at the atomic level.

Freestanding bilayer microscope for single-molecule imaging of membrane proteins.

Pérez-Mitta G, Sezgin Y, Wang W, MacKinnon R.

Sci Adv. 2024 Jun 21;10(25):eado4722.

doi: 10.1126/sciadv.ado4722. Epub 2024 Jun 21.

PMID: 38905330.

Integral membrane proteins (IMPs) constitute a large fraction of organismal proteomes, playing fundamental roles in physiology and disease. Despite their importance, the mechanisms underlying dynamic features of IMPs, such as anomalous diffusion, protein-protein interactions, and protein clustering, remain largely unknown due to the high complexity of cell membrane environments. Available methods for in vitro studies are insufficient to study IMP dynamics systematically. This publication introduces the freestanding bilayer microscope (FBM), which combines the advantages of freestanding bilayers with single-particle tracking. The FBM, based on planar lipid bilayers, enables the study of IMP dynamics with single-molecule resolution and unconstrained diffusion. This paper benchmarks the FBM against total internal reflection fluorescence imaging on supported bilayers and is used here to estimate ion channel open probability and to examine the diffusion behavior of an ion channel in phase-separated bilayers. The FBM emerges as a powerful tool to examine membrane protein/lipid organization and dynamics to understand cell membrane processes.

Ligand Screening of Membrane Proteins Embedded in Nanodiscs: How to Manage Non-Specific Interactions in Weak Affinity Chromatography?

Vidal FX, Deloche A, Zeder-Lutz G, Hideux M, Wagner R, Dugas V, Demesmay C.

Molecules. 2024 Jun 13;29(12):2814.

doi: 10.3390/molecules29122814.

PMID: 38930880.

Miniaturized weak affinity chromatography is emerging as an interesting alternative to conventional biophysical tools for performing fragment-screening studies in the context of fragment-based drug discovery. In order to push back the analytical limits, it is necessary not only to control non-specific interactions with chromatographic support, but also to adapt this methodology by comparing the results obtained on an affinity column to a control column. The work presented in this study focused on fragment screening that targets a model membrane protein, the adenosine A2A receptor, embedded in nanodiscs (NDs) as biomimetic membranes. By studying the retention behavior of test fragment mixtures on supports modified with different types of NDs, we were able to determine the contribution of ND-related non-specific interactions, in particular the electrostatic effect of anionic phospholipids and the hydrophobic effect of neutral phospholipids. Different strategies for the preparation of control columns (empty NDs, orthosteric site blocking) were investigated and are presented for the first time. With these two types of control columns, the screening enabled the identification of two new fragments of AA2AR, which were confirmed by competition experiments and whose Kd values, estimated directly during the screening or after the competition experiments in frontal mode, were in good agreement.

Numerical Model for Electrogenic Transport by the ATP-dependent Potassium Pump KdpFABC.

Hussein A, Zhang X, Stokes DL.

Biophys Rep (N Y). 2024 Jun 29:100169.

doi: 10.1016/j.bpr.2024.100169. Epub ahead of print.

PMID: 38950825.

In vitro assays of ion transport are an essential tool for understanding molecular mechanisms associated with ATP-dependent pumps. Because ion transport is generally electrogenic, principles of electrophysiology are applicable, but conventional tools like patch clamp are ineffective due to relatively low turnover rates of the pumps. Instead, assays have been developed to measure either voltage or current generated by transport activity of a population of molecules either in cell-derived membrane fragments or after reconstituting purified protein into proteoliposomes. In order to understand the nuances of these assays and to characterize effects of various operational parameters, we have developed a numerical model to simulate data produced by two relevant assays: fluorescence from voltage sensitive dyes and current recorded by capacitive coupling on solid supported membranes. Parameters of the model, which has been implemented in Python, are described along with underlying principles of the computational algorithm. Experimental data from KdpFABC, a K+ pump associated with P-type ATPases, are presented and model parameters have been adjusted to mimic these data. In addition, effects of key parameters such as non-selective leak conductance and turnover rate are demonstrated. Finally, simulated data are used to illustrate the effects of capacitive coupling on measured current and to compare alternative methods for quantification of raw data.

Microbio

Repurposing approved drugs as potential efflux pump inhibitors in multidrug-resistant Pseudomonas aeruginosa.

de Melo Guedes GM, Pereira VC, Freitas AS, Honório de Souza PR, Chacon Parra AL, Brasil JA, de Medeiros Guedes RF, Pereira de Sousa PC, Aguiar Cordeiro R, Gadelha Rocha MF, Costa Sidrim JJ, Souza Collares Maia Castelo Branco D.

Future Microbiol. 2024;19(6):495-508.

doi: 10.2217/fmb-2023-0208. Epub 2024 Apr 17.

PMID: 38629920.

Aim: To evaluate the action of promethazine, fluoxetine and carbonyl cyanide 3-chlorophenylhydrazone as efflux pump inhibitors (EPIs) against multidrug-resistant Pseudomonas aeruginosa.

Methods: The effect of the compounds was evaluated in planktonic cells and bacterial biofilms. Accumulation tests were performed with ethidium bromide to prove their action as EPIs. Then, they were associated with antimicrobials.

Results: Effect on planktonic cells and biofilms was found. Assays with ethidium bromide indicate their action as EPIs. Significant reductions in the metabolic activity of biofilms were observed after the association with the antimicrobials, especially for meropenem.

Conclusion: It is possible to prove the action of these compounds as EPIs for P. aeruginosa and demonstrate the relevance of efflux pumps in antimicrobial resistance.

Mode of the Interaction of Efflux Inhibitor Phenylalanyl-arginyl-β-naphtylamide with Bacterial Cells.

Sakalauskaitė S, Mikalayeva V, Sutkuvienė S, Daugelavičius R.

Biomedicines. 2024 Jun 14;12(6):1324.

doi: 10.3390/biomedicines12061324.

PMID: 38927531.

An increased efflux activity is one of the major reasons for bacterial antibiotic resistance. The usage of efflux pump inhibitors could be a promising approach to restoring the activity of inefficient antibiotics. The interaction of the RND family efflux pump inhibitor phenylalanyl-arginyl-β-naphthylamide (PAβN) with Salmonella enterica ser. Typhimurium cells was assayed using traditional microbiological techniques and a novel PAβN-selective electrode. Monitoring the PAβN concentration in the medium using the electrode enabled the real-time measurements of this compound’s interaction with bacterial cells. We showed that S. Typhimurium cells accumulate a high amount of PAβN because of its high affinity to lipopolysaccharides (LPSs), the major constituent of the outer layer of the outer membrane, and does not affect the functioning of the plasma membrane. EDTA enhanced the binding of PAβN to S. Typhimurium cells and the purified E. coli LPSs, but the energization of the cells by glucose does not affect the cell-bound amount of this inhibitor. Polycationic antibiotic Polymyxin B released both the cells accumulated and the suspended LPS-bound PAβN.

Role of bacterial multidrug efflux pumps during infection.

Laborda P, Molin S, Johansen HK, Martínez JL, Hernando-Amado S.

World J Microbiol Biotechnol. 2024 Jun 1;40(7):226.

doi: 10.1007/s11274-024-04042-7.

PMID: 38822187.

Multidrug efflux pumps are protein complexes located in the cell envelope that enable bacteria to expel, not only antibiotics, but also a wide array of molecules relevant for infection. Hence, they are important players in microbial pathogenesis. On the one hand, efflux pumps can extrude exogenous compounds, including host-produced antimicrobial molecules. Through this extrusion, pathogens can resist antimicrobial agents and evade host defenses. On the other hand, efflux pumps also have a role in the extrusion of endogenous compounds, such as bacterial intercommunication signaling molecules, virulence factors or metabolites. Therefore, efflux pumps are involved in the modulation of bacterial behavior and virulence, as well as in the maintenance of the bacterial homeostasis under different stresses found within the host. This review delves into the multifaceted roles that efflux pumps have, shedding light on their impact on bacterial virulence and their contribution to bacterial infection. These observations suggest that strategies targeting bacterial efflux pumps could both reinvigorate the efficacy of existing antibiotics and modulate the bacterial pathogenicity to the host. Thus, a comprehensive understanding of bacterial efflux pumps can be pivotal for the development of new effective strategies for the management of infectious diseases.

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.

Highly inclined light sheet allows volumetric super-resolution imaging of efflux pumps distribution in bacterial biofilms.

Vignolini T, Capitanio M, Caldini C, Gardini L, Pavone FS.

Sci Rep. 2024 Jun 5;14(1):12902.

doi: 10.1038/s41598-024-63729-x.

PMID: 38839922.

Bacterial biofilms are highly complex communities in which isogenic bacteria display different gene expression patterns and organize in a three-dimensional mesh gaining enhanced resistance to biocides. The molecular mechanisms behind such increased resistance remain mostly unknown, also because of the technical difficulties in biofilm investigation at the sub-cellular and molecular level. In this work we focus on the AcrAB-TolC protein complex, a multidrug efflux pump found in Enterobacteriaceae, whose overexpression is associated with most multiple drug resistance (MDR) phenotypes occurring in Gram-negative bacteria. We propose an optical method to quantify the expression level of the AcrAB-TolC pump within the biofilm volume at the sub-cellular level, with single-molecule sensitivity. Through a combination of super-resolution PALM with single objective light sheet and precision genome editing, we can directly quantify the spatial distribution of endogenous AcrAB-TolC pumps expressed in both planktonic bacteria and, importantly, within the bacterial biofilm volume. We observe a gradient of pump density within the biofilm volume and over the course of biofilm maturation. Notably, we propose an optical method that could be broadly employed to achieve volumetric super-resolution imaging of thick samples.

Non-interchangeable functions of efflux transporters of Pseudomonas aeruginosa in survival under infection-associated stress.

Adamiak JW, Ajmal L, Zgurskaya HI.

J Bacteriol. 2024 Jun 14:e0005424.

doi: 10.1128/jb.00054-24. Epub ahead of print.

PMID: 38874367.

Pseudomonas aeruginosa is a challenging opportunistic pathogen due to its intrinsic and acquired mechanisms of antibiotic resistance. A large repertoire of efflux transporters actively expels antibiotics, toxins, and metabolites from cells and enables growth of P. aeruginosa in diverse environments. In this study, we analyzed the roles of representative efflux pumps from the Resistance-Nodulation-Division (RND), Major Facilitator Superfamily (MFS), and Small Multidrug Resistance (SMR) families of proteins in the susceptibility of P. aeruginosa to antibiotics and bacterial growth under stresses imposed by human hosts during bacterial infections: an elevated temperature, osmotic stress, low iron, bile salts, and acidic pH. We selected five RND pumps MexAB-OprM, MexEF-OprN, MexCD-OprJ, MuxABC-OpmB, and TriABC-OpmH that differ in their substrate specificities and expression profiles, two MFS efflux pumps PA3136-3137 and PA5158-5160 renamed here into MfsAB and MfsCD-OpmG, respectively, and an SMR efflux transporter PA1540-1541 (MdtJI). We found that the most promiscuous RND pumps such as MexEF-OprN and MexAB-OprM are integrated into diverse survival mechanisms and enable P. aeruginosa growth under various stresses. MuxABC-OpmB and TriABC-OpmH pumps with narrower substrate spectra are beneficial only in the presence of the iron chelator 2,2′-dipyridyl and bile salts, respectively. MFS pumps do not contribute to antibiotic efflux but play orthogonal roles in acidic pH, low iron, and in the presence of bile salts. In contrast, MdtJI protects against polycationic antibiotics but does not contribute to survival under stress. Thus, efflux pumps play specific, non-interchangeable functions in P. aeruginosa cell physiology and bacterial survival under stresses.

Special collection to commemorate 40 years of antimicrobial efflux.

Kumar A, Blair JMA.

Microbiology (Reading). 2024 Jun;170(6).

doi: 10.1099/mic.0.001466.

PMID: 38885034.

To mark the 40 year anniversary of the seminal description of antibiotic efflux pumps in bacteria [1] and the 30 year milestone of the characterization of the resistance-nodulation-division (RND) family of efflux pumps [2, 3], we introduce this special collection on ‘Antimicrobial Efflux’[4]. Efflux pumps have long been recognized as key players in both acquired and intrinsic bacterial resistance to antibiotics and other antimicrobial agents. Over the years, our understanding of these pumps has deepened, revealing their intricate mechanisms, regulatory networks, and diverse physiological roles beyond drug resistance.

Miscellaneous

Lego bricks are making science more accessible

MIT Technology Review

https://www.technologyreview.com/2024/06/25/1093639/lego-bricks-science-accessibility/

Scientists use the iconic colorful bricks to build everything from bioprinters to microscopes—increasing the accessibility of science in the process.

The strategy behind one of the most successful labs in the world.

Gebel L, Velu C, Vidal-Puig A.

Nature. 2024 Jun;630(8018):813-816.

doi: 10.1038/d41586-024-02085-2.

PMID: 38926627.

One UK institute has produced a dozen Nobel laureates and biomedical breakthroughs across the board. How does Cambridge’s Laboratory of Molecular Biology do it?

long story short: promote scientific diversity, Foster long-term loyalty, effective managing scarce resources, establish feedback between scientific questions and engineering-based technology solutions, prioritize long-term goals over performance metrics.

https://www.nature.com/articles/d41586-024-02085-2?utm_source=Live+Audience&utm_campaign=b94550364f-nature-briefing-daily-20240627&utm_medium=email&utm_term=0_b27a691814-b94550364f-50537092

Should students use emojis in their thesis’s summary for the general public ?

NATURE BRIEFING 28 June 2024

https://www.nature.com/articles/d41586-024-02152-8

Last week, food-systems scholar Madhura Rao started a lively discussion on social media when she said students should feel free to use emojis in their thesis’s summary for the general public. 

https://x.com/madhurarrao/status/1802787319306752142

 The majority of readers who replied to our poll said emojis shouldn’t be allowed in scientific writing. Many felt that using symbols with unclear meaning takes away from the precision expected in research. “Including emoji’s in a thesis summary for the general public is like writing half of it in Klingon,” says pathologist Jacqueline Bentel. “Understood and appreciated by a vocal (and important!) subset of people, but misunderstood and alienating for the majority.”  Others suggested that, in some cases, emojis can help to convey feelings or ideas sometime more easily than words. “A text becomes unreadable 📚 if it looks 👀 like👍🏼 this, but I count on the intelligent student to decide which words, phrases or emojis they use when displaying their own work to the public,” says molecular biologist Yarin Livneh.

AI & robotics briefing: How AI referees are shaking up football.

Krämer K.

Nature. 2024 Jun 18.

doi: 10.1038/d41586-024-02064-7. Epub ahead of print.

PMID: 38898255.

Video assistant referees (VAR), a semi-automated AI system, will support human referees at this year’s UEFA Euro football tournament. Ten cameras above the pitch track 29 locations on each player’s body in real time, and the ball contains a sensor that notes its location and movement, 500 times per second. “One major application is detecting violations of the offside rule,” explains sports physicist John Eric Goff. The technology isn’t without detractors: inconsistencies in how referees apply VAR and the time they sometimes take to make decisions has fueled discontent. “Things such as fouls and yellow or red cards still require human decision-making,” adds Goff.