RESUMEN
Understanding structural dynamics of biomolecules at the single-molecule level is vital to advancing our knowledge of molecular mechanisms. Currently, there are few techniques that can capture dynamics at the sub-nanometre scale and in physiologically relevant conditions. Atomic force microscopy (AFM)1 has the advantage of analysing unlabelled single molecules in physiological buffer and at ambient temperature and pressure, but its resolution limits the assessment of conformational details of biomolecules2. Here we present localization AFM (LAFM), a technique developed to overcome current resolution limitations. By applying localization image reconstruction algorithms3 to peak positions in high-speed AFM and conventional AFM data, we increase the resolution beyond the limits set by the tip radius, and resolve single amino acid residues on soft protein surfaces in native and dynamic conditions. LAFM enables the calculation of high-resolution maps from either images of many molecules or many images of a single molecule acquired over time, facilitating single-molecule structural analysis. LAFM is a post-acquisition image reconstruction method that can be applied to any biomolecular AFM dataset.
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Microscopía de Fuerza Atómica/métodos , Microscopía de Fuerza Atómica/normas , Algoritmos , Aminoácidos/química , Anexina A5/química , Anexina A5/ultraestructura , Acuaporinas/química , Acuaporinas/ultraestructura , Canales de Cloruro/química , Canales de Cloruro/ultraestructura , Conjuntos de Datos como Asunto , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestructura , Humanos , Concentración de Iones de Hidrógeno , Simulación de Dinámica MolecularRESUMEN
In response to acidic pH, the widely expressed proton-activated chloride (PAC) channel opens and conducts anions across cellular membranes. By doing so, PAC plays an important role in both cellular physiology (endosome acidification) and diseases associated with tissue acidosis (acid-induced cell death). Despite the available structural information, how proton binding in the extracellular domain (ECD) leads to PAC channel opening remains largely unknown. Here, through comprehensive mutagenesis and electrophysiological studies, we identified several critical titratable residues, including two histidine residues (H130 and H131) and an aspartic acid residue (D269) at the distal end of the ECD, together with the previously characterized H98 at the transmembrane domain-ECD interface, as potential pH sensors for human PAC. Mutations of these residues resulted in significant changes in pH sensitivity. Some combined mutants also exhibited large basal PAC channel activities at neutral pH. By combining molecular dynamics simulations with structural and functional analysis, we further found that the ß12 strand at the intersubunit interface and the associated "joint region" connecting the upper and lower ECDs allosterically regulate the proton-dependent PAC activation. Our studies suggest a distinct pH-sensing and gating mechanism of this new family of ion channels sensitive to acidic environment.
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Canales de Cloruro , Cloruros , Protones , Ácido Aspártico/química , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Cloruros/metabolismo , Fenómenos Electrofisiológicos , Histidina/química , Humanos , Concentración de Iones de Hidrógeno , MutagénesisRESUMEN
There is substantial evidence for extensive nonvesicular sterol transport in cells. For example, lipid transfer by the steroidogenic acute regulator-related proteins (StarD) containing a StarT domain has been shown to involve several pathways of nonvesicular trafficking. Among the soluble StarT domain-containing proteins, StarD4 is expressed in most tissues and has been shown to be an effective sterol transfer protein. However, it was unclear whether the lipid composition of donor or acceptor membranes played a role in modulating StarD4-mediated transport. Here, we used fluorescence-based assays to demonstrate a phosphatidylinositol phosphate (PIP)-selective mechanism by which StarD4 can preferentially extract sterol from liposome membranes containing certain PIPs (especially, PI(4,5)P2 and to a lesser degree PI(3,5)P2). Monophosphorylated PIPs and other anionic lipids had a smaller effect on sterol transport. This enhancement of transport was less effective when the same PIPs were present in the acceptor membranes. Furthermore, using molecular dynamics (MD) simulations, we mapped the key interaction sites of StarD4 with PIP-containing membranes and identified residues that are important for this interaction and for accelerated sterol transport activity. We show that StarD4 recognizes membrane-specific PIPs through specific interaction with the geometry of the PIP headgroup as well as the surrounding membrane environment. Finally, we also observed that StarD4 can deform membranes upon longer incubations. Taken together, these results suggest a mechanism by which PIPs modulate cholesterol transfer activity via StarD4.
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Proteínas de Transporte de Membrana , Esteroles , Transporte Biológico , Liposomas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Fosfatos de Fosfatidilinositol , Esteroles/metabolismoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
G-protein-coupled receptors (GPCRs) are involved in numerous physiological processes and are the most frequent targets of approved drugs. The explosion in the number of new three-dimensional (3D) molecular structures of GPCRs (3D-GPCRome) over the last decade has greatly advanced the mechanistic understanding and drug design opportunities for this protein family. Molecular dynamics (MD) simulations have become a widely established technique for exploring the conformational landscape of proteins at an atomic level. However, the analysis and visualization of MD simulations require efficient storage resources and specialized software. Here we present GPCRmd (http://gpcrmd.org/), an online platform that incorporates web-based visualization capabilities as well as a comprehensive and user-friendly analysis toolbox that allows scientists from different disciplines to visualize, analyze and share GPCR MD data. GPCRmd originates from a community-driven effort to create an open, interactive and standardized database of GPCR MD simulations.
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Simulación de Dinámica Molecular , Receptores Acoplados a Proteínas G/química , Programas Informáticos , Metaboloma , Modelos Moleculares , Conformación ProteicaRESUMEN
G-protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiology and disease intervention, yet the molecular mechanisms responsible for ligand-dependent signalling responses remain poorly understood. In class A GPCRs, receptor activation and G-protein coupling entail outward movements of transmembrane helix 6 (TM6). Here, using single-molecule fluorescence resonance energy transfer imaging, we examine TM6 movements in the ß2 adrenergic receptor (ß2AR) upon exposure to orthosteric ligands with different efficacies, in the absence and presence of the Gs heterotrimer. We show that partial and full agonists differentially affect TM6 motions to regulate the rate at which GDP-bound ß2AR-Gs complexes are formed and the efficiency of nucleotide exchange leading to Gs activation. These data also reveal transient nucleotide-bound ß2AR-Gs species that are distinct from known structures, and provide single-molecule perspectives on the allosteric link between ligand- and nucleotide-binding pockets that shed new light on the G-protein activation mechanism.
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Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Imagen Individual de Molécula , Agonistas de Receptores Adrenérgicos beta 2/química , Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Sitio Alostérico , Membrana Celular/metabolismo , Clenbuterol/química , Clenbuterol/metabolismo , Clenbuterol/farmacología , Activación Enzimática/efectos de los fármacos , Epinefrina/química , Epinefrina/metabolismo , Epinefrina/farmacología , Transferencia Resonante de Energía de Fluorescencia , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Guanosina Difosfato/metabolismo , Humanos , Cinética , Ligandos , Modelos Moleculares , Movimiento/efectos de los fármacos , Estabilidad Proteica , Receptores Adrenérgicos beta 2/químicaRESUMEN
In proteins where conformational changes are functionally important, the number of accessible states and their dynamics are often difficult to establish. Here we describe a novel 19F-NMR spectroscopy approach to probe dynamics of large membrane proteins. We labeled a glutamate transporter homolog with a 19F probe via cysteine chemistry and with a Ni2+ ion via chelation by a di-histidine motif. We used distance-dependent enhancement of the longitudinal relaxation of 19F nuclei by the paramagnetic metal to assign the observed resonances. We identified one inward- and two outward-facing states of the transporter, in which the substrate-binding site is near the extracellular and intracellular solutions, respectively. We then resolved the structure of the unanticipated second outward-facing state by cryo-EM. Finally, we showed that the rates of the conformational exchange are accessible from measurements of the metal-enhanced longitudinal relaxation of 19F nuclei.
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Sistema de Transporte de Aminoácidos X-AG/química , Espectroscopía de Resonancia Magnética , Sistema de Transporte de Aminoácidos X-AG/genética , Sistema de Transporte de Aminoácidos X-AG/metabolismo , Microscopía por Crioelectrón , Cisteína/química , Flúor , Histidina/química , Modelos Moleculares , Mutación , Níquel/química , Conformación Proteica , Dominios Proteicos , Pyrococcus horikoshii/químicaRESUMEN
Neurotransmitter:sodium symporters (NSSs) in the SLC6 family terminate neurotransmission by coupling the thermodynamically favorable transport of ions to the thermodynamically unfavorable transport of neurotransmitter back into presynaptic neurons. Results from many structural, functional, and computational studies on LeuT, a bacterial NSS homolog, have provided critical insight into the mechanism of sodium-coupled transport, but the mechanism underlying substrate-specific transport rates is still not understood. We present a combination of molecular dynamics simulations, single-molecule fluorescence resonance energy transfer (smFRET) imaging, and measurements of Na+ binding and substrate transport that reveals an allosteric substrate specificity mechanism. In this mechanism, residues F259 and I359 in the substrate binding pocket couple the binding of substrate to Na+ release from the Na2 site by allosterically modulating the stability of a partially open, inward-facing state. We propose a model for transport selectivity in which residues F259 and I359 act as a volumetric sensor that inhibits the transport of bulky amino acids.
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Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/metabolismo , Regulación Alostérica , Transporte Biológico , Glicina/metabolismo , Mutación/genética , Fenilalanina/metabolismo , Estabilidad Proteica , Rotación , Sodio/metabolismo , Especificidad por SustratoRESUMEN
Cell penetration after recognition of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus by the ACE2 receptor and the fusion of its viral envelope membrane with cellular membranes are the early steps of infectivity. A region of the Spike protein of the virus, identified as the "fusion peptide" (FP), is liberated at its N-terminal site by a specific cleavage occurring in concert with the interaction of the receptor-binding domain of the Spike. Studies have shown that penetration is enhanced by the required binding of Ca2+ ions to the FPs of coronaviruses, but the mechanisms of membrane insertion and destabilization remain unclear. We have predicted the preferred positions of Ca2+ binding to the SARS-CoV-2-FP, the role of Ca2+ ions in mediating peptide-membrane interactions, the preferred mode of insertion of the Ca2+-bound SARS-CoV-2-FP, and consequent effects on the lipid bilayer from extensive atomistic molecular dynamics simulations and trajectory analyses. In a systematic sampling of the interactions of the Ca2+-bound peptide models with lipid membranes, SARS-CoV-2-FP penetrated the bilayer and disrupted its organization only in two modes involving different structural domains. In one, the hydrophobic residues F833/I834 from the middle region of the peptide are inserted. In the other, more prevalent mode, the penetration involves residues L822/F823 from the LLF motif, which is conserved in CoV-2-like viruses, and is achieved by the binding of Ca2+ ions to the D830/D839 and E819/D820 residue pairs. FP penetration is shown to modify the molecular organization in specific areas of the bilayer, and the extent of membrane binding of the SARS-CoV-2 FP is significantly reduced in the absence of Ca2+ ions. These findings provide novel mechanistic insights regarding the role of Ca2+ in mediating SARS-CoV-2 fusion and provide a detailed structural platform to aid the ongoing efforts in rational design of compounds to inhibit SARS-CoV-2 cell entry.
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Calcio/metabolismo , Membrana Celular/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , SARS-CoV-2/metabolismo , Secuencia de Aminoácidos , Permeabilidad de la Membrana Celular , Lípidos de la Membrana/química , Simulación de Dinámica Molecular , Presión , Probabilidad , Estabilidad Proteica , Proteínas Recombinantes de Fusión/química , Agua/químicaRESUMEN
The metalloprotease ADAM17 (a disintegrin and metalloprotease 17) regulates EGF-receptor and TNFα signaling, thereby not only protecting the skin and intestinal barrier, but also contributing to autoimmunity. ADAM17 can be rapidly activated by many stimuli through its transmembrane domain (TMD), with the seven membrane-spanning inactive Rhomboids (iRhom) 1 and 2 implicated as candidate regulatory partners. However, several alternative models of ADAM17 regulation exist that do not involve the iRhoms, such as regulation through disulfide bond exchange or through interaction with charged phospholipids. Here, we report that a non-activatable mutant of ADAM17 with the TMD of betacellulin (BTC) can be rescued by restoring residues from the ADAM17 TMD, but only in Adam17-/- cells, which contain iRhoms, not in iRhom1/2-/- cells. We also provide the first evidence that the extracellular juxtamembrane domains (JMDs) of ADAM17 and iRhom2 regulate the stimulation and substrate selectivity of ADAM17. Interestingly, a point mutation in the ADAM17 JMD identified in a patient with Tetralogy of Fallot, a serious heart valve defect, affects the substrate selectivity of ADAM17 toward Heparin-binding epidermal growth factor like growth factor (HB-EGF), a crucial regulator of heart valve development in mice. These findings provide new insights into the regulation of ADAM17 through an essential interaction with the TMD1 and JMD1 of iRhom2.
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Proteína ADAM17/metabolismo , Proteínas Portadoras/metabolismo , Mutación , Proteína ADAM17/química , Proteína ADAM17/genética , Animales , Proteínas Portadoras/química , Proteínas Portadoras/genética , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos C57BL , Dominios Proteicos , Especificidad por Sustrato , Tetralogía de Fallot/genéticaRESUMEN
Glutamate transporters terminate neurotransmission by clearing synaptically released glutamate from the extracellular space, allowing repeated rounds of signalling and preventing glutamate-mediated excitotoxicity. Crystallographic studies of a glutamate transporter homologue from the archaeon Pyrococcus horikoshii, GltPh, showed that distinct transport domains translocate substrates into the cytoplasm by moving across the membrane within a central trimerization scaffold. Here we report direct observations of these 'elevator-like' transport domain motions in the context of reconstituted proteoliposomes and physiological ion gradients using single-molecule fluorescence resonance energy transfer (smFRET) imaging. We show that GltPh bearing two mutations introduced to impart characteristics of the human transporter exhibits markedly increased transport domain dynamics, which parallels an increased rate of substrate transport, thereby establishing a direct temporal relationship between transport domain motion and substrate uptake. Crystallographic and computational investigations corroborated these findings by revealing that the 'humanizing' mutations favour structurally 'unlocked' intermediate states in the transport cycle exhibiting increased solvent occupancy at the interface between the transport domain and the trimeric scaffold.
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Sistemas de Transporte de Aminoácidos Acídicos/química , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Ácido Aspártico/metabolismo , Pyrococcus horikoshii/química , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos Acídicos/genética , Transporte Biológico , Cristalografía por Rayos X , Detergentes , Transferencia Resonante de Energía de Fluorescencia , Humanos , Cinética , Ligandos , Modelos Moleculares , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Movimiento , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación/genética , Estabilidad Proteica , Estructura Terciaria de Proteína , Proteolípidos/metabolismo , Sodio/metabolismo , Solventes , TermodinámicaRESUMEN
Crystal structures of the neurotransmitter:sodium symporter MhsT revealed occluded inward-facing states with one substrate (Trp) bound in the primary substrate (S1) site and a collapsed extracellular vestibule, which in LeuT contains the second substrate (S2) site. In n-dodecyl-ß-d-maltoside, the detergent used to prepare MhsT for crystallization, the substrate-to-protein binding stoichiometry was determined by using scintillation proximity to be 1 Trp:MhsT. Here, using the same experimental approach, as well as equilibrium dialysis, we report that in n-decyl-ß-d-maltoside, or after reconstitution in lipid, MhsT, like LeuT, can simultaneously bind two Trp substrate molecules. Trp binding to the S2 site sterically blocks access to a substituted Cys at position 33 in the S2 site, as well as access to the deeper S1 site. Mutation of either the S1 or S2 site disrupts transport, consistent with previous studies in LeuT showing that substrate binding to the S2 site is an essential component of the transport mechanism.
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Proteínas Bacterianas/química , Lactococcus lactis/química , Simportadores/química , Cristalografía por Rayos X , Humanos , Dominios ProteicosRESUMEN
Central among the tools and approaches used for ligand discovery and design are Molecular Dynamics (MD) simulations, which follow the dynamic changes in molecular structure in response to the environmental condition, interactions with other proteins, and the effects of ligand binding. The need for, and successes of, MD simulations in providing this type of essential information are well documented, but so are the challenges presented by the size of the resulting datasets encoding the desired information. The difficulty of extracting information on mechanistically important state-to-state transitions in response to ligand binding and other interactions is compounded by these being rare events in the MD trajectories of complex molecular machines, such as G-protein-coupled receptors (GPCRs). To address this problem, we have developed a protocol for the efficient detection of such events. We show that the novel Rare Event Detection (RED) protocol reveals functionally relevant and pharmacologically discriminating responses to the binding of different ligands to the 5-HT2AR orthosteric site in terms of clearly defined, structurally coherent, and temporally ordered conformational transitions. This information from the RED protocol offers new insights into specific ligand-determined functional mechanisms encoded in the MD trajectories, which opens a new and rigorously reproducible path to understanding drug activity with application in drug discovery.
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Aprendizaje Automático , Receptores Acoplados a Proteínas G/química , Humanos , Ligandos , Simulación de Dinámica Molecular , Conformación ProteicaRESUMEN
Intracellular transport of chloride by members of the CLC transporter family involves a coupled exchange between a Cl- anion and a proton (H+), which makes the transport function dependent on ambient pH. Transport activity peaks at pH 4.5 and stalls at neutral pH. However, a structure of the WT protein at acidic pH is not available, making it difficult to assess the global conformational rearrangements that support a pH-dependent gating mechanism. To enable modeling of the CLC-ec1 dimer at acidic pH, we have applied molecular dynamics simulations (MD) featuring a new force field modification scheme-termed an Equilibrium constant pH approach (ECpH). The ECpH method utilizes linear interpolation between the force field parameters of protonated and deprotonated states of titratable residues to achieve a representation of pH-dependence in a narrow range of physiological pH values. Simulations of the CLC-ec1 dimer at neutral and acidic pH comparing ECpH-MD to canonical MD, in which the pH-dependent protonation is represented by a binary scheme, substantiates the better agreement of the conformational changes and the final model with experimental data from NMR, cross-link and AFM studies, and reveals structural elements that support the gate-opening at pH 4.5, including the key glutamates Gluin and Gluex.
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Antiportadores/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Concentración de Iones de Hidrógeno , Microscopía de Fuerza Atómica , Simulación de Dinámica Molecular , Conformación Proteica , ProtonesRESUMEN
Recent discoveries about functional mechanisms of proteins in the TMEM16 family of phospholipid scramblases have illuminated the dual role of the membrane as both the substrate and a mechanistically responsive environment in the wide range of physiological processes and genetic disorders in which they are implicated. This is highlighted in the review of recent findings from our collaborative investigations of molecular mechanisms of TMEM16 scramblases that emerged from iterative functional, structural, and computational experimentation. In the context of this review, we present new MD simulations and trajectory analyses motivated by the fact that new structural information about the TMEM16 scramblases is emerging from cryo-EM determinations in lipid nanodiscs. Because the functional environment of these proteins in in vivo and in in vitro is closer to flat membranes, we studied comparatively the responses of the membrane to the TMEM16 proteins in flat membranes and nanodiscs. We find that bilayer shapes in the nanodiscs are very different from those observed in the flat membrane systems, but the function-related slanting of the membrane observed at the nhTMEM16 boundary with the protein is similar in the nanodiscs and in the flat bilayers. This changes, however, in the bilayer composed of longer-tail lipids, which is thicker near the phospholipid translocation pathway, which may reflect an enhanced tendency of the long tails to penetrate the pathway and create, as shown previously, a nonconductive environment. These findings support the correspondence between the mechanistic involvement of the lipid environment in the flat membranes, and the nanodiscs. © 2019 Wiley Periodicals, Inc.
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Anoctaminas/química , Lípidos de la Membrana/química , Proteínas de Transferencia de Fosfolípidos/química , Anoctaminas/metabolismo , Lípidos de la Membrana/metabolismo , Simulación de Dinámica Molecular , Proteínas de Transferencia de Fosfolípidos/metabolismoRESUMEN
Because lipid bilayers can bend and stretch in ways similar to thin elastic sheets, physical models of bilayer deformation have utilized mechanical constants such as the moduli for bending rigidity (κC) and area compressibility (KA). However, the use of these models to quantify the energetics of membrane deformation associated with protein-membrane interactions, and the membrane response to stress is often hampered by the shortage of experimental data suitable for the estimation of the mechanical constants of various lipid mixtures. Although computational tools such as molecular dynamics simulations can provide alternative means to estimate KA values, current approaches suffer significant technical limitations. Here, we present a novel, to our knowledge, computational framework that allows for a direct estimation of KA values for individual bilayer leaflets. The theory is based on the concept of elasticity and derives KA from real-space analysis of local thickness fluctuations sampled in molecular dynamics simulations. We explore and validate the model on a large set of single and multicomponent bilayers of different lipid compositions and sizes, simulated at different temperatures. The calculated bilayer compressibility moduli agree with values estimated previously from experiments and those obtained from a standard computational method based on a series of constrained tension simulations. We further validate our framework in a comparison with an existing polymer brush model and confirm the polymer brush model's predicted linear relationship with proportionality coefficient of 24, using elastic parameters calculated from the simulation trajectories. The robustness of the results that emerge from the method allows us to revisit the origins of the bilayer mechanical (compressible) thickness and in particular its dependence on acyl-chain unsaturation and the presence of cholesterol.
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Membrana Celular/metabolismo , Fuerza Compresiva , Membrana Dobles de Lípidos/metabolismo , Simulación de Dinámica Molecular , Fenómenos Biomecánicos , Colesterol/metabolismoRESUMEN
Unlike most transmembrane proteins, phospholipids can migrate from one leaflet of the membrane to the other. Because this spontaneous lipid translocation (flip-flop) tends to be very slow, cells facilitate the process with enzymes that catalyze the transmembrane movement and thereby regulate the transbilayer lipid distribution. Nonenzymatic membrane-spanning proteins with unrelated primary functions have also been found to accelerate lipid flip-flop in a nonspecific manner and by various hypothesized mechanisms. Using deuterated phospholipids, we examined the acceleration of flip-flop by gramicidin channels, which have well-defined structures and known functions, features that make them ideal candidates for probing the protein-membrane interactions underlying lipid flip-flop. To study compositionally and isotopically asymmetric proteoliposomes containing gramicidin, we expanded a recently developed protocol for the preparation and characterization of lipid-only asymmetric vesicles. Channel incorporation, conformation, and function were examined with small angle x-ray scattering, circular dichroism, and a stopped-flow spectrofluorometric assay, respectively. As a measure of lipid scrambling, we used differential scanning calorimetry to monitor the effect of gramicidin on the melting transition temperatures of the two bilayer leaflets. The two calorimetric peaks of the individual leaflets merged into a single peak over time, suggestive of scrambling, and the effect of the channel on the transbilayer lipid distribution in both symmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and asymmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles was quantified from proton NMR measurements. Our results show that gramicidin increases lipid flip-flop in a complex, concentration-dependent manner. To determine the molecular mechanism of the process, we used molecular dynamics simulations and further computational analysis of the trajectories to estimate the extent of membrane deformation. Together, the experimental and computational approaches were found to constitute an effective means for studying the effects of transmembrane proteins on lipid distribution in both symmetric and asymmetric model membranes.
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Gramicidina/metabolismo , Liposomas/metabolismo , Fosfolípidos/metabolismo , Cinética , Liposomas/química , Simulación de Dinámica MolecularRESUMEN
A disintegrin and metalloproteinase 17 (ADAM17) controls the release of the pro-inflammatory cytokine tumor necrosis factor α (TNFα, also known as TNF) and is crucial for protecting the skin and intestinal barrier by proteolytic activation of epidermal growth factor receptor (EGFR) ligands. The seven-membrane-spanning protein called inactive rhomboid 2 (Rhbdf2; also known as iRhom2) is required for ADAM17-dependent TNFα shedding and crosstalk with the EGFR, and a point mutation (known as sinecure, sin) in the first transmembrane domain (TMD) of Rhbdf2 (Rhbdf2sin) blocks TNFα shedding, yet little is known about the underlying mechanism. Here, we used a structure-function analysis informed by structural modeling to evaluate the interaction between the TMD of ADAM17 and the first TMD of Rhbdf2, and the role of this interaction in Rhbdf2-ADAM17-dependent shedding. Moreover, we show that double mutant mice that are homozygous for Rhbdf2sin/sin and lack Rhbdf1 closely resemble Rhbdf1/2-/- double knockout mice, highlighting the severe functional impact of the Rhbdf2sin/sin mutation on ADAM17 during mouse development. Taken together, these findings provide new mechanistic and conceptual insights into the critical role of the TMDs of ADAM17 and Rhbdf2 in the regulation of the ADAM17 and EGFR, and ADAM17 and TNFα signaling pathways.
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Proteína ADAM17/química , Proteína ADAM17/metabolismo , Proteínas Portadoras/metabolismo , Modelos Moleculares , Proteolisis , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Animales , Células de la Médula Ósea/citología , Proteínas Portadoras/química , Proteínas Portadoras/genética , Membrana Celular/metabolismo , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Placa de Crecimiento/metabolismo , Válvulas Cardíacas/metabolismo , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Ratones Mutantes , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación/genética , Unión Proteica , Relación Estructura-ActividadRESUMEN
BACKGROUND: Much of the structure-based mechanistic understandings of the function of SLC6A neurotransmitter transporters emerged from the study of their bacterial LeuT-fold homologs. It has become evident, however, that structural differences such as the long N- and C-termini of the eukaryotic neurotransmitter transporters are involved in an expanded set of functional properties to the eukaryotic transporters. These functional properties are not shared by the bacterial homologs, which lack the structural elements that appeared later in evolution. However, mechanistic insights into some of the measured functional properties of the eukaryotic transporters that have been suggested to involve these structural elements are sparse or merely descriptive. RESULTS: To learn how the structural elements added in evolution enable mechanisms of the eukaryotic transporters in ways not shared with their bacterial LeuT-like homologs, we focused on the human dopamine transporter (hDAT) as a prototype. We present the results of a study employing large-scale molecular dynamics simulations and comparative Markov state model analysis of experimentally determined properties of the wild-type and mutant hDAT constructs. These offer a quantitative outline of mechanisms in which a rich spectrum of interactions of the hDAT N-terminus and C-terminus contribute to the regulation of transporter function (e.g., by phosphorylation) and/or to entirely new phenotypes (e.g., reverse uptake (efflux)) that were added in evolution. CONCLUSIONS: The findings are consistent with the proposal that the size of eukaryotic neurotransmitter transporter termini increased during evolution to enable more functions (e.g., efflux) not shared with the bacterial homologs. The mechanistic explanations for the experimental findings about the modulation of function in DAT, the serotonin transporter, and other eukaryotic transporters reveal separate roles for the distal and proximal segments of the much larger N-terminus in eukaryotic transporters compared to the bacterial ones. The involvement of the proximal and distal segments - such as the role of the proximal segment in sustaining transport in phosphatidylinositol 4,5-bisphosphate-depleted membranes and of the distal segment in modulating efflux - may represent an evolutionary adaptation required for the function of eukaryotic transporters expressed in various cell types of the same organism that differ in the lipid composition and protein complement of their membrane environment.
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Proteínas de Transporte de Neurotransmisores/química , Proteínas de Transporte de Neurotransmisores/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Evolución Molecular , Humanos , Simulación de Dinámica Molecular , Fosforilación , Conformación Proteica , Procesamiento Proteico-PostraduccionalRESUMEN
G protein-coupled receptors (GPCRs) play a key role in many cellular signaling mechanisms, and must select among multiple coupling possibilities in a ligand-specific manner in order to carry out a myriad of functions in diverse cellular contexts. Much has been learned about the molecular mechanisms of ligand-GPCR complexes from Molecular Dynamics (MD) simulations. However, to explore ligand-specific differences in the response of a GPCR to diverse ligands, as is required to understand ligand bias and functional selectivity, necessitates creating very large amounts of data from the needed large-scale simulations. This becomes a Big Data problem for the high dimensionality analysis of the accumulated trajectories. Here we describe a new machine learning (ML) approach to the problem that is based on transforming the analysis of GPCR function-related, ligand-specific differences encoded in the MD simulation trajectories into a representation recognizable by state-of-the-art deep learning object recognition technology. We illustrate this method by applying it to recognize the pharmacological classification of ligands bound to the 5-HT2A and D2 subtypes of class-A GPCRs from the serotonin and dopamine families. The ML-based approach is shown to perform the classification task with high accuracy, and we identify the molecular determinants of the classifications in the context of GPCR structure and function. This study builds a framework for the efficient computational analysis of MD Big Data collected for the purpose of understanding ligand-specific GPCR activity.