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Membrane-induced interactions play an important role in organizing membrane proteins. Measurements of the interactions between two and three membrane deforming objects have revealed their nonadditive nature. They are thought to lead to complex many-body effects, however, experimental evidence is lacking. We here present an experimental method to measure many-body effects in membrane-mediated interactions using colloidal spheres placed between a deflated giant unilamellar vesicles and a planar substrate. The confined colloidal particles cause a large deformation of the membrane while not being physicochemically attached to it and interact through it. Two particles attract with a maximum force of 0.2 pN. For three particles, compact equilateral triangles were preferred over linear arrangements. We use numerical energy minimization to establish that the attraction stems from a reduction in the membrane-deformation energy caused by the particles. Confining up to 36 particles, we find a preference for hexagonally close packed clusters. However, with increasing number of particles the order of the confined particles decreases, at the same time, diffusivity of the particles increases. Our experiments show that the nonadditive nature of membrane-mediated interactions affects the interactions and arrangements and ultimately leads to spherical aggregates with liquid-like order of potential importance for cellular processes.
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Arc-shaped BIN/Amphiphysin/Rvs (BAR) domain proteins generate curvature by binding to membranes and induce membrane tubulation at sufficiently large protein coverages. For the amphiphysin N-BAR domain, Le Roux et al., Nat. Commun. 2021, 12, 6550, measured a threshold coverage of 0.44 ± 0.097 for nanotubules emerging from the supported lipid bilayer. In this article, we systematically investigate membrane tubulation induced by arc-shaped protein-like particles with coarse-grained modeling and simulations and determine the threshold coverages at different particle-particle interaction strengths and membrane spontaneous curvatures. In our simulations, the binding of arc-shaped particles induces a membrane shape transition from spherical vesicles to tubules at a particle threshold coverage of about 0.5, which is rather robust to variations of the direct attractive particle interactions or spontaneous membrane curvature in the coarse-grained model. Our study suggests that threshold coverages of around or slightly below 0.5 are a general requirement for membrane tubulation by arc-shaped BAR domain proteins.
Assuntos
Bicamadas Lipídicas , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Simulação de Dinâmica Molecular , Domínios Proteicos , Membrana Celular/química , Membrana Celular/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismoRESUMO
Several bacterial toxins and viruses can deform membranes through multivalent binding to lipids for clathrin-independent endocytosis. However, it remains unclear, how membrane deformation and endocytic internalization are mechanistically linked. Here we show that many lipid-binding virions induce membrane deformation and clathrin-independent endocytosis, suggesting a common mechanism based on multivalent lipid binding by globular particles. We create a synthetic cellular system consisting of a lipid-anchored receptor in the form of GPI-anchored anti-GFP nanobodies and a multivalent globular binder exposing 180 regularly-spaced GFP molecules on its surface. We show that these globular, 40 nm diameter, particles bind to cells expressing the receptor, deform the plasma membrane upon adhesion and become endocytosed in a clathrin-independent manner. We explore the role of the membrane adhesion energy in endocytosis by using receptors with affinities varying over 7 orders of magnitude. Using this system, we find that once a threshold in adhesion energy is overcome to allow for membrane deformation, endocytosis occurs reliably. Multivalent, binding-induced membrane deformation by globular binders is thus sufficient for internalization to occur and we suggest it is the common, purely biophysical mechanism for lipid-binding mediated endocytosis of toxins and pathogens.
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Comunicação Celular , Endocitose , Membrana Celular/metabolismo , Clatrina/metabolismo , LipídeosRESUMO
The conformational ensembles of G protein-coupled receptors (GPCRs) include inactive and active states. Spectroscopy techniques, including NMR, show that agonists, antagonists and other ligands shift the ensemble toward specific states depending on the pharmacological efficacy of the ligand. How receptors recognize ligands and the kinetic mechanism underlying this population shift is poorly understood. Here, we investigate the kinetic mechanism of neurotensin recognition by neurotensin receptor 1 (NTS1) using 19F-NMR, hydrogen-deuterium exchange mass spectrometry and stopped-flow fluorescence spectroscopy. Our results indicate slow-exchanging conformational heterogeneity on the extracellular surface of ligand-bound NTS1. Numerical analysis of the kinetic data of neurotensin binding to NTS1 shows that ligand recognition follows an induced-fit mechanism, in which conformational changes occur after neurotensin binding. This approach is applicable to other GPCRs to provide insight into the kinetic regulation of ligand recognition by GPCRs.
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Neurotensina , Receptores de Neurotensina , Neurotensina/metabolismo , Receptores de Neurotensina/metabolismo , Ligantes , Receptores Acoplados a Proteínas G/metabolismo , Ligação ProteicaRESUMO
Several bacterial toxins and viruses can deform membranes through multivalent binding to lipids for clathrin-independent endocytosis. However, it remains unclear, how membrane deformation and endocytic internalization are mechanistically linked. Here we show that many lipid-binding virions induce membrane deformation and clathrin-independent endocytosis, suggesting a common mechanism based on multivalent lipid binding by globular particles. We create a synthetic cellular system consisting of a lipid-anchored receptor in the form of GPI-anchored anti-GFP nanobodies and a multivalent globular binder exposing 180 regularly-spaced GFP molecules on its surface. We show that these globular, 40 nm diameter, particles bind to cells expressing the receptor, deform the plasma membrane upon adhesion and become endocytosed in a clathrin-independent manner. We explore the role of the membrane adhesion energy in endocytosis by using receptors with affinities varying over 7 orders of magnitude. Using this system, we find that once a threshold in adhesion energy is overcome to allow for membrane deformation, endocytosis occurs reliably. Multivalent, binding-induced membrane deformation by globular binders is thus sufficient for internalization to occur and we suggest it is the common, purely biophysical mechanism for lipid-binding mediated endocytosis of toxins and pathogens.
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Binding forces between biomolecules are ubiquitous in nature but sometimes as weak as a few pico-Newtons (pN). In many cases, the binding partners are attached to biomembranes with the help of a lipid anchor. One important example are glycolipids that promote membrane adhesion through weak carbohydrate-carbohydrate binding between adjacent membranes. Here, we use molecular dynamics (MD) simulations to quantify the forces generated by bonds involving membrane-anchored molecules. We introduce a method in which the protrusion of the lipid anchors from the membrane acts as the force sensor. Our results with two different glycolipids reveal binding forces of up to 20 pN and corroborate the recent notion that carbohydrate-carbohydrate interactions are generic rather than specific.
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Fenômenos Mecânicos , Simulação de Dinâmica Molecular , Glicolipídeos , MembranasRESUMO
Molecular dynamics simulations of immune receptor and ligand proteins in their native membrane environment allow to determine the orientational and structural variability of the proteins and protein complexes. The simulations complement the static, "membrane-free" structural information obtained from cryo-EM structures of transmembrane proteins in detergent micelles or from crystal structures of extracellular protein domains. Here we describe how to set up and perform simulations of transmembrane receptors, ligands, and receptor-ligand complexes.
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Simulação de Dinâmica Molecular , Receptores Imunológicos , Ligantes , Membrana Celular/metabolismo , Domínios Proteicos , Receptores Imunológicos/metabolismoRESUMO
T cells use their T-cell receptors (TCRs) to discriminate between lower-affinity self and higher-affinity foreign peptide major-histocompatibility-complexes (pMHCs) based on the TCR/pMHC off-rate. It is now appreciated that T cells generate mechanical forces during this process but how force impacts the TCR/pMHC off-rate remains debated. Here, we measured the effect of mechanical force on the off-rate of multiple TCR/pMHC interactions. Unexpectedly, we found that lower-affinity TCR/pMHCs with faster solution off-rates were more resistant to mechanical force (weak slip or catch bonds) than higher-affinity interactions (strong slip bonds). This was confirmed by molecular dynamics simulations. Consistent with these findings, we show that the best-characterized catch bond, involving the OT-I TCR, has a low affinity and an exceptionally fast solution off-rate. Our findings imply that reducing forces on the TCR/pMHC interaction improves antigen discrimination, and we suggest a role for the adhesion receptors CD2 and LFA-1 in force-shielding the TCR/pMHC interaction.
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Receptores de Antígenos de Linfócitos T , Linfócitos T , Receptores de Antígenos de Linfócitos T/metabolismo , Complexo Principal de Histocompatibilidade , Peptídeos , Simulação de Dinâmica Molecular , Ligação ProteicaRESUMO
Partner recognition in protein binding is critical for all biological functions, and yet, delineating its mechanism is challenging, especially when recognition happens within microseconds. We present a theoretical and experimental framework based on straight-forward nuclear magnetic resonance relaxation dispersion measurements to investigate protein binding mechanisms on sub-millisecond timescales, which are beyond the reach of standard rapid-mixing experiments. This framework predicts that conformational selection prevails on ubiquitin's paradigmatic interaction with an SH3 (Src-homology 3) domain. By contrast, the SH3 domain recognizes ubiquitin in a two-state binding process. Subsequent molecular dynamics simulations and Markov state modeling reveal that the ubiquitin conformation selected for binding exhibits a characteristically extended C-terminus. Our framework is robust and expandable for implementation in other binding scenarios with the potential to show that conformational selection might be the design principle of the hubs in protein interaction networks.
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Proteínas de Transporte , Domínios de Homologia de src , Proteínas de Transporte/metabolismo , Ligação Proteica , Conformação Proteica , Ubiquitina/metabolismoRESUMO
Glycolipids mediate stable membrane adhesion of potential biological relevance. In this article, we investigate the trans- and cis-interactions of glycolipids in molecular dynamics simulations and relate these interactions to the glycolipid-induced average separations of membranes obtained from neutron scattering experiments. We find that the cis-interactions between glycolipids in the same membrane leaflet tend to strengthen the trans-interactions between glycolipids in apposing leaflets. The trans-interactions of the glycolipids in our simulations require local membrane separations that are significantly smaller than the average membrane separations in the neutron scattering experiments, which indicates an important role of membrane shape fluctuations in glycolipid trans-binding. Simulations at the experimentally measured average membrane separations provide a molecular picture of the interplay between glycolipid attraction and steric repulsion of the fluctuating membranes probed in the experiments.
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We investigate the structural and orientational variability of the membrane-embedded T cell receptor (TCR) - CD3 complex in extensive atomistic molecular dynamics simulations based on the recent cryo-EM structure determined by Dong et al., 2019. We find that the TCR extracellular (EC) domain is highly variable in its orientation by attaining tilt angles relative to the membrane normal that range from 15° to 55°. The tilt angle of the TCR EC domain is both coupled to a rotation of the domain and to characteristic changes throughout the TCR - CD3 complex, in particular in the EC interactions of the Cß FG loop of the TCR, as well as in the orientation of transmembrane helices. The concerted motions of the membrane-embedded TCR - CD3 complex revealed in our simulations provide atomistic insights on conformational changes of the complex in response to tilt-inducing forces on antigen-bound TCRs.
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Complexo CD3/metabolismo , Membrana Celular/metabolismo , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T/metabolismo , Complexo CD3/ultraestrutura , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Complexo Receptor-CD3 de Antígeno de Linfócitos T/ultraestrutura , Receptores de Antígenos de Linfócitos T alfa-beta/ultraestrutura , Relação Estrutura-Atividade , Linfócitos T/imunologia , Linfócitos T/ultraestruturaRESUMO
T cells are sensitive to 1 to 10 foreign-peptide-MHC complexes among a vast majority of self-peptide-MHC complexes, and discriminate selectively between peptide-MHC complexes that differ not much in their binding affinity to T-cell receptors (TCRs). Quantitative models that aim to explain this sensitivity and selectivity largely focus on single TCR/peptide-MHC complexes, but T cell adhesion involves a multitude of different complexes. In this article, we demonstrate in a three-dimensional computational model of T-cell adhesion that the cooperative stabilization of close-contact zones is sensitive to one to three foreign-peptide-MHC complexes and occurs at a rather sharp threshold affinity of these complexes, which implies selectivity. In these close-contact zones with lateral extensions of hundred to several hundred nanometers, few TCR/foreign-peptide-MHC complexes and many TCR/self-peptide-MHC complexes are segregated from LFA-1/ICAM-1 complexes that form at larger membrane separations. Previous high-resolution microscopy experiments indicate that the sensitivity and selectivity in the formation of closed-contact zones reported here are relevant for T-cell recognition, because the stabilization of close-contact zones by foreign, agonist peptide-MHC complexes precedes T-cell signaling and activation in the experiments.
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Complexo Principal de Histocompatibilidade/imunologia , Linfócitos T/citologia , Adesão Celular , Membrana Celular/metabolismo , Simulação por Computador , Difusão , Humanos , Ativação Linfocitária , Modelos Teóricos , Peptídeos/química , Probabilidade , Ligação Proteica , Receptores de Antígenos de Linfócitos T/metabolismo , TermodinâmicaRESUMO
Biological membranes are shaped by various proteins that either generate inward or outward membrane curvature. In this article, we investigate the membrane morphologies induced by mixtures of arc-shaped particles with coarse-grained modeling and simulations. The particles bind to the membranes either with their inward, concave side or their outward, convex side and, thus, generate membrane curvature of opposite sign. We find that small fractions of convex-binding particles can stabilize three-way junctions of membrane tubules, as suggested for the protein lunapark in the endoplasmic reticulum of cells. For comparable fractions of concave-binding and convex-binding particles, we observe lines of particles of the same type, and diverse membrane morphologies with grooves and bulges induced by these particle lines. The alignment and segregation of the particles is driven by indirect, membrane-mediated interactions.
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Retículo Endoplasmático , Membrana Celular , MembranasRESUMO
Carbohydrates such as the trisaccharide motif LeX are key constituents of cell surfaces. Despite intense research, the interactions between carbohydrates of apposing cells or membranes are not well understood. In this article, we investigate carbohydrate-carbohydrate interactions in membrane adhesion as well as in solution with extensive atomistic molecular dynamics simulations that exceed the simulation times of previous studies by orders of magnitude. For LeX, we obtain association constants of soluble carbohydrates, adhesion energies of lipid-anchored carbohydrates, and maximally sustained forces of carbohydrate complexes in membrane adhesion that are in good agreement with experimental results in the literature. Our simulations thus appear to provide a realistic, detailed picture of LeX-LeX interactions in solution and during membrane adhesion. In this picture, the LeX-LeX interactions are fuzzy, i.e. LeX pairs interact in a large variety of short-lived, bound conformations. For the synthetic tetrasaccharide Lac 2, which is composed of two lactose units, we observe similarly fuzzy interactions and obtain association constants of both soluble and lipid-anchored variants that are comparable to the corresponding association constants of LeX. The fuzzy, weak carbohydrate-carbohydrate interactions quantified in our simulations thus appear to be a generic feature of small, neutral carbohydrates such as LeX and Lac 2.
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Carboidratos , Antígenos CD15 , Conformação Molecular , Simulação de Dinâmica Molecular , TrissacarídeosRESUMO
The HIV-1 protease is a major target of inhibitor drugs in AIDS therapies. The therapies are impaired by mutations of the HIV-1 protease that can lead to resistance to protease inhibitors. These mutations are classified into major mutations, which usually occur first and clearly reduce the susceptibility to protease inhibitors, and minor, accessory mutations that occur later and individually do not substantially affect the susceptibility to inhibitors. Major mutations are predominantly located in the active site of the HIV-1 protease and can directly interfere with inhibitor binding. Minor mutations, in contrast, are typically located distal to the active site. A central question is how these distal mutations contribute to resistance development. In this article, we present a systematic computational investigation of stability changes caused by major and minor mutations of the HIV-1 protease. As most small single-domain proteins, the HIV-1 protease is only marginally stable. Mutations that destabilize the folded, active state of the protease therefore can shift the conformational equilibrium towards the unfolded, inactive state. We find that the most frequent major mutations destabilize the HIV-1 protease, whereas roughly half of the frequent minor mutations are stabilizing. An analysis of protease sequences from patients in treatment indicates that the stabilizing minor mutations are frequently correlated with destabilizing major mutations, and that highly resistant HIV-1 proteases exhibit significant fractions of stabilizing mutations. Our results thus indicate a central role of minor mutations in balancing the marginal stability of the protease against the destabilization induced by the most frequent major mutations.
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Farmacorresistência Viral/genética , Inibidores da Protease de HIV/química , Protease de HIV/química , HIV-1/efeitos dos fármacos , Mutação , Domínio Catalítico , Estabilidade Enzimática , Protease de HIV/genética , Protease de HIV/metabolismo , Inibidores da Protease de HIV/metabolismo , HIV-1/química , HIV-1/enzimologia , Humanos , Ligação Proteica , Relação Estrutura-Atividade , TermodinâmicaRESUMO
We present a detailed investigation of the effect of the crystallographic structure of the HIV-1 protease (PR) on the binding energy of different classes of inhibitors obtained from docking simulations. The crystal structures of 222 HIV-1 proteases (in wild-type and mutant forms) and 202 inhibitors were downloaded from appropriate databases. A cross-docking approach (docking of all 202 inhibitors to all 222â¯PR structures) using Autodock Vina was implemented. The protease structures were clustered using a Kohonen self-organization map analysis of the data matrix of docking energies. The obtained clusters of PRs were correlated with the x-y-z coordinates of the PR structures to identify structural segments underlying this clustering. The PR structures were clustered into 4 classes. One of these classes exhibits rather strong binding with almost all inhibitors, while another class exhibits rather weak binding. The remaining two classes are intermediate in binding strength. The selectivity ratio indices for the carbon-alpha atoms of the PR structures indicate that conformational motion of residues outside the binding pocket contributes significantly to the discrimination of the 4 classes.
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Protease de HIV/química , Simulação de Acoplamento Molecular , Análise Discriminante , Análise dos Mínimos Quadrados , Análise de Componente Principal , Conformação Proteica , TermodinâmicaRESUMO
The intricate shapes of biological membranes such as tubules and membrane stacks are induced by proteins. In this article, we systematically investigate the membrane shapes induced by arc-shaped scaffolds such as proteins and protein complexes with coarse-grained modeling and simulations. We find that arc-shaped scaffolds induce membrane tubules at membrane coverages larger than a threshold of â¼40%, irrespective of their arc angle. The membrane morphologies at intermediate coverages below this tubulation threshold, in contrast, strongly depend on the arc angle. Scaffolds with arc angles of about 60°, akin to N-BAR domains, do not change the membrane shape at coverages below the tubulation threshold, whereas scaffolds with arc angles larger than about 120° induce double-membrane stacks at intermediate coverages. The scaffolds stabilize the curved membrane edges that connect the membrane stacks, as suggested for complexes of reticulon proteins. Our results provide general insights on the determinants of membrane shaping by arc-shaped scaffolds.
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Membrana Celular/química , Simulação de Dinâmica Molecular , Estresse Mecânico , Proteínas de Membrana/química , Domínios ProteicosRESUMO
Cell-cell interactions that result from membrane proteins binding weakly in trans can cause accumulations in cis that suggest cooperativity and thereby an acute sensitivity to environmental factors. The ubiquitous 'marker of self' protein CD47 binds weakly to SIRPα on macrophages, which leads to accumulation of SIRPα (also known as SHPS-1, CD172A and SIRPA) at phagocytic synapses and ultimately to inhibition of engulfment of 'self' cells - including cancer cells. We reconstituted this macrophage checkpoint with GFP-tagged CD47 on giant vesicles generated from plasma membranes and then imaged vesicles adhering to SIRPα immobilized on a surface. CD47 diffusion is impeded near the surface, and the binding-unbinding events reveal cooperative interactions as a concentration-dependent two-dimensional affinity. Membrane fluctuations out-of-plane link cooperativity to membrane flexibility with suppressed fluctuations in the vicinity of bound complexes. Slight acidity (pH 6) stiffens membranes, diminishes cooperative interactions and also reduces 'self' signaling of cancer cells in phagocytosis. Sensitivity of cell-cell interactions to microenvironmental factors - such as the acidity of tumors and other diseased or inflamed sites - can thus arise from the collective cooperative properties of flexible membranes.This article has an associated First Person interview with the first author of the paper.
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Acidose/metabolismo , Antígenos de Diferenciação/metabolismo , Antígeno CD47/metabolismo , Macrófagos/metabolismo , Receptores Imunológicos/metabolismo , Biomarcadores/metabolismo , Humanos , Neoplasias/metabolismo , Fagocitose/fisiologia , Ligação Proteica/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Besides direct protein-protein interactions, indirect interactions mediated by membranes play an important role for the assembly and cooperative function of proteins in membrane shaping and adhesion. The intricate shapes of biological membranes are generated by proteins that locally induce membrane curvature. Indirect curvature-mediated interactions between these proteins arise because the proteins jointly affect the bending energy of the membranes. These curvature-mediated interactions are attractive for crescent-shaped proteins and are a driving force in the assembly of the proteins during membrane tubulation. Membrane adhesion results from the binding of receptor and ligand proteins that are anchored in the apposing membranes. The binding of these proteins strongly depends on nanoscale shape fluctuations of the membranes, leading to a fluctuation-mediated binding cooperativity. A length mismatch between receptor-ligand complexes in membrane adhesion zones causes repulsive curvature-mediated interactions that are a driving force for the length-based segregation of proteins during membrane adhesion.