RESUMO
Eukaryotic cells can undergo different forms of programmed cell death, many of which culminate in plasma membrane rupture as the defining terminal event1-7. Plasma membrane rupture was long thought to be driven by osmotic pressure, but it has recently been shown to be in many cases an active process, mediated by the protein ninjurin-18 (NINJ1). Here we resolve the structure of NINJ1 and the mechanism by which it ruptures membranes. Super-resolution microscopy reveals that NINJ1 clusters into structurally diverse assemblies in the membranes of dying cells, in particular large, filamentous assemblies with branched morphology. A cryo-electron microscopy structure of NINJ1 filaments shows a tightly packed fence-like array of transmembrane α-helices. Filament directionality and stability is defined by two amphipathic α-helices that interlink adjacent filament subunits. The NINJ1 filament features a hydrophilic side and a hydrophobic side, and molecular dynamics simulations show that it can stably cap membrane edges. The function of the resulting supramolecular arrangement was validated by site-directed mutagenesis. Our data thus suggest that, during lytic cell death, the extracellular α-helices of NINJ1 insert into the plasma membrane to polymerize NINJ1 monomers into amphipathic filaments that rupture the plasma membrane. The membrane protein NINJ1 is therefore an interactive component of the eukaryotic cell membrane that functions as an in-built breaking point in response to activation of cell death.
Assuntos
Moléculas de Adesão Celular Neuronais , Morte Celular , Membrana Celular , Fatores de Crescimento Neural , Animais , Humanos , Camundongos , Moléculas de Adesão Celular Neuronais/química , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Moléculas de Adesão Celular Neuronais/ultraestrutura , Membrana Celular/metabolismo , Membrana Celular/patologia , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/ultraestrutura , Mutagênese Sítio-Dirigida , Biopolímeros/química , Biopolímeros/genética , Biopolímeros/metabolismoRESUMO
The Bcl-2 family controls apoptosis by direct interactions of pro- and anti-apoptotic proteins. The principle mechanism is binding of the BH3 domain of pro-apoptotic proteins to the hydrophobic groove of anti-apoptotic siblings, which is therapeutically exploited by approved BH3-mimetic anti-cancer drugs. Evidence suggests that also the transmembrane domain (TMD) of Bcl-2 proteins can mediate Bcl-2 interactions. We developed a highly-specific split luciferase assay enabling the analysis of TMD interactions of pore-forming apoptosis effectors BAX, BAK, and BOK with anti-apoptotic Bcl-2 proteins in living cells. We confirm homotypic interaction of the BAX-TMD, but also newly identify interaction of the TMD of anti-apoptotic BCL-2 with the TMD of BOK, a peculiar pro-apoptotic Bcl-2 protein. BOK-TMD and BCL-2-TMD interact at the endoplasmic reticulum. Molecular dynamics simulations confirm dynamic BOK-TMD and BCL-2-TMD dimers and stable heterotetramers. Mutation of BCL-2-TMD at predicted key residues abolishes interaction with BOK-TMD. Also, inhibition of BOK-induced apoptosis by BCL-2 depends specifically on their TMDs. Thus, TMDs of Bcl-2 proteins are a relevant interaction interface for apoptosis regulation and provide a novel potential drug target.
RESUMO
Evolution turned aquaporins (AQPs) into the most efficient facilitators of passive water flow through cell membranes at no expense of solute discrimination. In spite of a plethora of solved AQP structures, many structural details remain hidden. Here, by combining extensive sequence- and structural-based analysis of a unique set of 20 non-redundant high-resolution structures and molecular dynamics simulations of four representatives, key aspects of AQP stability, gating, selectivity, pore geometry, and oligomerization, with a potential impact on channel functionality, are identified. The general view of AQPs possessing a continuous open water pore is challenged and it is depicted that AQPs' selectivity is not exclusively shaped by pore-lining residues but also by the relative arrangement of transmembrane helices. Moreover, this analysis reveals that hydrophobic interactions constitute the main determinant of protein thermal stability. Finally, a numbering scheme of the conserved AQP scaffold is established, facilitating direct comparison of, for example, disease-causing mutations and prediction of potential structural consequences. Additionally, the results pave the way for the design of optimized AQP water channels to be utilized in biotechnological applications.
Assuntos
Aquaporinas , Aquaporinas/química , Aquaporinas/genética , Aquaporinas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , ÁguaRESUMO
BACKGROUND: Lipid-protein interactions stabilize protein oligomers, shape their structure, and modulate their function. Whereas in vitro experiments already account for the functional importance of lipids by using natural lipid extracts, in silico methods lack behind by embedding proteins in single component lipid bilayers. However, to accurately complement in vitro experiments with molecular details at very high spatio-temporal resolution, molecular dynamics simulations have to be performed in natural(-like) lipid environments. RESULTS: To enable more accurate MD simulations, we have prepared four membrane models of E. coli polar lipid extract, a typical model organism, each at all-atom (CHARMM36) and coarse-grained (Martini3) representations. These models contain all main lipid headgroup types of the E. coli inner membrane, i.e., phosphatidylethanolamines, phosphatidylglycerols, and cardiolipins, symmetrically distributed between the membrane leaflets. The lipid tail (un)saturation and propanylation stereochemistry represent the bacterial lipid tail composition of E. coli grown at 37∘C until 3/4 of the log growth phase. The comparison of the Simple three lipid component models to the complex 14-lipid component model Avanti over a broad range of physiologically relevant temperatures revealed that the balance of lipid tail unsaturation and propanylation in different positions and inclusion of lipid tails of various length maintain realistic values for lipid mobility, membrane area compressibility, lipid ordering, lipid volume and area, and the bilayer thickness. The only Simple model that was able to satisfactory reproduce most of the structural properties of the complex Avanti model showed worse agreement of the activation energy of basal water permeation with the here performed measurements. The Martini3 models reflect extremely well both experimental and atomistic behavior of the E. coli polar lipid extract membranes. Aquaporin-1 embedded in our native(-like) membranes causes partial lipid ordering and membrane thinning in its vicinity. Moreover, aquaporin-1 attracts and temporarily binds negatively charged lipids, mainly cardiolipins, with a distinct cardiolipin binding site in the crevice at the contact site between two monomers, most probably stabilizing the tetrameric protein assembly. CONCLUSIONS: The here prepared and validated membrane models of E. coli polar lipids extract revealed that lipid tail complexity, in terms of double bond and cyclopropane location and varying lipid tail length, is key to stabilize membrane properties over a broad temperature range. In addition, they build a solid basis for manifold future simulation studies on more realistic lipid membranes bridging the gap between simulations and experiments.
Assuntos
Cardiolipinas/química , Escherichia coli/química , Bicamadas Lipídicas/química , Fosfatidiletanolaminas/química , Fosfatidilgliceróis/química , Membrana Celular/química , Simulação de Dinâmica MolecularRESUMO
Chemokine receptors, a subclass of G protein coupled receptors (GPCRs), play essential roles in the human immune system, they are involved in cancer metastasis as well as in HIV-infection. A plethora of studies show that homo- and heterodimers or even higher order oligomers of the chemokine receptors CXCR4, CCR5, and CCR2 modulate receptor function. In addition, membrane cholesterol affects chemokine receptor activity. However, structural information about homo- and heterodimers formed by chemokine receptors and their interplay with cholesterol is limited. Here, we report homo- and heterodimer configurations of the chemokine receptors CXCR4, CCR5, and CCR2 at atomistic detail, as obtained from thousands of molecular dynamics simulations. The observed homodimerization patterns were similar for the closely related CC chemokine receptors, yet they differed significantly between the CC receptors and CXCR4. Despite their high sequence identity, cholesterol modulated the CC homodimer interfaces in a subtype-specific manner. Chemokine receptor heterodimers display distinct dimerization patterns for CXCR4/CCR5 and CXCR4/CCR2. Furthermore, associations between CXCR4 and CCR5 reveal an increased cholesterol-sensitivity as compared to CXCR4/CCR2 heterodimerization patterns. This work provides a first comprehensive structural overview over the complex interaction network between chemokine receptors and indicates how heterodimerization and the interaction with the membrane environment diversifies the function of closely related GPCRs.
Assuntos
Receptores de Quimiocinas/química , Receptores de Quimiocinas/genética , Receptores Acoplados a Proteínas G/genética , Animais , Quimiocinas/metabolismo , Colesterol/metabolismo , Simulação por Computador , Dimerização , Humanos , Simulação de Dinâmica Molecular , Receptores CCR2/química , Receptores CCR2/metabolismo , Receptores CCR2/ultraestrutura , Receptores CCR5/química , Receptores CCR5/metabolismo , Receptores CCR5/ultraestrutura , Receptores CXCR4/química , Receptores CXCR4/metabolismo , Receptores CXCR4/ultraestrutura , Transdução de SinaisRESUMO
The vesicular protein synaptobrevin II (sybII) constitutes a central component of the SNARE complex, which mediates vesicle fusion in neuronal exocytosis. Previous studies revealed that the transmembrane domain (TMD) of sybII is playing a critical role in the fusion process and is involved in all distinct fusion stages from priming to fusion pore opening. Here, we analyzed sequence-dependent effects of sybII and of mutants of sybII on both structure and flexibility of the protein and the interactions with a phospholipid bilayer by means of microsecond atomistic simulations. The sybII TMD was found to direct the folding of both the juxtamembrane helix and of the connecting linker and thus to influence both the intrinsic helicity and flexibility. Fusion active peptides revealed two helical segments, one for the juxtamembrane region and one for the TMD, connected by a flexible linker. In contrast, a fusion-inactive poly-leucine TMD mutant assumes a structure with a comparably rigid linker that is suggested to hinder the formation of the trans-SNARE complex during fusion. Kinking of the TMD at the central glycine together with anchoring of the TMD via conserved tryptophans and a lysine in position 94 likely yields an enhanced flexibility of sybII for different membrane thickness. All studied peptides were found to deform the outer membrane layer by altering the lipid head group orientation, causing partial membrane dehydration and enhancing lipid protrusions. These effects weaken the integrity of the outer membrane layer and are attributed mainly to the highly charged linker and JM regions of sybII.
Assuntos
Membrana Celular/química , Bicamadas Lipídicas/química , Proteínas SNARE/química , Proteína 2 Associada à Membrana da Vesícula/química , Motivos de Aminoácidos/genética , Animais , Membrana Celular/metabolismo , Exocitose , Glicina/química , Bicamadas Lipídicas/metabolismo , Fusão de Membrana/genética , Proteínas de Membrana/química , Simulação de Dinâmica Molecular , Estrutura Terciária de Proteína , Ratos , Proteínas SNARE/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
G protein coupled receptors (GPCRs) allow for the transmission of signals across biological membranes. For a number of GPCRs, this signaling was shown to be coupled to prior dimerization of the receptor. The chemokine receptor type 4 (CXCR4) was reported before to form dimers and their functionality was shown to depend on membrane cholesterol. Here, we address the dimerization pattern of CXCR4 in pure phospholipid bilayers and in cholesterol-rich membranes. Using ensembles of molecular dynamics simulations, we show that CXCR4 dimerizes promiscuously in phospholipid membranes. Addition of cholesterol dramatically affects the dimerization pattern: cholesterol binding largely abolishes the preferred dimer motif observed for pure phospholipid bilayers formed mainly by transmembrane helices 1 and 7 (TM1/TM5-7) at the dimer interface. In turn, the symmetric TM3,4/TM3,4 interface is enabled first by intercalating cholesterol molecules. These data provide a molecular basis for the modulation of GPCR activity by its lipid environment.
Assuntos
Colesterol/química , Bicamadas Lipídicas/química , Modelos Químicos , Multimerização Proteica , Receptores CXCR4/química , Receptores CXCR4/ultraestrutura , Sítios de Ligação , Simulação por Computador , Cinética , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/ultraestrutura , Relação Estrutura-AtividadeRESUMO
SNARE complexes have been shown to act cooperatively to enable the synaptic vesicle fusion in neuronal transmission at millisecond timescale. It has previously been suggested that the oligomerization of SNARE complexes required for cooperative action in fusion is mediated by interactions between transmembrane domains (TMDs). We study the oligomerization of synaptobrevin TMD using ensembles of molecular dynamics (MD) simulations at coarse-grained resolution for both the wild-type (WT) and selected mutants. Trimerization and tetramerization of the sybII WT and mutants displayed distinct kinetics depending both on the rate of dimerization and the availability of alternative binding interfaces. Interestingly, the tetramerization kinetics and propensity for the sybII W89A-W90A mutant was significantly increased as compared with the WT; the tryptophans in WT sybII impose sterical restraints on oligomer packing, thereby maintaining an appropriate plasticity and accessibility of sybII to the binding of its cognate SNARE partners during membrane fusion. Higher-order oligomeric models (ranging from pentamer to octamer), built by incremental addition of peptides to smaller oligomers, revealed substantial stability and high compactness. These larger sybII oligomers may induce membrane deformation, thereby possibly facilitating fast fusion exocytosis.
Assuntos
Membrana Celular/química , Multimerização Proteica , Proteínas R-SNARE/química , Sequência de Aminoácidos , Cinética , Modelos Moleculares , Mutação , Conformação Proteica em alfa-Hélice , Estrutura Quaternária de Proteína , Proteínas R-SNARE/genéticaRESUMO
A system based on two designed peptides, namely the cationic peptide K, (KIAALKE)3, and its complementary anionic counterpart called peptide E, (EIAALEK)3, has been used as a minimal model for membrane fusion, inspired by SNARE proteins. Although the fact that docking of separate vesicle populations via the formation of a dimeric E/K coiled-coil complex can be rationalized, the reasons for the peptides promoting fusion of vesicles cannot be fully explained. Therefore it is of significant interest to determine how the peptides aid in overcoming energetic barriers during lipid rearrangements leading to fusion. In this study, investigations of the peptides' interactions with neutral PC/PE/cholesterol membranes by fluorescence spectroscopy show that tryptophan-labeled K∗ binds to the membrane (KK∗ â¼6.2 103 M-1), whereas E∗ remains fully water-solvated. 15N-NMR spectroscopy, depth-dependent fluorescence quenching, CD-spectroscopy experiments, and MD simulations indicate a helix orientation of K∗ parallel to the membrane surface. Solid-state 31P-NMR of oriented lipid membranes was used to study the impact of peptide incorporation on lipid headgroup alignment. The membrane-immersed K∗ is found to locally alter the bilayer curvature, accompanied by a change of headgroup orientation relative to the membrane normal and of the lipid composition in the vicinity of the bound peptide. The NMR results were supported by molecular dynamics simulations, which showed that K reorganizes the membrane composition in its vicinity, induces positive membrane curvature, and enhances the lipid tail protrusion probability. These effects are known to be fusion relevant. The combined results support the hypothesis for a twofold role of K in the mechanism of membrane fusion: 1) to bring opposing membranes into close proximity via coiled-coil formation and 2) to destabilize both membranes thereby promoting fusion.
Assuntos
Bicamadas Lipídicas/metabolismo , Fusão de Membrana , Peptídeos/química , Peptídeos/metabolismo , Sequência de Aminoácidos , Membrana Celular/química , Membrana Celular/metabolismo , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação ProteicaRESUMO
Synaptic vesicle fusion requires assembly of the SNARE complex composed of SNAP-25, syntaxin-1, and synaptobrevin-2 (sybII) proteins. The SNARE proteins found in vesicle membranes have previously been shown to dimerize via transmembrane (TM) domain interactions. While syntaxin homodimerization is supposed to promote the transition from hemifusion to complete fusion, the role of synaptobrevin's TM domain association in the fusion process remains poorly understood. Here, we combined coarse-grained and atomistic simulations to model the homodimerization of the sybII transmembrane domain and of selected TM mutants. The wild-type helix is shown to form a stable, right-handed dimer with the most populated helix-helix interface, including key residues predicted in a previous mutagenesis study. In addition, two alternative binding interfaces were discovered, which are essential to explain the experimentally observed higher-order oligomerization of sybII. In contrast, only one dimerization interface was found for a fusion-inactive poly-Leu mutant. Moreover, the association kinetics found for this mutant is lower as compared to the wild-type. These differences in dimerization between the wild-type and the poly-Leu mutant are suggested to be responsible for the reported differences in fusogenic activity between these peptides. This study provides molecular insight into the role of TM sequence specificity for peptide aggregation in membranes.
Assuntos
Proteínas R-SNARE/metabolismo , Sequência de Aminoácidos , Dimerização , Bicamadas Lipídicas/metabolismo , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutação , Estrutura Secundária de Proteína , Proteínas R-SNARE/genéticaRESUMO
Facilitated water permeation through narrow biological channels is fundamental for all forms of life. Despite its significance in health and disease as well as for biotechnological applications, the energetics of water permeation are still elusive. Gibbs free energy of activation is composed of an enthalpic and an entropic component. Whereas the enthalpic contribution is readily accessible via temperature dependent water permeability measurements, estimation of the entropic contribution requires information on the temperature dependence of the rate of water permeation. Here, we estimate, by means of accurate activation energy measurements of water permeation through Aquaporin-1 and by determining the accurate single channel permeability, the entropic barrier of water permeation through a narrow biological channel. Thereby the calculated value for [Formula: see text] = 2.01 ± 0.82 J/(mol·K) links the activation energy of 3.75 ± 0.16 kcal/mol with its efficient water conduction rate of ~1010 water molecules/second. This is a first step in understanding the energetic contributions in various biological and artificial channels exhibiting vastly different pore geometries.
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Gasdermins execute programmatory cell death, known as pyroptosis, by forming medium-sized membrane pores. Recently, the molecular structure of those pores as well as the diversity in their shape and size have been revealed by cryoTEM and atomic force microscopy, respectively. Even though a growth of smaller to larger oligomers and reshaping from slits to rings could be documented, the initiation of the gasdermin pore formation remains a mystery. In one hypothesis, gasdermin monomers insert into membranes before associating into oligomeric pores. In the other hypothesis, gasdermin oligomers preassemble on the membrane surface prior to membrane insertion. Here, by studying the behavior of monomeric membrane-inserted gasdermin-A3 (GSDMA3), we unveil that a monomeric gasdermin prefers the membrane-adsorbed over the membrane-inserted state. Our results thus support the hypothesis of oligomers preassembling on the membrane surface before membrane penetration. At the same time, our simulations of small membrane-inserted arcs of GSDMA3 suggest that the inserting oligomer can be small and does not have to comprise a full ring of approximately 26-30 subunits. Moreover, our simulations have revealed an astonishingly large impact of salt-bridge formation and protein surroundings on the transmembrane passage of charged residues, reducing the energetic cost by up to 53% as compared to their free forms. The here observed free energy barrier of mere 5.6 kcal/mol for the membrane insertion of monomeric GSDMA3 explains the surprising ability of gasdermins to spontaneously self-insert into cellular membranes.
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The water permeability of aquaporins (AQPs) varies by more than an order of magnitude even though the pore structure, geometry, as well as the channel lining residues are highly conserved. However, channel gating by pH, divalent ions or phosphorylation was only shown for a minority of AQPs. Structural and in silico indications of water flux modulation by flexible side chains of channel lining residues have not been experimentally confirmed yet. Hence, the aquaporin "open state" is still considered to be a continuously open pore with water molecules permeating in a single-file fashion. Using protein mutations outside the selectivity filter in the aqua(glycerol)facilitator GlpF of Escherichia coli we, to the best of our knowledge, for the first time, modulate the position of the highly conserved Arg in the selectivity filter. This in turn enhances or reduces the unitary water permeability of GlpF as shown in silico by molecular dynamics (MD) simulations and in vitro with purified and reconstituted GlpF. This finding suggests that AQP water permeability can indeed be regulated by lipid bilayer asymmetry and the transmembrane potential. Strikingly, our long-term MD simulations reveal that not only the conserved Arg in the selectivity filter, but the position and dynamics of multiple other pore lining residues modulate water passage through GlpF. This finding is expected to trigger a wealth of future investigations on permeability and regulation of AQPs among others with the aim to tune water permeability for biotechnological applications.
Assuntos
Aquaporinas , Proteínas de Escherichia coli , Aquaporinas/química , Aquaporinas/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Glicerol/metabolismo , Bicamadas Lipídicas/química , Permeabilidade , Água/químicaRESUMO
Gasdermins are main effectors of pyroptosis, an inflammatory form of cell death. Released by proteolysis, the N-terminal gasdermin domain assembles large oligomers to punch lytic pores into the cell membrane. While the endpoint of this reaction, the fully formed pore, has been well characterized, the assembly and pore-forming mechanisms remain largely unknown. To resolve these mechanisms, we characterize mouse gasdermin-A3 by high-resolution time-lapse atomic force microscopy. We find that gasdermin-A3 oligomers assemble on the membrane surface where they remain attached and mobile. Once inserted into the membrane gasdermin-A3 grows variable oligomeric stoichiometries and shapes, each able to open transmembrane pores. Molecular dynamics simulations resolve how the membrane-inserted amphiphilic ß-hairpins and the structurally adapting hydrophilic head domains stabilize variable oligomeric conformations and open the pore. The results show that without a vertical collapse gasdermin pore formation propagates along a set of multiple parallel but connected reaction pathways to ensure a robust cellular response.
Assuntos
Piroptose , Animais , Morte Celular , Membrana Celular/metabolismo , Camundongos , Microscopia de Força Atômica , Proteínas de Ligação a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros , Piroptose/fisiologiaRESUMO
In cell membranes, G protein-coupled receptors (GPCRs) interact with cholesterol, which modulates their assembly, stability, and conformation. Previous studies have shown how cholesterol modulates the structural properties of GPCRs at ambient temperature. Here, we characterized the mechanical, kinetic, and energetic properties of the human ß2-adrenergic receptor (ß2AR) in the presence and absence of the cholesterol analog cholesteryl hemisuccinate (CHS) at room temperature (25°C), at physiological temperature (37°C), and at high temperature (42°C). We found that CHS stabilized various structural regions of ß2AR differentially, which changed nonlinearly with temperature. Thereby, the strongest effects were observed for structural regions that are important for receptor signaling. Moreover, at 37°C, but not at 25° or 42°C, CHS caused ß2AR to increase and stabilize conformational substates to adopt to basal activity. These findings indicate that the nonlinear, temperature-dependent action of CHS in modulating the structural and functional properties of this GPCR is optimized for 37°C.
Assuntos
Colesterol , Colesterol/metabolismo , Humanos , Cinética , Modelos Moleculares , TemperaturaRESUMO
G protein-coupled receptors (GPCRs) are the largest class of human membrane proteins that bind extracellular ligands at their orthosteric binding pocket to transmit signals to the cell interior. Ligand binding evokes conformational changes in GPCRs that trigger the binding of intracellular interaction partners (G proteins, G protein kinases, and arrestins), which initiate diverse cellular responses. It has become increasingly evident that the preference of a GPCR for a certain intracellular interaction partner is modulated by a diverse range of factors, e.g., ligands or lipids embedding the transmembrane receptor. Here, by means of molecular dynamics simulations of the ß2-adrenergic receptor and ß-arrestin2, we study how membrane lipids and receptor phosphorylation regulate GPCR-arrestin complex conformation and dynamics. We find that phosphorylation drives the receptor's intracellular loop 3 (ICL3) away from a native negatively charged membrane surface to interact with arrestin. If the receptor is embedded in a neutral membrane, the phosphorylated ICL3 attaches to the membrane surface, which widely opens the receptor core. This opening, which is similar to the opening in the G protein-bound state, weakens the binding of arrestin. The loss of binding specificity is manifested by shallower arrestin insertion into the receptor core and higher dynamics of the receptor-arrestin complex. Our results show that receptor phosphorylation and the local membrane composition cooperatively fine-tune GPCR-mediated signal transduction. Moreover, the results suggest that deeper understanding of complex GPCR regulation mechanisms is necessary to discover novel pathways of pharmacological intervention.
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Cells employ highly conserved families of insertases and translocases to insert and fold proteins into membranes. How insertases insert and fold membrane proteins is not fully known. To investigate how the bacterial insertase YidC facilitates this process, we here combine single-molecule force spectroscopy and fluorescence spectroscopy approaches, and molecular dynamics simulations. We observe that within 2 ms, the cytoplasmic α-helical hairpin of YidC binds the polypeptide of the membrane protein Pf3 at high conformational variability and kinetic stability. Within 52 ms, YidC strengthens its binding to the substrate and uses the cytoplasmic α-helical hairpin domain and hydrophilic groove to transfer Pf3 to the membrane-inserted, folded state. In this inserted state, Pf3 exposes low conformational variability such as typical for transmembrane α-helical proteins. The presence of YidC homologues in all domains of life gives our mechanistic insight into insertase-mediated membrane protein binding and insertion general relevance for membrane protein biogenesis.
Assuntos
Algoritmos , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Simulação de Dinâmica Molecular , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Cinética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Microscopia de Força Atômica , Microscopia Confocal , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Espectrometria de FluorescênciaRESUMO
Double resonance spectroscopy of clusters of guanine with aspartic acid reveals geometries similar to patterns exhibited in DNA base pairs. In the spectral region of 32,800 cm(-1) to 35,500 cm(-1) we observe five isomers of guanine-aspartic acid clusters and assign their structures based on IR-UV hole-burning spectra and wave function theory calculations at the MP2/cc-pVDZ and MP2/cc-pVTZ levels. The calculations employed both harmonic and one-dimensional scan anharmonic approximations. Three of the isomers are similar, assigned to structures containing three hydrogen bonds and 9-enolguanine. We assign the fourth isomer to a structure containing a 9-keto tautomer of guanine and forming a triply bonded structure similar to a base pairing interaction. The fifth isomer dissociates with proton transfer upon excitation or ionization. This is the first set of experiments and high-level ab initio calculations of the isolated, microscopic interactions of an amino acid and a nucleobase, the building blocks of nucleic acids and proteins.
Assuntos
Ácido Aspártico/química , Guanina/química , Espectrofotometria Infravermelho/métodos , Espectrofotometria Ultravioleta/métodos , Ligação de Hidrogênio , Modelos Moleculares , Sondas MolecularesRESUMO
Lipids and proteins, as essential components of biological cell membranes, exhibit a significant degree of freedom for different kinds of motions including lateral long-range mobility. Due to their interactions, they not only preserve the cellular membrane but also contribute to many important cellular functions as e.g., signal transport or molecular exchange of the cell with its surrounding. Many of these processes take place on a short time (up to some nanoseconds) and length scale (up to some nanometers) which is perfectly accessible by quasielastic neutron scattering (QENS) experiments and molecular dynamics (MD) simulations. In order to probe the influence of a peptide, a transmembrane sequence of the transferrin receptor (TFRC) protein, on the dynamics of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) large unilamellar vesicles (LUVs) on a nanosecond time scale, high-resolution QENS experiments and complementary MD simulations have been utilized. By using different scattering contrasts in the experiment (chain-deuterated lipids and protonated lipids, respectively), a model could be developed which allows to examine the lipid and peptide dynamics separately. The experimental results revealed a restricted lipid lateral mobility in the presence of the TFRC transmembrane peptides. Also the apparent self-diffusion coefficient of the lateral movement of the peptide molecules could be determined quantitatively for the probed short-time regime. The findings could be confirmed very precisely by MD simulations. Furthermore, the article presents an estimation for the radius of influence of the peptides on the lipid long-range dynamics which could be determined by consistently combining results from experiment and simulation.
RESUMO
The structure of dithienobicyclo[4.4.1]undeca-3,8-diene-11-one ethylene glycol ketal (database code RESVAN) was determined using the wave function theory (WFT) as well as density functional theory (DFT) methods combined with various Gaussian AO basis sets. The apparently most accurate procedure, employing the CCSD(T)/complete basis set (CBS), provides an S-S distance and an angle between the two thiophene rings which differ considerably from experimental values. The best agreement with the experimental data among all WFT methods was surprisingly obtained at the MP3/aug-cc-pVDZ and MP3/CBS(B) levels (the correction term to CBS was obtained by the aug-cc-pVDZ basis set). The very good results obtained by the CCSD(T)/6-31G* method are clearly a consequence of fortunate error compensation. MP2 calculations, even with a small basis set, overestimate the attraction between the thiophene rings, and the worst agreement with experimental data was found in full MP2/QZVP method optimizations (i.e., a strong distortion of the thiophene rings was observed). The SCS(MI)-MP2 and SCS-MP2 methods exhibit improvement over the MP2 procedure. All standard DFT approaches fail to predict reasonable S-S distances. The lack of intramolecular London dispersion energy results in too great distance between the thiophene rings. Much better agreement with experiment was obtained if advanced DFT methods, covering dispersion effects, were used. The best results were obtained at the TPSS-D/TZVP, M06-L/TZVP and B2PLYP-D/def2-TZVP levels. When a larger basis (LP in the case of TPSS functional) or more advanced versions of the new Truhlar functionals (M06-2X) was used, the agreement with experiment deteriorated. The accurate description of this molecule is highly functional/basis dependent and this dependence is hardly predictable. To estimate effects of neighboring molecules in the experimental crystal structure, an optimization in the electric field of the 26 closest RESVAN molecules was performed, which, however, leads to only moderate (<0.05 A) changes of the S-S distance.