RESUMO
Atypical Chemokine Receptor 3 (ACKR3) belongs to the G protein-coupled receptor family but it does not signal through G proteins. The structural properties that govern the functional selectivity and the conformational dynamics of ACKR3 activation are poorly understood. Here, we combined hydrogen/deuterium exchange mass spectrometry, site-directed mutagenesis, and molecular dynamics simulations to examine the binding mode and mechanism of action of ACKR3 ligands of different efficacies. Our results show that activation or inhibition of ACKR3 is governed by intracellular conformational changes of helix 6, intracellular loop 2, and helix 7, while the DRY motif becomes protected during both processes. Moreover, we identified the binding sites and the allosteric modulation of ACKR3 upon ß-arrestin 1 binding. In summary, this study highlights the structure-function relationship of small ligands, the binding mode of ß-arrestin 1, the activation dynamics, and the atypical dynamic features in ACKR3 that may contribute to its inability to activate G proteins.
Assuntos
Simulação de Dinâmica Molecular , Ligação Proteica , Receptores CXCR , Humanos , Receptores CXCR/metabolismo , Receptores CXCR/genética , Sítios de Ligação , Conformação Proteica , beta-Arrestina 1/metabolismo , beta-Arrestina 1/genética , Ligantes , Células HEK293 , Mutagênese Sítio-Dirigida , Regulação Alostérica , Relação Estrutura-AtividadeRESUMO
The TOPOVIL complex catalyzes the formation of DNA double strand breaks (DSB) that initiate meiotic homologous recombination, an essential step for chromosome segregation and genetic diversity during gamete production. TOPOVIL is composed of two subunits (SPO11 and TOPOVIBL) and is evolutionarily related to the archaeal TopoVI topoisomerase complex. SPO11 is the TopoVIA subunit orthologue and carries the DSB formation catalytic activity. TOPOVIBL shares homology with the TopoVIB ATPase subunit. TOPOVIBL is essential for meiotic DSB formation, but its molecular function remains elusive, partly due to the lack of biochemical studies. Here, we purified TOPOVIBLΔC25 and characterized its structure and mode of action in vitro. Our structural analysis revealed that TOPOVIBLΔC25 adopts a dynamic conformation in solution and our biochemical study showed that the protein remains monomeric upon incubation with ATP, which correlates with the absence of ATP binding. Moreover, TOPOVIBLΔC25 interacted with DNA, with a preference for some geometries, suggesting that TOPOVIBL senses specific DNA architectures. Altogether, our study identified specific TOPOVIBL features that might help to explain how TOPOVIL function evolved toward a DSB formation activity in meiosis.
RESUMO
Atypical chemokine receptor 1 (ACKR1) is a G protein-coupled receptor (GPCR) targeted by Staphylococcus aureus bicomponent pore-forming leukotoxins to promote bacterial growth and immune evasion. Here, we have developed an integrative molecular pharmacology and structural biology approach in order to characterize the effect of leukotoxins HlgA and HlgB on ACKR1 structure and function. Interestingly, using cell-based assays and native mass spectrometry, we found that both components HlgA and HlgB compete with endogenous chemokines through a direct binding with the extracellular domain of ACKR1. Unexpectedly, hydrogen/deuterium exchange mass spectrometry analysis revealed that toxin binding allosterically modulates the intracellular G protein-binding domain of the receptor, resulting in dissociation and/or changes in the architecture of ACKR1-Gαi1 protein complexes observed in living cells. Altogether, our study brings important molecular insights into the initial steps of leukotoxins targeting a host GPCR.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Staphylococcus aureus/fisiologia , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Dimerização , Sistema do Grupo Sanguíneo Duffy/isolamento & purificação , Sistema do Grupo Sanguíneo Duffy/metabolismo , Exotoxinas/metabolismo , Humanos , Espectrometria de Massas/métodos , Ligação Proteica , Receptores de Superfície Celular/isolamento & purificação , Receptores de Superfície Celular/metabolismo , Células Sf9RESUMO
Adiponectin receptors (ADIPORs) are integral membrane proteins that control glucose and lipid metabolism by mediating, at least in part, a cellular ceramidase activity that catalyses the hydrolysis of ceramide to produce sphingosine and a free fatty acid (FFA). The crystal structures of the two receptor subtypes, ADIPOR1 and ADIPOR2, show a similar overall seven-transmembrane-domain architecture with large unoccupied cavities and a zinc binding site within the seven transmembrane domain. However, the molecular mechanisms by which ADIPORs function are not known. Here we describe the crystal structure of ADIPOR2 bound to a FFA molecule and show that ADIPOR2 possesses intrinsic basal ceramidase activity that is enhanced by adiponectin. We also identify a ceramide binding pose and propose a possible mechanism for the hydrolytic activity of ADIPOR2 using computational approaches. In molecular dynamics simulations, the side chains of residues coordinating the zinc rearrange quickly to promote the nucleophilic attack of a zinc-bound hydroxide ion onto the ceramide amide carbonyl. Furthermore, we present a revised ADIPOR1 crystal structure exhibiting a seven-transmembrane-domain architecture that is clearly distinct from that of ADIPOR2. In this structure, no FFA is observed and the ceramide binding pocket and putative zinc catalytic site are exposed to the inner membrane leaflet. ADIPOR1 also possesses intrinsic ceramidase activity, so we suspect that the two distinct structures may represent key steps in the enzymatic activity of ADIPORs. The ceramidase activity is low, however, and further studies will be required to characterize fully the enzymatic parameters and substrate specificity of ADIPORs. These insights into ADIPOR function will enable the structure-based design of potent modulators of these clinically relevant enzymes.
Assuntos
Ceramidas/química , Ceramidas/metabolismo , Receptores de Adiponectina/química , Receptores de Adiponectina/metabolismo , Adiponectina/metabolismo , Adiponectina/farmacologia , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Ácidos Graxos não Esterificados/química , Ácidos Graxos não Esterificados/metabolismo , Humanos , Hidrólise/efeitos dos fármacos , Hidróxidos/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Domínios Proteicos , Zinco/metabolismoRESUMO
Sphingolipid metabolism is tightly controlled by enzymes to regulate essential processes in human physiology. The central metabolite is ceramide, a pro-apoptotic lipid catabolized by ceramidase enzymes to produce pro-proliferative sphingosine-1-phosphate. Alkaline ceramidases are transmembrane enzymes that recently attracted attention for drug development in fatty liver diseases. However, due to their hydrophobic nature, no specific small molecule inhibitors have been reported. We present the discovery and mechanism of action of the first drug-like inhibitors of alkaline ceramidase 3 (ACER3). In particular, we chemically engineered novel fluorescent ceramide substrates enabling screening of large compound libraries and characterized enzyme:inhibitor interactions using mass spectrometry and MD simulations. In addition to revealing a new paradigm for inhibition of lipid metabolising enzymes with non-lipidic small molecules, our data lay the ground for targeting ACER3 in drug discovery efforts.
Assuntos
CeramidasesRESUMO
Small molecules are known to stabilize membrane proteins and to modulate their function and oligomeric state, but such interactions are often hard to precisely define. Here we develop and apply a high-resolution, Orbitrap mass spectrometry-based method for analyzing intact membrane protein-ligand complexes. Using this platform, we resolve the complexity of multiple binding events, quantify small molecule binding and reveal selectivity for endogenous lipids that differ only in acyl chain length.
Assuntos
Lipídeos/química , Espectrometria de Massas/métodos , Proteínas de Membrana/metabolismo , Fragmentos de Peptídeos/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Humanos , Modelos Moleculares , Ligação ProteicaRESUMO
Mass spectrometry (MS) provides an impressive array of information about the structure, function and interactions of proteins. In recent years, many new developments have been in the field of native MS and these exemplify a new coming of age of this field. In this mini review, we connect the latest methodological and instrumental developments in native MS to the new insights these have enabled. We highlight the prominence of an increasingly common strategy of using hybrid approaches, where multiple MS-based techniques are used in combination, and integrative approaches, where MS is used alongside other techniques such as ion-mobility spectrometry. We also review how the emergence of a native top-down approach, which combines native MS with top-down proteomics into a single experiment, is the pièce de résistance of structural mass spectrometry's coming of age. Finally, we outline key developments that have enabled membrane protein native MS to shift from being extremely challenging to routine, and how this technique is uncovering inaccessible details of membrane protein-lipid interactions.
Assuntos
Espectrometria de Massas/métodos , Proteínas/química , Proteínas/metabolismo , Sítios de Ligação , Ligantes , Ligação Proteica , Conformação Proteica , ProteômicaRESUMO
The phylum Apicomplexa encompasses deadly pathogens such as malaria and Cryptosporidium. Apicomplexa cell division is mechanistically divergent from that of their mammalian host, potentially representing an attractive source of drug targets. Depending on the species, apicomplexan parasites can modulate the output of cell division, producing two to thousands of daughter cells at once. The inherent flexibility of their cell division mechanisms allows these parasites to adapt to different niches, facilitating their dissemination. Toxoplasma gondii tachyzoites divide using a unique form of cell division called endodyogeny. This process involves a single round of DNA replication, closed nuclear mitosis, and assembly of two daughter cells within a mother. In higher Eukaryotes, the four-subunit chromosomal passenger complex (CPC) (Aurora kinase B (ARKB)/INCENP/Borealin/Survivin) promotes chromosome bi-orientation by detaching incorrect kinetochore-microtubule attachments, playing an essential role in controlling cell division fidelity. Herein, we report the characterization of the Toxoplasma CPC (Aurora kinase 1 (Ark1)/INCENP1/INCENP2). We show that the CPC exhibits dynamic localization in a cell cycle-dependent manner. TgArk1 interacts with both TgINCENPs, with TgINCENP2 being essential for its translocation to the nucleus. While TgINCENP1 appears to be dispensable, interfering with TgArk1 or TgINCENP2 results in pronounced division and growth defects. Significant anti-cancer drug development efforts have focused on targeting human ARKB. Parasite treatment with low doses of hesperadin, a known inhibitor of human ARKB at higher concentrations, phenocopies the TgArk1 and TgINCENP2 mutants. Overall, our study provides new insights into the mechanisms underpinning cell cycle control in Apicomplexa, and highlights TgArk1 as potential drug target.
Assuntos
Segregação de Cromossomos , Cromossomos/genética , Fuso Acromático/metabolismo , Toxoplasma/genética , Animais , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Cromossomos/metabolismo , Replicação do DNA/genética , Expressão Gênica , Interações Hospedeiro-Parasita , Humanos , Microscopia Eletrônica de Transmissão , Mitose/genética , Toxoplasma/fisiologia , Toxoplasma/ultraestrutura , Toxoplasmose/parasitologiaRESUMO
Antimicrobial peptides (AMPs) are considered as potential therapeutic sources of future antibiotics because of their broad-spectrum activities and alternative mechanisms of action compared to conventional antibiotics. Although AMPs present considerable advantages over conventional antibiotics, their clinical and commercial development still have some limitations, because of their potential toxicity, susceptibility to proteases, and high cost of production. To overcome these drawbacks, the use of peptides mimics is anticipated to avoid the proteolysis, while the identification of minimalist peptide sequences retaining antimicrobial activities could bring a solution for the cost issue. We describe here new polycationic ß-amino acids combining these two properties, that we used to design small dipeptides that appeared to be active against Gram-positive and Gram-negative bacteria, selective against prokaryotic versus mammalian cells, and highly stable in human plasma. Moreover, the in vivo data activity obtained in septic mice reveals that the bacterial killing effect allows the control of the infection and increases the survival rate of cecal ligature and puncture (CLP)-treated mice.
Assuntos
Anti-Infecciosos/administração & dosagem , Anti-Infecciosos/síntese química , Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Peptídeos Catiônicos Antimicrobianos/síntese química , Sepse/tratamento farmacológico , Aminoácidos/química , Animais , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Modelos Animais de Doenças , Desenho de Fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Mimetismo Molecular , Proteólise , Sepse/etiologia , Sepse/microbiologiaRESUMO
Among non-invasive cell delivery strategies, cell-penetrating peptide (CPP) vectors represent interesting new tools. To get fundamental knowledge about the still debated internalisation mechanisms of these peptides, we modified the membrane content of cells, typically by hydrolysis of sphingomyelin or depletion of cholesterol from the membrane outer leaflet. We quantified and visualised the effect of these viable cell surface treatments on the internalisation efficiency of different CPPs, among which the most studied Tat, R9, penetratin and analogues, that all carry the N-terminal biotin-Gly4 tag cargo. Under these cell membrane treatments, only penetratin and R6W3 underwent a massive glycosaminoglycan (GAG)-dependent entry in cells. Internalisation of the other peptides was only slightly increased, similarly in the absence or the presence of GAGs for R9, and only in the presence of GAGs for Tat and R6L3. Ceramide formation (or cholesterol depletion) is known to lead to the reorganisation of membrane lipid domains into larger platforms, which can serve as a trap and cluster receptors. These results show that GAG clustering, enhanced by formation of ceramide, is efficiently exploited by penetratin and R6W3, which contains Trp residues in their sequence but not Tat, R9 and R6L3. Hence, these data shed new lights on the differences in the internalisation mechanism and pathway of these peptides that are widely used in delivery of cargo molecules.
Assuntos
Peptídeos Penetradores de Células/metabolismo , Colesterol/metabolismo , Glicosaminoglicanos/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Peptídeos Penetradores de Células/química , Ceramidas/análise , Cricetinae , Cricetulus , Endocitose , Microscopia Confocal , Dados de Sequência Molecular , Esfingomielinas/metabolismo , Triptofano/químicaRESUMO
The realization that the lipid environment is crucial for maintaining the structure and function of membrane proteins prompts new methods to understand lipid interactions. One such method, mass spectrometry, is emerging with the potential to monitor different modes of lipid binding to membrane protein complexes. Initial studies monitored the addition of lipids and deduced the kinetic and thermodynamic effects of lipid binding to proteins. Recent efforts however have focused on identifying lipids already present, explicitly in plugs, annular rings, or cavities. Lipids that bind within these orifices to membrane proteins will have higher residence times than those in the bulk lipid bilayer and consequently can be quantified and characterized by mass spectrometry. In special cases, lipids identified within cavities have been proposed as substrates following activity assays. Alternatively, a gas-phase unfolding protocol can be used to distinguish lipids that are important for stability. These lipids can subsequently be added during crystallization for the characterization of lipid-bound protein complexes. Overall therefore this Perspective provides an overview of recent advances in mass spectrometry, with a particular focus on the distinction of the various modes of lipid binding, and their implications for structure and function as well as new directions that lie ahead.
Assuntos
Espectrometria de Massas/métodos , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Animais , Humanos , Lipídeos de Membrana/química , Proteínas de Membrana/química , Modelos Moleculares , Ligação ProteicaRESUMO
Deciphering the structural requirements and mechanisms for internalization of cell-penetrating peptides (CPPs) is required to improve their delivery efficiency. Herein, a unique role of tryptophan (Trp) residues in the interaction and structuring of cationic CPP sequences with glycosaminoglycans (GAGs) has been characterized, in relation with cell internalization. Using isothermal titration calorimetry, circular dichroism, NMR, mass spectrometry, and phase-contrast microscopy, we compared the interaction of 7 basic CPPs with 5 classes of GAGs. We found that the affinity of CPPs for GAGs increases linearly with the number of Trp residues, from 30 nM for a penetratin analog with 1 Trp residue to 1.5 nM for a penetratin analog with 6 Trp residues for heparin (HI); peptides with Trp residues adopt a predominantly ß-strand structure in complex with HI and form large, stable ß-sheet aggregates with GAGs; and in the absence of any cytotoxicity effect, the quantity of peptide internalized into CHO cells increased 2 times with 1 Trp residue, 10 times with 2 Trp residues, and 20 times with 3 Trp residues, compared with +6 peptides with no Trp residues. Therefore, Trp residues represent molecular determinants in basic peptide sequences not only for direct membrane translocation but also for efficient endocytosis through GAGs.
Assuntos
Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismo , Endocitose/fisiologia , Glicosaminoglicanos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Transporte Biológico Ativo , Células CHO , Membrana Celular/metabolismo , Peptídeos Penetradores de Células/genética , Cricetinae , Cricetulus , Glicosaminoglicanos/química , Modelos Biológicos , Dados de Sequência Molecular , Estrutura Molecular , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Secundária de Proteína , Termodinâmica , Triptofano/químicaRESUMO
Amphipols (APols) are amphipathic polymers with the ability to substitute detergents to keep membrane proteins (MPs) soluble and functional in aqueous solutions. APols also protect MPs against denaturation. Here, we have examined the ability of APol-trapped MPs to be analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). For that purpose, we have used ionic and nonionic APols and as model proteins (i) the transmembrane domain of Escherichia coli outer membrane protein A, a ß-barrel, eubacterial MP, (ii) Halobacterium salinarum bacteriorhodopsin, an α-helical archaebacterial MP with a single cofactor, and (iii, iv) two eukaryotic MP complexes comprising multiple subunits and many cofactors, cytochrome b(6)f from the chloroplast of the green alga Chlamydomonas reinhardtii and cytochrome bc(1) from beef heart mitochondria. We show that these MP/APol complexes can be readily analyzed by MALDI-TOF-MS; most of the subunits and some lipids and cofactors were identified. APols alone, even ionic ones, had no deleterious effects on MS signals and were not detected in mass spectra. Thus, the combination of MP stabilization by APols and MS analyses provides an interesting new approach to investigating supramolecular interactions in biological membranes.
Assuntos
Interações Hidrofóbicas e Hidrofílicas , Proteínas Imobilizadas/análise , Proteínas Imobilizadas/química , Proteínas de Membrana/análise , Proteínas de Membrana/química , Polímeros/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Animais , Bovinos , Detergentes/química , Proteínas Imobilizadas/metabolismo , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Solubilidade , Tripsina/metabolismoRESUMO
Staphylococcus aureus (SA) leukocidin ED (LukED) belongs to a family of bicomponent pore forming toxins that play important roles in SA immune evasion and nutrient acquisition. LukED targets specific G protein-coupled chemokine receptors to lyse human erythrocytes (red blood cells) and leukocytes (white blood cells). The first recognition step of receptors is critical for specific cell targeting and lysis. The structural and molecular bases for this mechanism are not well understood but could constitute essential information to guide antibiotic development. Here, we characterized the interaction of LukE with chemokine receptors ACKR1, CCR2, and CCR5 using a combination of structural, pharmacological, and computational approaches. First, crystal structures of LukE in complex with a small molecule mimicking sulfotyrosine side chain (p-cresyl sulfate) and with peptides containing sulfotyrosines issued from receptor sequences revealed the location of receptor sulfotyrosine binding sites in the toxins. Then, by combining previous and novel experimental data with protein docking, classical and accelerated weight histogram (AWH) molecular dynamics we propose models of the ACKR1-LukE and CCR5-LukE complexes. This work provides novel insights into chemokine receptor recognition by leukotoxins and suggests that the conserved sulfotyrosine binding pocket could be a target of choice for future drug development.
Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Humanos , Evasão da Resposta Imune , Leucocidinas/metabolismo , Receptores de Quimiocinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Staphylococcus aureus/genéticaRESUMO
The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that mediates a broad spectrum of (patho)physiological processes in response to numerous substances including pollutants, natural products and metabolites. However, the scarcity of structural data precludes understanding of how AHR is activated by such diverse compounds. Our 2.85 Å structure of the human indirubin-bound AHR complex with the chaperone Hsp90 and the co-chaperone XAP2, reported herein, reveals a closed conformation Hsp90 dimer with AHR threaded through its lumen and XAP2 serving as a brace. Importantly, we disclose the long-awaited structure of the AHR PAS-B domain revealing a unique organisation of the ligand-binding pocket and the structural determinants of ligand-binding specificity and promiscuity of the receptor. By providing structural details of the molecular initiating event leading to AHR activation, our study rationalises almost forty years of biochemical data and provides a framework for future mechanistic studies and structure-guided drug design.
Assuntos
Proteínas de Choque Térmico HSP90 , Peptídeos e Proteínas de Sinalização Intracelular , Receptores de Hidrocarboneto Arílico , Humanos , Microscopia Crioeletrônica , Citosol/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligantes , Receptores de Hidrocarboneto Arílico/metabolismoRESUMO
Comprehensive characterization of physicochemical properties of monoclonal antibodies (mAbs) is a critical process to ensure their quality, efficacy, and safety. For this purpose, mAb analysis at different levels (bottom-up, middle-up) is a common approach that includes rather complex multistep sample preparation (reduction, digestion). To ensure high analysis performance, the development of fully integrated methodologies is highly valuable. Capillary zone electrophoresis is a particularly well-adapted technique for the multistep implementation of analytical strategies from sample preparation to detection. This feature was employed to develop novel integrated methodologies for the analysis of mAb at the middle-up level. Multiple in-line reactions (simultaneous reduction and digestion) were performed for the first time in the separation capillary. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was used as an effective reducing agent under a broad pH range and IdeS (Immunoglobulin degrading enzyme from Streptococcus) as a highly specific enzyme for mAb digestion. Transverse diffusion of laminar flow profile (TDLFP) was applied for reactants mixing. Both in-line sample preparation and separation parameters were optimized under non-denaturing and denaturing conditions. The developed in-line methodologies provided good reproducibility and higher peak efficiencies comparing with off-line assays. They were successfully applied to different mAbs.
Assuntos
Anticorpos Monoclonais/análise , Eletroforese Capilar/métodos , Difusão , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
In this study, we have investigated the lipids surrounding AqpZ, and the effects of a destabilizing mutation W14A (Schmidt and Sturgis, 2017) on lipid protein interactions. In a first approach, we used Styrene Maleic Acid copolymer to prepare AqpZ containing nanodiscs, and these were analyzed for their lipid content, investigating both the lipid head-group and acyl-chain compositions. These results were complemented by native mass spectrometry of purified AqpZ in the presence of lipids, to give insights of variations in lipid binding at the surface of AqpZ. In an effort to gain molecular insights, to aid interpretation of these results, we performed a series of coarse grained molecular dynamics simulations of AqpZ, in mixed lipid membranes, and correlated our observations with the experimental measurements. These various results are then integrated to give a clearer picture of the lipid environment of AqpZ, both in the native membrane, and in lipid nanodiscs. We conclude that AqpZ contains a lipid binding-site, at the interface between the monomers of the tetramer, that is specific for cardiolipin. Almost all the cardiolipin, in AqpZ containing nanodiscs, is probably associated with this site. The SMA 3:1 nanodiscs we obtained contain a rather high proportion of lipid, and in the case of nanodiscs containing AqpZ cardiolipin is depleted. This is possibly because, in the membrane, there is little cardiolipin not associated with binding sites on the surface of the different membrane proteins. Surprisingly, we see no evidence for lipid sorting based on acyl chain length, even in the presence of a large hydrophobic mismatch, suggesting that conformational restrictions are energetically less costly than lipid sorting.
Assuntos
Aquaporinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Lipídeos/química , Membrana Celular/metabolismo , Lipídeos/isolamento & purificação , Simulação de Dinâmica Molecular , Nanopartículas/química , Nanopartículas/ultraestrutura , Fosfolipídeos/isolamento & purificaçãoRESUMO
With the recent success in determining membrane protein structures, further detailed understanding of the identity and function of the bound lipidome is essential. Using an approach that combines high-energy native mass spectrometry (HE-nMS) and solution-phase lipid profiling, this protocol can be used to determine the identity of the endogenous lipids that directly interact with a protein. Furthermore, this method can identify systems in which such lipid binding has a major role in regulating the oligomeric assembly of membrane proteins. The protocol begins with recording of the native mass spectrum of the protein of interest, under successive delipidation conditions, to determine whether delipidation leads to disruption of the oligomeric state. Subsequently, we propose using a bipronged strategy: first, an HE-nMS platform is used that allows dissociation of the detergent micelle at the front end of the instrument. This allows for isolation of the protein-lipid complex at the quadrupole and successive fragmentation at the collision cell, which leads to identification of the bound lipid masses. Next, simultaneous coupling of this with in-solution LC-MS/MS-based identification of extracted lipids reveals the complete identity of the interacting lipidome that copurifies with the proteins. Assimilation of the results of these two sets of experiments divulges the complete identity of the set of lipids that directly interact with the membrane protein of interest, and can further delineate its role in maintaining the oligomeric state of the protein. The entire procedure takes 2 d to complete.
Assuntos
Espectrometria de Massas/métodos , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Ligação Proteica , Multimerização ProteicaRESUMO
Studying the mechanisms of entry of cell-penetrating peptides (CPPs) requires reliable methods to measure their cellular uptake efficiency, monitor their metabolic stability, and identify their intracellular localization. We describe here a protocol based on the direct detection of peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which allows the absolute quantification of the intact internalized species and the analysis of their intracellular degradation. This protocol can be easily applied to the simultaneous quantification of different species, for example mixtures of CPPs.
Assuntos
Peptídeos Penetradores de Células/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Transporte Biológico , Soluções Tampão , Células CHO , Técnicas de Cultura de Células/métodos , Peptídeos Penetradores de Células/análise , Cricetulus , Marcação por Isótopo/métodosRESUMO
Lipids are critical components of membranes that could affect the properties of membrane proteins, yet the precise compositions of lipids surrounding membrane-embedded protein complexes is often difficult to discern. Here we report that, for the heterodimeric ABC transporter TmrAB, the extent of delipidation can be controlled by timed exposure to detergent. We subsequently characterize the cohort of endogenous lipids that are extracted in contact with the membrane protein complex, and show that with prolonged delipidation the number of neutral lipids is reduced in favour of their negatively charged counterparts. We show that lipid A is retained by the transporter and that the extent of its binding decreases during the catalytic cycle, implying that lipid A release is linked to adenosine tri-phosphate hydrolysis. Together, these results enable us to propose that a subset of annular lipids is invariant in composition, with negatively charged lipids binding tightly to TmrAB, and imply a role for this exporter in glycolipid translocation.