MP
Inhibition of M. tuberculosis and human ATP synthase by BDQ and TBAJ-587.
Zhang Y, Lai Y, Zhou S, Ran T, Zhang Y, Zhao Z, Feng Z, Yu L, Xu J, Shi K, Wang J, Pang Y, Li L, Chen H, Guddat LW, Gao Y, Liu F, Rao Z, Gong H.
Nature. 2024 Jul;631(8020):409-414.
doi: 10.1038/s41586-024-07605-8. Epub 2024 Jul 3.
PMID: 38961288.
Bedaquiline (BDQ), a first-in-class diarylquinoline anti-tuberculosis drug, and its analogue, TBAJ-587, prevent the growth and proliferation of Mycobacterium tuberculosis by inhibiting ATP synthase1,2. However, BDQ also inhibits human ATP synthase3. At present, how these compounds interact with either M. tuberculosis ATP synthase or human ATP synthase is unclear. Here we present cryogenic electron microscopy structures of M. tuberculosis ATP synthase with and without BDQ and TBAJ-587 bound, and human ATP synthase bound to BDQ. The two inhibitors interact with subunit a and the c-ring at the leading site, c-only sites and lagging site in M. tuberculosis ATP synthase, showing that BDQ and TBAJ-587 have similar modes of action. The quinolinyl and dimethylamino units of the compounds make extensive contacts with the protein. The structure of human ATP synthase in complex with BDQ reveals that the BDQ-binding site is similar to that observed for the leading site in M. tuberculosis ATP synthase, and that the quinolinyl unit also interacts extensively with the human enzyme. This study will improve researchers’ understanding of the similarities and differences between human ATP synthase and M. tuberculosis ATP synthase in terms of the mode of BDQ binding, and will allow the rational design of novel diarylquinolines as anti-tuberculosis drugs.
N&V:
Blueprints for ATP machinery will aid tuberculosis drug design.
Cook GM, McNeil MB.
Nature. 2024 Jul;631(8020):278-280.
doi: 10.1038/d41586-024-02094-1.
PMID: 38961207.
Membrane protein MHZ3 regulates the on-off switch of ethylene signaling in rice.
Li XK, Huang YH, Zhao R, Cao WQ, Lu L, Han JQ, Zhou Y, Zhang X, Wu WA, Tao JJ, Wei W, Zhang WK, Chen SY, Ma B, Zhao H, Yin CC, Zhang JS.
Nat Commun. 2024 Jul 16;15(1):5987.
doi: 10.1038/s41467-024-50290-4.
PMID: 39013913.
Ethylene regulates plant growth, development, and stress adaptation.
Here: rapid phospho-shift of rice OsCTR2 in response to ethylene as a sensitive readout for signal activation => MHZ3, previously identified as a stabilizer of ETHYLENE INSENSITIVE 2 (OsEIN2), is crucial for maintaining OsCTR2 phosphorylation.
=> dual role of MHZ3 in fine-tuning ethylene signaling activation, providing insights into the initial stages of the ethylene signaling cascade.
High-resolution in situ structures of mammalian respiratory supercomplexes.
Zheng W, Chai P, Zhu J, Zhang K.
Nature. 2024 Jul;631(8019):232-239.
doi: 10.1038/s41586-024-07488-9. Epub 2024 May 29.
PMID: 38811722.
cf. BioRxiV preprint mentionned in the 20240415_membrane digest
Insights into Allosteric Inhibition of the AcrB Efflux Pump: Role of Distinct Binding Pockets, Protomer Preferences, and Crosstalk Disruption.
Roy RK, Bera A, Patra N.
J Chem Inf Model. 2024 Jul 16.
doi: 10.1021/acs.jcim.4c00306. Epub ahead of print.
PMID: 39011748.
AcrB, a key component in bacterial efflux processes, exhibits distinct binding pockets that influence inhibitor interactions. In addition to the well-known distal binding pocket within the periplasmic domain, a noteworthy pocket amidst the transmembrane (TM) helices serves as an alternate binding site for inhibitors. The bacterial efflux mechanism involves a pivotal functional rotation of the TM protein, inducing conformational changes in each protomer and propelling drugs toward the outer membrane domain. Surprisingly, inhibitors binding to the TM domain display a preference for L protomers over T protomers. Metadynamics simulations elucidate that Lys940 in the TM domain of AcrB can adopt two conformations in L protomers, whereas the energy barrier for such transitions is higher in T protomers. This phenomenon results in stable inhibitor binding in l protomers. Upon a detailed analysis of unbinding pathways using random accelerated molecular dynamics and umbrella sampling, we have identified three distinct routes for ligand exit from the allosteric site, specifically involving regions within the TM domains─TM4, TM5, and TM10. To explore allosteric crosstalk, we focused on the following key residues: Val452 from the TM domain and Ala831 from the porter domain. Surprisingly, our findings reveal that inhibitor binding disrupts this communication. The shortest path connecting Val452 and Ala831 increases upon inhibitor binding, suggesting sabotage of the natural interdomain communication dynamics. This result highlights the intricate interplay between inhibitor binding and allosteric signaling within our studied system.
An oligopeptide permease, OppABCD, requires an iron-sulfur cluster domain for functionality.
Yang X, Hu T, Liang J, Xiong Z, Lin Z, Zhao Y, Zhou X, Gao Y, Sun S, Yang X, Guddat LW, Yang H, Rao Z, Zhang B.
Nat Struct Mol Biol. 2024 Jul;31(7):1072-1082. doi: 10.1038/s41594-024-01256-z. Epub 2024 Mar 28. PMID: 38548954.
Oligopeptide permease, OppABCD, belongs to the type I ABC transporter family. Its role is to import oligopeptides into bacteria for nutrient uptake and to modulate the host immune response. OppABCD consists of a cluster C substrate-binding protein (SBP), OppA, membrane-spanning OppB and OppC subunits, and an ATPase, OppD, that contains two nucleotide-binding domains (NBDs). Here, using cryo-electron microscopy, we determined the high-resolution structures of Mycobacterium tuberculosis OppABCD in the resting state, oligopeptide-bound pre-translocation state, AMPPNP-bound pre-catalytic intermediate state and ATP-bound catalytic intermediate state. The structures show an assembly of a cluster C SBP with its ABC translocator and a functionally required [4Fe-4S] cluster-binding domain in OppD. Moreover, the ATP-bound OppABCD structure has an outward-occluded conformation, although no substrate was observed in the transmembrane cavity. Here, we reveal an oligopeptide recognition and translocation mechanism of OppABCD, which provides a perspective on how this and other type I ABC importers facilitate bulk substrate transfer across the lipid bilayer.
The mechanism of mammalian proton-coupled peptide transporters.
Lichtinger SM, Parker JL, Newstead S, Biggin PC.
Elife. 2024 Jul 23;13:RP96507.
doi: 10.7554/eLife.96507.
PMID: 39042711.
Proton-coupled oligopeptide transporters (POTs) are of great pharmaceutical interest owing to their promiscuous substrate binding site that has been linked to improved oral bioavailability of several classes of drugs. Members of the POT family are conserved across all phylogenetic kingdoms and function by coupling peptide uptake to the proton electrochemical gradient. Cryo-EM structures and alphafold models have recently provided new insights into different conformational states of two mammalian POTs, SLC15A1, and SLC15A2. Nevertheless, these studies leave open important questions regarding the mechanism of proton and substrate coupling, while simultaneously providing a unique opportunity to investigate these processes using molecular dynamics (MD) simulations. Here, we employ extensive unbiased and enhanced-sampling MD to map out the full SLC15A2 conformational cycle and its thermodynamic driving forces. By computing conformational free energy landscapes in different protonation states and in the absence or presence of peptide substrate, we identify a likely sequence of intermediate protonation steps that drive inward-directed alternating access. These simulations identify key differences in the extracellular gate between mammalian and bacterial POTs, which we validate experimentally in cell-based transport assays. Our results from constant-PH MD and absolute binding free energy (ABFE) calculations also establish a mechanistic link between proton binding and peptide recognition, revealing key details underpining secondary active transport in POTs. This study provides a vital step forward in understanding proton-coupled peptide and drug transport in mammals and pave the way to integrate knowledge of solute carrier structural biology with enhanced drug design to target tissue and organ bioavailability.
Membrane
Faster but Not Sweeter: A Model of Escherichia coli Re-level Lipopolysaccharide for Martini 3 and a Martini 2 Version with Accelerated Kinetics
Astrid F. Brandner Dheeraj Prakaash Alexandre Blanco González Fergus Waterhouse and Syma Khalid.
J. Chem. Theory Comput. 2024, July 15 2024
https://doi.org/10.1021/acs.jctc.4c00374
Lipopolysaccharide (LPS) is a complex glycolipid molecule that is the main lipidic component of the outer leaflet of the outer membrane of Gram-negative bacteria. It has very limited lateral motion compared to phospholipids, which are more ubiquitous in biological membranes, including in the inner leaflet of the outer membrane of Gram-negative bacteria. The slow-moving nature of LPS can present a hurdle for molecular dynamics simulations, given that the (pragmatically) accessible timescales to simulations are currently limited to microseconds, during which LPS displays some conformational dynamics but hardly any lateral diffusion. Thus, it is not feasible to observe phenomena such as insertion of molecules, including antibiotics/antimicrobials, directly into the outer membrane from the extracellular side nor to observe LPS dissociating from proteins via molecular dynamics using currently available models at the atomistic and more coarse-grained levels of granularity. Here, we present a model of deep rough LPS compatible with the Martini 2 coarse-grained force field with scaled down nonbonded interactions to enable faster diffusion. We show that the faster-diffusing LPS model is able to reproduce the salient biophysical properties of the standard models, but due to its faster lateral motion, molecules are able to penetrate deeper into membranes containing the faster model. We show that the fast ReLPS model is able to reproduce experimentally determined patterns of interaction with outer membrane proteins while also allowing for LPS to associate and dissociate with proteins within microsecond timescales. We also complete the Martini 3 LPS toolkit for Escherichia coli by presenting a (standard) model of deep rough LPS for this force field.
Analyzing lipid distributions and curvature in molecular dynamics simulations of complex membranes.
Cino EA, Ramirez-Echemendia DP, Hu S, Tieleman DP.
Methods Enzymol. 2024;701:579-601.
doi: 10.1016/bs.mie.2024.03.014. Epub 2024 Apr 4.
PMID: 39025583.
We describe methods to analyze lipid distributions and curvature in membranes with complex lipid mixtures and embedded membrane proteins. We discuss issues involved in these analyses, available tools to calculate curvature preferences of lipids and proteins, and focus on tools developed in our group for visual analysis of lipid-protein interactions and the analysis of membrane curvature.
Building complex membranes with Martini 3.
Ozturk TN, König M, Carpenter TS, Pedersen KB, Wassenaar TA, Ingólfsson HI, Marrink SJ.
Methods Enzymol. 2024;701:237-285.
doi: 10.1016/bs.mie.2024.03.010. Epub 2024 Apr 9.
PMID: 39025573.
The Martini model is a popular force field for coarse-grained simulations. Membranes have always been at the center of its development, with the latest version, Martini 3, showing great promise in capturing more and more realistic behavior. In this chapter we provide a step-by-step tutorial on how to construct starting configurations, run initial simulations and perform dedicated analysis for membrane-based systems of increasing complexity, including leaflet asymmetry, curvature gradients and embedding of membrane proteins.
Elucidating the Role of Lipid Interactions in Stabilizing the Membrane Protein KcsA.
Qiao P, Odenkirk MT, Zheng W, Wang Y, Chen J, Xu W, Baker ES.
Biophys J. 2024 Jul 18:S0006-3495(24)00478-8.
doi: 10.1016/j.bpj.2024.07.019. Epub ahead of print.
PMID: 39030907.
The significant effects of lipid binding on the functionality of potassium channel KcsA have been validated by brilliant studies. However, the specific interactions between lipids and KcsA, such as binding parameters for each binding event, have not been fully elucidated. In this study, we employed native mass spectrometry to investigate the binding of lipids to KcsA and their effects on the channel. The tetrameric structure of KcsA remains intact even in the absence of lipid binding. However, the subunit architecture of the E71A mutant, which is constantly open at low pH, relies on tightly associated copurified lipids. Furthermore, we observed that lipids exhibit weak binding to KcsA at high pH when the channel is at a closed/inactivation state in the absence of permeant cation K+. This feeble interaction potentially facilitates the association of K+ ions, leading to the transition of the channel to a resting closed/open state. Interestingly, both anionic and zwitterionic lipids strongly bind to KcsA at low pH when the channel is in an open/inactivation state. We also investigated the binding patterns of KcsA with natural lipids derived from E. coli and S. lividans. Interestingly, lipids from E. coli exhibited much stronger binding affinity compared to the lipids from S. lividans. Among the natural lipids from S. lividans, free fatty acids and triacylglycerols demonstrated the tightest binding to KcsA, whereas no detectable binding events were observed with natural PA lipids. These findings suggest that the lipid association pattern in S. lividans, the natural host for KcsA, warrants further investigation. In conclusion, our study sheds light on the role of lipids in stabilizing KcsA and highlights the importance of specific lipid-protein interactions in modulating its conformational states.
Molecules
Design, Synthesis, and Evaluation of a New Chemotype Fluorescent Ligand for the P2Y2 Receptor.
Knight R, Kilpatrick LE, Hill SJ, Stocks MJ.
ACS Med Chem Lett. 2024 Jun 12;15(7):1127-1135.
doi: 10.1021/acsmedchemlett.4c00211.
PMID: 39015271.
P2Y2R homology model used to design new antagonist scaffolds. One antagonist scaffold retained millimolar affinity for the P2Y2R and upon further functionalization, affinity was improved over 100-fold.
+ development of new chemotype P2Y2R fluorescent ligands, one of which demonstrated micromolar affinity for the P2Y2R in isolated cell membranes and distinct pharmacology from an existing P2Y2R fluorescent antagonist.
Structure and Conformation Determine Gas-Phase Infrared Spectra of Detergents.
Kirschbaum C, Greis K, Gewinner S, Schöllkopf W, Meijer G, von Helden G, Pagel K, Urner LH.
Chempluschem. 2024 Jun 21:e202400340.
doi: 10.1002/cplu.202400340. Epub ahead of print.
PMID: 39031638.
Native mass spectrometry of membrane proteins relies on non-ionic detergents which protect the protein during transfer from solution into the gas phase. Once in the gas phase, the detergent micelle must be efficiently removed, which is usually achieved by collision-induced dissociation (CID). Recently, infrared multiple photon dissociation (IRMPD) has emerged as an alternative activation method for the analysis of membrane proteins, which has led to a growing interest in detergents that efficiently absorb infrared light. Here we investigate whether the absorption properties of synthetic detergents can be tailored by merging structural motifs of existing detergents into new hybrid detergents. We combine gas-phase infrared ion spectroscopy with density functional theory to investigate and rationalize the absorption properties of three established detergents and two hybrid detergents with fused headgroups. We show that, although the basic intramolecular interactions in the parent and hybrid detergents are similar, the three-dimensional structures differ significantly and so do the infrared spectra. Our results outline a roadmap for guiding the synthesis of tailored detergents with computational chemistry for future mass spectrometry applications.
Quantifying Induced Dipole Effects in Small Molecule Permeation in a Model Phospholipid Bilayer.
Montgomery JM, Lemkul JA.
J Phys Chem B. 2024 Jul 22.
doi: 10.1021/acs.jpcb.4c01634. Epub ahead of print.
PMID: 39038441.
The cell membrane functions as a semipermeable barrier that governs the transport of materials into and out of cells. The bilayer features a distinct dielectric gradient due to the amphiphilic nature of its lipid components. This gradient influences various aspects of small molecule permeation and the folding and functioning of membrane proteins. Here, we employ polarizable molecular dynamics simulations to elucidate the impact of the electronic environment on the permeation process. We simulated eight distinct amino-acid side chain analogs within a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer using the Drude polarizable force field (FF). Our approach includes both unbiased and umbrella sampling simulations. By using a polarizable FF, we sought to investigate explicit dipole responses in relation to local electric fields along the membrane normal. We evaluate molecular dipole moments, which exhibit variation based on their localization within the membrane, and compare the outcomes with analogous simulations using the nonpolarizable CHARMM36 FF. This comparative analysis aims to discern characteristic differences in the free energy surfaces of permeation for the various amino-acid analogs. Our results provide the first systematic quantification of the impact of employing an explicitly polarizable FF in this context compared to the fixed-charge convention inherent to nonpolarizable FFs, which may not fully capture the influence of the membrane dielectric gradient.
Design, Synthesis, and Characterization of New δ Opioid Receptor-Selective Fluorescent Probes and Applications in Single-Molecule Microscopy of Wild-Type Receptors.
Drakopoulos A, Koszegi Z, Seier K, Hübner H, Maurel D, Sounier R, Granier S, Gmeiner P, Calebiro D, Decker M.
J Med Chem. 2024 Jul 23.
doi: 10.1021/acs.jmedchem.4c00627. Epub ahead of print.
PMID: 39044606.
The delta opioid receptor (δOR or DOR) is a G protein-coupled receptor (GPCR) showing a promising profile as a drug target for nociception and analgesia. Herein, we design and synthesize new fluorescent antagonist probes with high δOR selectivity that are ideally suited for single-molecule microscopy (SMM) applications in unmodified, untagged receptors. Using our new probes, we investigated wild-type δOR localization and mobility at low physiological receptor densities for the first time. Furthermore, we investigate the potential formation of δOR homodimers, as such a receptor organization might exhibit distinct pharmacological activity, potentially paving the way for innovative pharmacological therapies. Our findings indicate that the majority of δORs labeled with these probes exist as freely diffusing monomers on the cell surface in a simple cell model. This discovery advances our understanding of OR behavior and offers potential implications for future therapeutic research.
Methods
Vesicle Picker: A tool for efficient identification of membrane protein complexes in vesicles.
Ryan Karimi, Claire E Coupland, John L Rubinstein
bioRxiv 2024.07.15.603622;
doi:https://doi.org/10.1101/2024.07.15.603622
Particle selection from electron micrographs of lipid vesicles can be challenging due to the strong signal contributed from the lipid bilayer.
Here: ”Vesicle Picker” = open-source program for automatic identification of vesicles in cryo-EM micrographs. Designed to interface with cryoSPARC.
Fluorescence-Based Proteoliposome Methods to Monitor Redox-Active Transition Metal Transmembrane Translocation by Metal Transporters.
Pope MA, Curtis RM, Gull H, Horadigala Gamage MA, Abeyrathna SS, Abeyrathna NS, Fahrni CJ, Meloni G.
Methods Mol Biol. 2024;2839:77-97.
doi: 10.1007/978-1-0716-4043-2_5.
PMID: 39008249.
Fluorescence-based proteoliposome methods to monitor redox-active metal transmembrane translocation upon reconstitution of purified metal transporters in artificial lipid bilayers (turn-on/-off iron or copper-dependent sensors encapsulated) => real-time transport assays.
Single-Molecule Imaging of Integral Membrane Protein Dynamics and Function.
Modak A, Kilic Z, Chattrakun K, Terry DS, Kalathur RC, Blanchard SC.
Annu Rev Biophys. 2024 Jul;53(1):427-453.
doi: 10.1146/annurev-biophys-070323-024308.
PMID: 39013028.
Review on the requirements for studyingMPs using smFRET+ overview of the technical and conceptual frameworks emerging from this area of investigation.
Miscellaneous
Thanks for everything
Science writer Tabitha Carvan is enchanted by the found poetry hidden in the acknowledgment sections of PhD theses.
Here is a tour of some of her favourites:
and this one, probably the most rock and roll:
”Finally, I wish to acknowledge the work of Dave Grohl who provided a fin soundtrack for much of this PhD study.
Andrew Sullivan
Competitive thermodynamics and non-linear bush fire behavior (2007)”
Karaoke-related stress soars after a good night of REM sleep
Nature 630, 11 (2024)
doi: https://doi-org.insb.bib.cnrs.fr/10.1038/d41586-024-01532-4
Everybody hurts — especially those who undergo rapid-eye movement (REM) in their sleep the night after a stressful experience (what a great catchphrase ! Stipe et al. fans will understand).
Previous research has found that disrupted sleep diminishes the ability to manage emotions. To understand why this might happen, Risto Halonen and his colleagues at the University of Helsinki investigated how 29 young adults responded, after a bad night’s sleep, to the memory of a stressful situation.
The volunteers participated in a round of karaoke, in which they were individually recorded singing Abba’s ‘Dancing Queen’. Before each of them went to sleep, the researchers played their recording back to them without any background music. Fourteen of the participants had their deep sleep disrupted and 15 had their REM sleep disrupted.
The participants’ sweatiness — a sign of stress — was measured before the karaoke and 12 hours after it. Sweat levels rose more in participants with intact REM sleep than in those with intact deep sleep. These findings suggest that REM sleep preserves a stress response by strengthening a stressful memory’s emotional charge, the authors write.
Dogs might have evolved to read your emotions.
Kreier F.
Nature. 2024 Jul 16.
doi: 10.1038/d41586-024-02320-w. Epub ahead of print.
PMID: 39014205.
Centuries of co-evolution seem to have led to dogs being able to sense our emotional states. When dogs were played the sounds of people crying or humming they appeared more stressed by the sad sounds than by the neutral one. And they’re not just learning from being around us. Pet pigs — who were also raised alongside people but whose species played a very different role in human societies — seemed to find the humming more upsetting.
The power of scientific collaborations and the future of structural biology.
Beltrao P.
Nat Struct Mol Biol. 2024 Jul 15.
doi: 10.1038/s41594-024-01358-8. Epub ahead of print.
PMID: 39009854.
The idea of a scientific discovery is often linked to the eureka moment of a lone scientist, which then transforms our thinking. However, scientific discoveries are never made by individuals in isolation. They build on the work of countless researchers, and often require interdisciplinary and collaborative teams of researchers.