RESUMEN
Hierarchical organization of integral membrane proteins (IMP) and lipids at the membrane is essential for regulating myriad downstream signaling. A quantitative understanding of these processes requires both detections of oligomeric organization of IMPs and lipids directly from intact membranes and determination of key membrane components and properties that regulate them. Addressing this, we have developed a platform that enables native mass spectrometry (nMS) analysis of IMP-lipid complexes directly from intact and customizable lipid membranes. Both the lipid composition and membrane properties (such as curvature, tension, and fluidity) of these bilayers can be precisely customized to a target membrane. Subsequent direct nMS analysis of these intact proteolipid vesicles can yield the oligomeric states of the embedded IMPs, identify bound lipids, and determine the membrane properties that can regulate the observed IMP-lipid organization. Applying this method, we show how lipid binding regulates neurotransmitter release and how membrane composition regulates the functional oligomeric state of a transporter.
Asunto(s)
Lípidos , Proteínas de la Membrana , Espectrometría de Masas/métodos , Transporte Biológico , Lípidos/química , Proteínas de la Membrana/química , Membrana Dobles de Lípidos/químicaRESUMEN
Computer simulation techniques form a versatile tool, a computational microscope, for exploring biological processes. This tool has been particularly effective in exploring different features of biological membranes. In recent years, thanks to elegant multiscale simulation schemes, some fundamental limitations of investigations by distinct simulation techniques have been resolved. As a result, we are now capable of exploring processes spanning multiple scales beyond the capacity of any single technique. In this perspective, we argue that mesoscale simulations require more attention and must be further developed to fill evident gaps in a quest toward simulating and modeling living cell membranes.
Asunto(s)
Simulación por Computador , Membrana CelularRESUMEN
Ion channels and G protein-coupled receptors (GPCRs) are regulated by lipids in their membrane environment. Structural studies combined with biophysical and molecular simulation investigations reveal interaction sites for specific lipids on membrane protein structures. For K channels, PIP2 plays a key role in regulating Kv and Kir channels. Likewise, several recent cryo-EM structures of TRP channels have revealed bound lipids, including PIP2 and cholesterol. Among the pentameric ligand-gated ion channel family, structural and biophysical studies suggest the M4 TM helix may act as a lipid sensor, e.g., forming part of the binding sites for neurosteroids on the GABAA receptor. Structures of GPCRs have revealed multiple cholesterol sites, which may modulate both receptor dynamics and receptor oligomerization. PIP2 also interacts with GPCRs and may modulate their interactions with G proteins. Overall, it is evident that multiple lipid binding sites exist on channels and receptors that modulate their function allosterically and are potential druggable sites.
Asunto(s)
Canales Iónicos/metabolismo , Lípidos/química , Receptores Acoplados a Proteínas G/metabolismo , Animales , Sitios de Unión , Colesterol/metabolismo , Simulación por Computador , Humanos , Canales Iónicos/química , Ligandos , Receptores Acoplados a Proteínas G/químicaRESUMEN
Protein-lipid interactions are a key element of the function of many integral membrane proteins. These potential interactions should be considered alongside the complexity and diversity of membrane lipid composition. Inward rectifier potassium channel (Kir) Kir2.2 has multiple interactions with plasma membrane lipids: Phosphatidylinositol (4, 5)-bisphosphate (PIP2) activates the channel; a secondary anionic lipid site has been identified, which augments the activation by PIP2; and cholesterol inhibits the channel. Molecular dynamics simulations are used to characterize in molecular detail the protein-lipid interactions of Kir2.2 in a model of the complex plasma membrane. Kir2.2 has been simulated with multiple, functionally important lipid species. From our simulations we show that PIP2 interacts most tightly at the crystallographic interaction sites, outcompeting other lipid species at this site. Phosphatidylserine (PS) interacts at the previously identified secondary anionic lipid interaction site, in a PIP2 concentration-dependent manner. There is interplay between these anionic lipids: PS interactions are diminished when PIP2 is not present in the membrane, underlining the need to consider multiple lipid species when investigating protein-lipid interactions.
Asunto(s)
Metabolismo de los Lípidos/genética , Lípidos/genética , Canales de Potasio de Rectificación Interna/genética , Animales , Aniones/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Simulación de Dinámica Molecular , Fosfatidilinositol 4,5-Difosfato/metabolismo , Potasio/metabolismo , Canales de Potasio de Rectificación Interna/metabolismoRESUMEN
Bacterial cell envelopes are compositionally complex and crowded and while highly dynamic in some areas, their molecular motion is very limited, to the point of being almost static in others. Therefore, it is no real surprise that studying them at high resolution across a range of temporal and spatial scales requires a number of different techniques. Details at atomistic to molecular scales for up to tens of microseconds are now within range for molecular dynamics simulations. Here we review how such simulations have contributed to our current understanding of the cell envelopes of Gram-negative bacteria.
Asunto(s)
Pared Celular , Bacterias Gramnegativas , Membrana Celular , Bacterias Gramnegativas/genética , Simulación de Dinámica MolecularRESUMEN
Gram-negative bacteria inhabit a broad range of ecological niches. For Escherichia coli, this includes river water as well as humans and animals, where it can be both a commensal and a pathogen. Intricate regulatory mechanisms ensure that bacteria have the right complement of ß-barrel outer membrane proteins (OMPs) to enable adaptation to a particular habitat. Yet no mechanism is known for replacing OMPs in the outer membrane, an issue that is further confounded by the lack of an energy source and the high stability and abundance of OMPs. Here we uncover the process underpinning OMP turnover in E. coli and show it to be passive and binary in nature, in which old OMPs are displaced to the poles of growing cells as new OMPs take their place. Using fluorescent colicins as OMP-specific probes, in combination with ensemble and single-molecule fluorescence microscopy in vivo and in vitro, as well as molecular dynamics simulations, we established the mechanism for binary OMP partitioning. OMPs clustered to form â¼0.5-µm diameter islands, where their diffusion is restricted by promiscuous interactions with other OMPs. OMP islands were distributed throughout the cell and contained the Bam complex, which catalyses the insertion of OMPs in the outer membrane. However, OMP biogenesis occurred as a gradient that was highest at mid-cell but largely absent at cell poles. The cumulative effect is to push old OMP islands towards the poles of growing cells, leading to a binary distribution when cells divide. Hence, the outer membrane of a Gram-negative bacterium is a spatially and temporally organized structure, and this organization lies at the heart of how OMPs are turned over in the membrane.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/citología , Escherichia coli/metabolismo , Polaridad Celular , Difusión , Escherichia coli/química , Escherichia coli/genética , Proteínas Ligadas a Lípidos/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Simulación de Dinámica Molecular , Complejos Multiproteicos/metabolismo , Unión Proteica , Transporte de ProteínasRESUMEN
Monolysocardiolipin (MLCL) is a three-tailed variant of cardiolipin (CL), the signature lipid of mitochondria. MLCL is not normally found in healthy tissue but accumulates in mitochondria of people with Barth syndrome (BTHS), with an overall increase in the MLCL:CL ratio. The reason for MLCL accumulation remains to be fully understood. The effect of MLCL build-up and decreased CL content in causing the characteristics of BTHS are also unclear. In both cases, an understanding of the nature of MLCL interaction with mitochondrial proteins will be key. Recent work has shown that MLCL associates less tightly than CL with proteins in the mitochondrial inner membrane, suggesting that MLCL accumulation is a result of CL degradation, and that the lack of MLCL-protein interactions compromises the stability of the protein-dense mitochondrial inner membrane, leading to a decrease in optimal respiration. There is some data on MLCL-protein interactions for proteins involved in the respiratory chain and in apoptosis, but there remains much to be understood regarding the nature of MLCL-protein interactions. Recent developments in structural, analytical and computational approaches mean that these investigations are now possible. Such an understanding will be key to further insights into how MLCL accumulation impacts mitochondrial membranes. In turn, these insights will help to support the development of therapies for people with BTHS and give a broader understanding of other diseases involving defective CL content.
Asunto(s)
Apoptosis , Cardiolipinas/metabolismo , Transporte de Electrón , Lisofosfolípidos/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Animales , Cardiolipinas/química , Microscopía por Crioelectrón , Dimerización , Humanos , Concentración de Iones de Hidrógeno , Lípidos/química , Mitocondrias/metabolismo , Simulación de Dinámica Molecular , Fosforilación Oxidativa , Unión ProteicaRESUMEN
Cardiolipin (CL) and its precursor phosphatidylglycerol (PG) are important anionic phospholipids widely distributed throughout all domains of life. They have key roles in several cellular processes by shaping membranes and modulating the activity of the proteins inserted into those membranes. They are synthesized by two main pathways, the so-called eukaryotic pathway, exclusively found in mitochondria, and the prokaryotic pathway, present in most bacteria and archaea. In the prokaryotic pathway, the first and the third reactions are catalyzed by phosphatidylglycerol phosphate synthase (Pgps) belonging to the transferase family and cardiolipin synthase (Cls) belonging to the hydrolase family, while in the eukaryotic pathway, those same reactions are catalyzed by unrelated homonymous enzymes: Pgps of the hydrolase family and Cls of the transferase family. Because of the enzymatic arrangement found in both pathways, it seems that the eukaryotic pathway evolved by convergence to the prokaryotic pathway. However, since mitochondria evolved from a bacterial endosymbiont, it would suggest that the eukaryotic pathway arose from the prokaryotic pathway. In this review, it is proposed that the eukaryote pathway evolved directly from a prokaryotic pathway by the neofunctionalization of the bacterial enzymes. Moreover, after the eukaryotic radiation, this pathway was reshaped by horizontal gene transfers or subsequent endosymbiotic processes.
Asunto(s)
Archaea/enzimología , Bacterias/enzimología , Cardiolipinas/biosíntesis , Eucariontes/enzimología , Fosfatidilgliceroles/metabolismo , Sitios de Unión , Vías Biosintéticas , Catálisis , Evolución Molecular , Transferencia de Gen Horizontal , Hidrolasas/metabolismo , Mitocondrias/metabolismo , Modelos Moleculares , Fosfolípidos/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , FilogeniaRESUMEN
The anionic lipid cardiolipin is an essential component of active ATP synthases. In metazoans, their rotors contain a ring of eight c-subunits consisting of inner and outer circles of N- and C-terminal α-helices, respectively. The beginning of the C-terminal α-helix contains a strictly conserved and fully trimethylated lysine residue in the lipid head-group region of the membrane. Larger rings of known structure, from c9-c15 in eubacteria and chloroplasts, conserve either a lysine or an arginine residue in the equivalent position. In computer simulations of hydrated membranes containing trimethylated or unmethylated bovine c8-rings and bacterial c10- or c11-rings, the head-groups of cardiolipin molecules became associated selectively with these modified and unmodified lysine residues and with adjacent polar amino acids and with a second conserved lysine on the opposite side of the membrane, whereas phosphatidyl lipids were attracted little to these sites. However, the residence times of cardiolipin molecules with the ring were brief and sufficient for the rotor to turn only a fraction of a degree in the active enzyme. With the demethylated c8-ring and with c10- and c11-rings, the density of bound cardiolipin molecules at this site increased, but residence times were not changed greatly. These highly specific but brief interactions with the rotating c-ring are consistent with functional roles for cardiolipin in stabilizing and lubricating the rotor, and, by interacting with the enzyme at the inlet and exit of the transmembrane proton channel, in participation in proton translocation through the membrane domain of the enzyme.
Asunto(s)
Cardiolipinas/metabolismo , Lisina/metabolismo , Simulación de Dinámica Molecular , ATPasas de Translocación de Protón/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Cardiolipinas/química , Bovinos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Lisina/química , Lisina/genética , Metilación , Unión Proteica , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Cardiolipin in eukaryotes is found in the mitochondrial inner membrane, where it interacts with membrane proteins and, although not essential, is necessary for the optimal activity of a number of proteins. One of them is the mitochondrial ADP/ATP carrier, which imports ADP into the mitochondrion and exports ATP. In the crystal structures, cardiolipin is bound to three equivalent sites of the ADP/ATP carrier, but its role is unresolved. Conservation of residues at these cardiolipin binding sites across other members of the mitochondrial carrier superfamily indicates cardiolipin binding is likely to be important for the function of all mitochondrial carriers. Multiscale simulations were performed in a cardiolipin-containing membrane to investigate the dynamics of cardiolipin around the yeast and bovine ADP/ATP carriers in a lipid bilayer and the properties of the cardiolipin-binding sites. In coarse-grain simulations, cardiolipin molecules bound to the carriers for longer periods of time than phosphatidylcholine and phosphatidylethanolamine lipids-with timescales in the tens of microseconds. Three long-lived cardiolipin binding sites overlapped with those in the crystal structures of the carriers. Other shorter-lived cardiolipin interaction sites were identified in both membrane leaflets. However, the timescales of the interactions were of the same order as phosphatidylcholine and phosphatidylethanolamine, suggesting that these sites are not specific for cardiolipin binding. The calculation of lipid binding times and the overlap of the cardiolipin binding sites between the structures and simulations demonstrate the potential of multiscale simulations to investigate the dynamics and behavior of lipids interacting with membrane proteins.
Asunto(s)
Cardiolipinas/metabolismo , Translocasas Mitocondriales de ADP y ATP/metabolismo , Dominios y Motivos de Interacción de Proteínas , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Cardiolipinas/química , Cardiolipinas/genética , Bovinos , Secuencia Conservada/genética , Mitocondrias/metabolismo , Translocasas Mitocondriales de ADP y ATP/genética , Membranas Mitocondriales/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica/genética , Dominios y Motivos de Interacción de Proteínas/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiaeRESUMEN
Alternative translation initiation (ATI) is a mechanism of producing multiple proteins from a single transcript, which in some cases regulates trafficking of proteins to different cellular compartments, including mitochondria. Application of a genome-wide computational screen predicts a cryptic mitochondrial targeting signal for 126 proteins in mouse and man that is revealed when an AUG codon located downstream from the canonical initiator methionine codon is used as a translation start site, which we term downstream ATI (dATI). Experimental evidence in support of dATI is provided by immunoblotting of endogenous truncated proteins enriched in mitochondrial cell fractions or of co-localization with mitochondria using immunocytochemistry. More detailed cellular localization studies establish mitochondrial targeting of a member of the cytosolic poly(A) binding protein family, PABPC5, and of the RNA/DNA helicase PIF1α. The mitochondrial isoform of PABPC5 co-immunoprecipitates with the mitochondrial poly(A) polymerase, and is markedly reduced in abundance when mitochondrial DNA and RNA are depleted, suggesting it plays a role in RNA metabolism in the organelle. Like PABPC5 and PIF1α, most of the candidates identified by the screen are not currently annotated as mitochondrial proteins, and so dATI expands the human mitochondrial proteome.
Asunto(s)
Codón Iniciador , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteoma/genética , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN Polimerasa gamma , ADN Mitocondrial/aislamiento & purificación , Proteínas de Unión al ADN/aislamiento & purificación , ADN Polimerasa Dirigida por ADN/aislamiento & purificación , Humanos , Ratones , Mitocondrias/enzimología , Proteínas Mitocondriales/análisis , Proteínas Mitocondriales/aislamiento & purificación , Proteínas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Mutación , Iniciación de la Cadena Peptídica Traduccional , Proteínas de Unión a Poli(A)/genética , Proteínas de Unión a Poli(A)/aislamiento & purificación , Proteínas de Unión a Poli(A)/metabolismo , Polinucleotido Adenililtransferasa/aislamiento & purificación , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , Proteoma/metabolismoRESUMEN
Spurred by advances in AI-driven modeling and experimental methods, molecular dynamics simulations are now acting as a platform to integrate these different approaches. This combination of methods is especially useful to understand ß-barrel proteins from the molecular level, e.g., identifying specific interactions with lipids or small molecules, up to assemblies comprised of hundreds of proteins and thousands of lipids. In this minireview, we will discuss recent advances, mainly from the last 5 years, in modeling ß-barrel proteins and their assemblies. These approaches require specific kinds of modeling and potentially different model resolutions that we will first describe in Subheading 1. We will then focus on different aspects of ß-barrel protein modeling: how different types of molecules can diffuse through ß-barrel proteins (Subheading 2); how lipids can interact with these proteins (Subheading 3); how ß-barrel proteins can interact with membrane partners (Subheading 4) or periplasmic extensions and partners (Subheading 5) to form large assemblies.
Asunto(s)
Proteínas de la Membrana , Simulación de Dinámica Molecular , Periplasma/metabolismo , Lípidos , Proteínas de la Membrana Bacteriana Externa/metabolismoRESUMEN
Cryo-electron microscopy (cryo-EM) enables the determination of membrane protein structures in native-like environments. Characterising how membrane proteins interact with the surrounding membrane lipid environment is assisted by resolution of lipid-like densities visible in cryo-EM maps. Nevertheless, establishing the molecular identity of putative lipid and/or detergent densities remains challenging. Here we present LipIDens, a pipeline for molecular dynamics (MD) simulation-assisted interpretation of lipid and lipid-like densities in cryo-EM structures. The pipeline integrates the implementation and analysis of multi-scale MD simulations for identification, ranking and refinement of lipid binding poses which superpose onto cryo-EM map densities. Thus, LipIDens enables direct integration of experimental and computational structural approaches to facilitate the interpretation of lipid-like cryo-EM densities and to reveal the molecular identities of protein-lipid interactions within a bilayer environment. We demonstrate this by application of our open-source LipIDens code to ten diverse membrane protein structures which exhibit lipid-like densities.
Asunto(s)
Proteínas de la Membrana , Simulación de Dinámica Molecular , Proteínas de la Membrana/química , Microscopía por Crioelectrón , Lípidos de la Membrana , Conformación ProteicaRESUMEN
The mitochondrial electron transport chain comprises a series of protein complexes embedded in the inner mitochondrial membrane that generate a proton motive force via oxidative phosphorylation, ultimately generating ATP. These protein complexes can oligomerize to form larger structures called supercomplexes. Cardiolipin (CL), a conical lipid, unique within eukaryotes to the inner mitochondrial membrane, has proven essential in maintaining the stability and function of supercomplexes. Monolysocardiolipin (MLCL) is a CL variant that accumulates in people with Barth syndrome (BTHS). BTHS is caused by defects in CL biosynthesis and characterised by abnormal mitochondrial bioenergetics and destabilised supercomplexes. However, the mechanisms by which MLCL causes pathogenesis remain unclear. Here, multiscale molecular dynamics characterise the interactions of CL and MLCL with yeast and mammalian mitochondrial supercomplexes containing complex III (CIII) and complex IV (CIV). Coarse-grained simulations reveal that both CL and MLCL bind to sites at the interface between CIII and CIV of the supercomplex. Free energy perturbation calculations show that MLCL interaction is weaker than that of CL and suggest that interaction with CIV drives this difference. Atomistic contact analyses show that, although interaction with CIII is similar for CL and MLCL, CIV makes more contacts with CL than MLCL, demonstrating that CL is a more successful "glue" between the two complexes. Simulations of the human CIII2CIV supercomplex show that this interface site is maintained between species. Our study suggests that MLCL accumulation in people with BTHS disrupts supercomplex stability by formation of relatively weak interactions at the interface lipid binding site.
RESUMEN
Lipids play important modulatory and structural roles for membrane proteins. Molecular dynamics simulations are frequently used to provide insights into the nature of these protein-lipid interactions. Systematic comparative analysis requires tools that provide algorithms for objective assessment of such interactions. We introduce PyLipID, a Python package for the identification and characterization of specific lipid interactions and binding sites on membrane proteins from molecular dynamics simulations. PyLipID uses a community analysis approach for binding site detection, calculating lipid residence times for both the individual protein residues and the detected binding sites. To assist structural analysis, PyLipID produces representative bound lipid poses from simulation data, using a density-based scoring function. To estimate residue contacts robustly, PyLipID uses a dual-cutoff scheme to differentiate between lipid conformational rearrangements while bound from full dissociation events. In addition to the characterization of protein-lipid interactions, PyLipID is applicable to analysis of the interactions of membrane proteins with other ligands. By combining automated analysis, efficient algorithms, and open-source distribution, PyLipID facilitates the systematic analysis of lipid interactions from large simulation data sets of multiple species of membrane proteins.
Asunto(s)
Proteínas de la Membrana , Simulación de Dinámica Molecular , Sitios de Unión , Ligandos , Membrana Dobles de Lípidos/química , Lípidos , Proteínas de la Membrana/químicaRESUMEN
Fibronectin Leucine-rich Repeat Transmembrane (FLRT 1-3) proteins are a family of broadly expressed single-spanning transmembrane receptors that play key roles in development. Their extracellular domains mediate homotypic cell-cell adhesion and heterotypic protein interactions with other receptors to regulate cell adhesion and guidance. These in trans FLRT interactions determine the formation of signaling complexes of varying complexity and function. Whether FLRTs also interact at the surface of the same cell, in cis, remains unknown. Here, molecular dynamics simulations reveal two dimerization motifs in the FLRT2 transmembrane helix. Single particle tracking experiments show that these Small-X3-Small motifs synergize with a third dimerization motif encoded in the extracellular domain to permit the cis association and co-diffusion patterns of FLRT2 receptors on cells. These results may point to a competitive switching mechanism between in cis and in trans interactions, which suggests that homotypic FLRT interaction mirrors the functionalities of classic adhesion molecules.
Asunto(s)
Moléculas de Adhesión Celular , Glicoproteínas de Membrana , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/metabolismo , Dimerización , Glicoproteínas de Membrana/química , Transducción de SeñalRESUMEN
Deregulation of protein kinases is associated with numerous diseases, making them important targets for drug discovery. The majority of drugs target the catalytic site of these proteins, but due to the high level of similarity within the ATP binding sites of protein kinases, it is often difficult to achieve the required pharmacological selectivity. In this study, we describe the identification and subsequent analysis of water patterns in the ATP binding sites of 171 protein kinase structures, comprising 19 different kinases from various branches of the kinome, and demonstrate that structurally similar binding sites often have significantly different water patterns. We show that the observed variations in water patterns of different, but structurally similar kinases can be exploited in the structure-based design of potent and selective kinase inhibitors.
Asunto(s)
Proteínas Quinasas/química , Agua/química , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Sitios de Unión , Análisis por Conglomerados , Bases de Datos de Proteínas , Humanos , Modelos Moleculares , Conformación Proteica , Proteínas Quinasas/metabolismo , Agua/metabolismoRESUMEN
GPCRs have been shown to form oligomers, which generate distinctive signaling outcomes. However, the structural nature of the oligomerization process remains uncertain. We have characterized oligomeric configurations of the adenosine A2a receptor (A2aR) by combining large-scale molecular dynamics simulations with Markov state models. These oligomeric structures may also serve as templates for studying oligomerization of other class A GPCRs. Our simulation data revealed that receptor activation results in enhanced oligomerization, more diverse oligomer populations, and a more connected oligomerization network. The active state conformation of the A2aR shifts protein-protein association interfaces to those involving intracellular loop ICL3 and transmembrane helix TM6. Binding of PIP2 to A2aR stabilizes protein-protein interactions via PIP2-mediated association interfaces. These results indicate that A2aR oligomerization is responsive to the local membrane lipid environment. This, in turn, suggests a modulatory effect on A2aR whereby a given oligomerization profile favors the dynamic formation of specific supramolecular signaling complexes.
Asunto(s)
Adenosina/metabolismo , Conformación Molecular , Receptor de Adenosina A2A/metabolismo , Sitios de Unión , Humanos , Simulación de Dinámica MolecularRESUMEN
Integral membrane proteins are localized and/or regulated by lipids present in the surrounding bilayer. While bacteria have relatively simple membranes, there is ample evidence that many bacterial proteins bind to specific lipids, especially the anionic lipid cardiolipin. Here, we apply molecular dynamics simulations to assess lipid binding to 42 different Escherichia coli inner membrane proteins. Our data reveal an asymmetry between the membrane leaflets, with increased anionic lipid binding to the inner leaflet regions of the proteins, particularly for cardiolipin. From our simulations, we identify >700 independent cardiolipin binding sites, allowing us to identify the molecular basis of a prototypical cardiolipin binding site, which we validate against structures of bacterial proteins bound to cardiolipin. This allows us to construct a set of metrics for defining a high-affinity cardiolipin binding site on bacterial membrane proteins, paving the way for a heuristic approach to defining other protein-lipid interactions.
Asunto(s)
Cardiolipinas , Escherichia coli , Proteínas Bacterianas/metabolismo , Cardiolipinas/química , Escherichia coli/metabolismo , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/metabolismo , Simulación de Dinámica MolecularRESUMEN
Voltage-gated sodium (Na v ) channels form the basis for the initiation of the action potential in excitable cells by allowing sodium ions to pass through the cell membrane. The Na v channel α subunit is known to function both with and without associated ß subunits. There is increasing evidence that these ß subunits have multiple roles that include not only influencing the voltage-dependent gating but also the ability to alter the spatial distribution of the pore-forming α subunit. Recent structural data has shown possible ways in which ß1 subunits may interact with the α subunit. However, the position of the ß1 subunit would not be compatible with a previous trimer structure of the ß3 subunit. Furthermore, little is currently known about the dynamic behavior of the ß subunits both as individual monomers and as higher order oligomers. Here, we use multiscale molecular dynamics simulations to assess the dynamics of the ß3, and the closely related, ß1 subunit. These findings reveal the spatio-temporal dynamics of ß subunits and should provide a useful framework for interpreting future low-resolution experiments such as atomic force microscopy.