RESUMEN
For the discovery of novel chemical matter generally endowed with bioactivity, strategies may be particularly efficient that combine previous insight about biological relevance, e.g., natural product (NP) structure, with methods that enable efficient coverage of chemical space, such as fragment-based design. We describe the de novo combination of different 5-membered NP-derived N-heteroatom fragments to structurally unprecedented "pseudo-natural products" in an efficient complexity-generating and enantioselective one-pot synthesis sequence. The pseudo-NPs inherit characteristic elements of NP structure but occupy areas of chemical space not covered by NP-derived chemotypes, and may have novel biological targets. Investigation of the pseudo-NPs in unbiased phenotypic assays and target identification led to the discovery of the first small-molecule ligand of the RHO GDP-dissociation inhibitor 1 (RHOGDI1), termed Rhonin. Rhonin inhibits the binding of the RHOGDI1 chaperone to GDP-bound RHO GTPases and alters the subcellular localization of RHO GTPases.
Asunto(s)
Productos Biológicos , Productos Biológicos/química , Ligandos , Proteínas de Unión al GTP rho , Inhibidor alfa de Disociación del Nucleótido Guanina rho , Inhibidores de la Disociación del Nucleótido Guanina rho-EspecíficoRESUMEN
Although the Ras protein has been seen as a potential target for cancer therapy for the past 30 years, there was a tendency to consider it undruggable until recently. This has changed with the demonstration that small molecules with a specificity for (disease related mutants of) Ras can indeed be found, and some of these molecules form covalent adducts. A subgroup of these molecules can be characterized as competing with binding of the natural ligands GTP and GDP. Because of the distinct properties of Ras and related GTPases, in particular the very high nucleotide affinities and associated very low dissociation rates, assays for characterizing such molecules are not trivial. This is compounded by the fact that Ras family GTPases tend to be thermally unstable in the absence of a bound nucleotide. Here, we show that instead of using the unstable nucleotide-free Ras, the protein can be isolated as a 1:1 complex with a modified nucleotide (GDP-ß-methyl ester) with low affinity to Ras. With this nucleotide analogue bound to the protein, testing of inhibitors is made experimentally more convenient and we present assays that allow the rapid assessment of the kinetic constants describing the inhibition process.
Asunto(s)
Bioensayo/métodos , Nucleótidos/análisis , Proteínas ras/antagonistas & inhibidores , Animales , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Cinética , Nucleótidos/farmacología , Espectrometría de Fluorescencia , Proteínas ras/metabolismoRESUMEN
Our understanding of the manner in which Rab proteins regulate intracellular vesicular transport has progressed remarkably in the last one or two decades by application of a wide spectrum of biochemical, biophysical and cell biological methods, augmented by the methods of chemical biology. Important additional insights have arisen from examination of the manner in which certain bacteria can manipulate vesicular transport mechanisms. The progress in these areas is summarized here.
Asunto(s)
Vesículas Transportadoras/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Bacterias/citología , Bacterias/metabolismo , Humanos , Proteínas de Unión al GTP rab/químicaRESUMEN
Prenylated Rab proteins exist in the cytosol as soluble, high-affinity complexes with GDI that need to be disrupted for membrane attachment and targeting of Rab proteins. The Legionella pneumophila protein DrrA displaces GDI from Rab1:GDI complexes, incorporating Rab1 into Legionella-containing vacuoles and activating Rab1 by exchanging GDP for GTP. Here, we present the crystal structure of a complex between the GEF domain of DrrA and Rab1 and a detailed kinetic analysis of this exchange. DrrA efficiently catalyzes nucleotide exchange and mimics the general nucleotide exchange mechanism of mammalian GEFs for Ras-like GTPases. We show that the GEF activity of DrrA is sufficient to displace prenylated Rab1 from the Rab1:GDI complex. Thus, apparent GDI displacement by DrrA is linked directly to nucleotide exchange, suggesting a basic model for GDI displacement and specificity of Rab localization that does not require discrete GDI displacement activity.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Legionella pneumophila/metabolismo , Proteínas de Unión al GTP rab1/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Inhibidores de Disociación de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Unión Proteica , Conformación Proteica , Proteínas de Unión al GTP rab1/química , Proteínas de Unión al GTP rab1/genéticaRESUMEN
Intracellular membrane trafficking requires correct and specific localization of Rab GTPases. The hypervariable C-terminal domain (HVD) of Rabs is posttranslationally modified by isoprenyl moieties that enable membrane association. A model asserting HVD-directed targeting has been contested in previous studies, but the role of the Rab HVD and the mechanism of Rab membrane targeting remain elusive. To elucidate the function of the HVD, we have substituted this region with an unnatural polyethylenglycol (PEG) linker by using oxime ligation. The PEGylated Rab proteins undergo normal prenylation, underlining the unique ability of the Rab prenylation machinery to process the Rab family with diverse C-terminal sequences. Through localization studies and functional analyses of semisynthetic PEGylated Rab1, Rab5, Rab7, and Rab35 proteins, we demonstrate that the role of the HVD of Rabs in membrane targeting is more complex than previously understood. The HVD of Rab1 and Rab5 is dispensable for membrane targeting and appears to function simply as a linker between the GTPase domain and the membrane. The N-terminal residues of the Rab7 HVD are important for late endosomal/lysosomal localization, apparently due to their involvement in interaction with the Rab7 effector Rab-interacting lysosomal protein. The C-terminal polybasic cluster of the Rab35 HVD is essential for plasma membrane (PM) targeting, presumably because of the electrostatic interaction with negatively charged lipids on the PM. Our findings suggest that Rab membrane targeting is dictated by a complex mechanism involving GEFs, GAPs, effectors, and C-terminal interaction with membranes to varying extents, and possibly other binding partners.
Asunto(s)
Membrana Celular/metabolismo , Variación Genética , Modelos Biológicos , Transporte de Proteínas/fisiología , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Animales , Perros , Células HeLa , Humanos , Células de Riñón Canino Madin Darby , Estructura Terciaria de ProteínaRESUMEN
The characterization of low-affinity protein complexes is challenging due to their dynamic nature. Here, we present a method to stabilize transient protein complexes inâ vivo by generating a covalent and conformationally flexible bridge between the interaction partners. A highly active pyrrolysyl tRNA synthetase mutant directs the incorporation of unnatural amino acids bearing bromoalkyl moieties (BrCnK) into proteins. We demonstrate for the first time that low-affinity protein complexes between BrCnK-containing proteins and their binding partners can be stabilized inâ vivo in bacterial and mammalian cells. Using this approach, we determined the crystal structure of a transient GDP-bound complex between a small G-protein and its nucleotide exchange factor. We envision that this approach will prove valuable as a general tool for validating and characterizing protein-protein interactions inâ vitro and inâ vivo.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Reguladores de Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/genética , Reguladores de Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/química , Proteínas Fluorescentes Verdes/química , Células HEK293 , Humanos , Modelos Moleculares , Mutación , Unión Proteica , Estabilidad ProteicaRESUMEN
The Legionella pneumophila protein AnkX that is injected into infected cells by a Type IV secretion system transfers a phosphocholine group from CDP-choline to a serine in the Rab1 and Rab35 GTPase Switch II regions. We show here that the consequences of phosphocholination on the interaction of Rab1/Rab35 with various partner proteins are quite distinct. Activation of phosphocholinated Rabs by GTP/GDP exchange factors (GEFs) and binding to the GDP dissociation inhibitor (GDI) are strongly inhibited, whereas deactivation by GTPase activating proteins (GAPs) and interactions with Rab-effector proteins (such as LidA and MICAL-3) are only slightly inhibited. We show that the Legionella protein lpg0696 has the ability to remove the phosphocholine group from Rab1. We present a model in which the action of AnkX occurs as an alternative to GTP/GDP exchange, stabilizing phosphocholinated Rabs in membranes in the GDP form because of loss of GDI binding ability, preventing interactions with cellular GTPase effectors, which require the GTP-bound form. Generation of the GTP form of phosphocholinated Rab proteins cannot occur due to loss of interaction with cellular GEFs.
Asunto(s)
Proteínas Bacterianas/metabolismo , Legionella pneumophila/metabolismo , Fosforilcolina/metabolismo , Proteínas de Unión al GTP rab/metabolismo , GTP Fosfohidrolasas/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismoRESUMEN
Most GTPases and many ATPases belong to the P-loop class of proteins with significant structural and mechanistic similarities. Here we compare and contrast the basic properties of the Ras family GTPases and myosin, and conclude that there are fundamental similarities but also distinct differences related to their specific roles. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 422-430, 2016.
Asunto(s)
Metabolismo Energético/fisiología , Miosinas , Proteínas ras , Animales , Humanos , Miosinas/química , Miosinas/metabolismo , Estructura Secundaria de Proteína , Relación Estructura-Actividad , Proteínas ras/química , Proteínas ras/metabolismoRESUMEN
Membrane trafficking is regulated by small Ras-like GDP/GTP binding proteins of the Rab subfamily (Rab GTPases) that cycle between membranes and cytosol depending on their nucleotide state. The GDP dissociation inhibitor (GDI) solubilizes prenylated Rab GTPases from and shuttles them between membranes in the form of a soluble cytosolic complex. We use attenuated total reflection-Fourier transform infrared spectroscopy to directly observe extraction of Rab GTPases from model membranes by GDI. In their native form, most Rab GTPases are doubly geranylgeranylated at the C terminus to achieve localization to the membrane. We find that monogeranylgeranylated Rab35 and Rab1b reversibly bind to a negatively charged model membrane. Correct folding and GTPase activity of the membrane-bound protein can be evaluated. The dissociation kinetics depends on the C-terminal sequence and charge of the GTPases. The attenuated total reflection experiments show that GDI genuinely accelerates the intrinsic Rab membrane dissociation. The extraction process is characterized and occurs in a nucleotide-dependent manner. Furthermore, we find that phosphocholination of Rab35, which is catalyzed by the Legionella pneumophila protein AnkX, interferes with the ability of GDI to extract Rab35 from the membrane. The attenuated total reflection-Fourier transform infrared spectroscopy approach enables label-free investigation of the interaction between GDI and Rab GTPases in a membrane environment. Thereby, GDI is revealed to actively extract monogeranylgeranylated membrane-bound Rab GTPases and, thus, is not merely a solubilization factor.
Asunto(s)
Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Legionella pneumophila/metabolismo , Membranas/metabolismo , Espectrofotometría Infrarroja/métodos , Vesículas Transportadoras/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Bovinos , Cinética , Fosforilcolina/metabolismo , Prenilación , Saccharomyces cerevisiae , Espectroscopía Infrarroja por Transformada de Fourier/métodosRESUMEN
The stable post-translational modification of proteins by adenylylation or uridylylation was discovered more than four decades ago as a mechanism to regulate the activity of enzymes. Although many other processes involving the covalent transfer of an AMP residue to an amino acid side chain have been identified since then, these are transient adenylylation events that essentially use the free energy of ATP hydrolysis to activate specific processes. Recently, new examples of stable adenylylation of small GTPases involved in signal transduction and regulation of cellular events were discovered, which appear to modulate downstream processes such as cytoskeletal rearrangement and vesicular trafficking. We present a survey of the historical and modern phases of research in this area, focusing on the common and differing aspects of protein adenylylation.
Asunto(s)
Adenina/metabolismo , Procesamiento Proteico-Postraduccional , Adenina/química , Adenosina Monofosfato/metabolismo , Animales , Glutamato Sintasa/química , Glutamato Sintasa/metabolismo , Humanos , Proteínas/metabolismoRESUMEN
The oculocerebrorenal syndrome of Lowe (OCRL), also called Lowe syndrome, is characterized by defects of the nervous system, the eye and the kidney. Lowe syndrome is a monogenetic X-linked disease caused by mutations of the inositol-5-phosphatase OCRL1. OCRL1 is a membrane-bound protein recruited to membranes via interaction with a variety of Rab proteins. The structural and kinetic basis of OCRL1 for the recognition of several Rab proteins is unknown. In this study, we report the crystal structure of the Rab-binding domain (RBD) of OCRL1 in complex with Rab8a and the kinetic binding analysis of OCRL1 with several Rab GTPases (Rab1b, Rab5a, Rab6a and Rab8a). In contrast to other effectors that bind their respective Rab predominantly via α-helical structure elements, the Rab-binding interface of OCRL1 consists mainly of the IgG-like ß-strand structure of the ASPM-SPD-2-Hydin domain as well as one α-helix. Our results give a deeper structural understanding of disease-causing mutations of OCRL1 affecting Rab binding.
Asunto(s)
Mutación/genética , Síndrome Oculocerebrorrenal/genética , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/genética , Proteínas de Unión al GTP rab/metabolismo , Membrana Celular/metabolismo , Cristalización , Cristalografía por Rayos X , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoprecipitación , Síndrome Oculocerebrorrenal/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas de Unión al GTP rab/genéticaRESUMEN
We present a comprehensive toolkit for Förster resonance energy transfer (FRET)-restrained modeling of biomolecules and their complexes for quantitative applications in structural biology. A dramatic improvement in the precision of FRET-derived structures is achieved by explicitly considering spatial distributions of dye positions, which greatly reduces uncertainties due to flexible dye linkers. The precision and confidence levels of the models are calculated by rigorous error estimation. The accuracy of this approach is demonstrated by docking a DNA primer-template to HIV-1 reverse transcriptase. The derived model agrees with the known X-ray structure with an r.m.s. deviation of 0.5 Å. Furthermore, we introduce FRET-guided 'screening' of a large structural ensemble created by molecular dynamics simulations. We used this hybrid approach to determine the formerly unknown configuration of the flexible single-strand template overhang.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Transcriptasa Inversa del VIH/química , Cartilla de ADN/química , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica MolecularRESUMEN
Legionella pneumophila is an intracellularly surviving pathogen that releases about 270 different proteins into the host cell during infection. A set of secreted proteins takes control of the vesicular trafficking regulator Rab1. Legionella LepB inactivates Rab1 by acting as a GTPase-activating protein (GAP). We present the crystal structure of the Rab1b:LepB complex together with a thorough biochemical analysis and show that the GAP domain of LepB consists of an unusual fold. LepB acts by an atypical RabGAP mechanism that is reminiscent of classical GAPs and therefore sets the protein apart from mammalian TBC-like GAPs. Surprisingly, LepB can function as a GAP for Rab3, Rab8, Rab13 and Rab35, too, suggesting that it has a broader cellular role than previously thought.
Asunto(s)
Proteínas Bacterianas/química , Legionella pneumophila/enzimología , Proteínas de Unión al GTP rab1/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Secuencia Conservada , Cristalografía por Rayos X , Guanosina Trifosfato/química , Interacciones Huésped-Patógeno , Humanos , Hidrólisis , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas de Unión al GTP rab/químicaRESUMEN
Intracellular vesicular trafficking is regulated by approximately 60 members of the Rab subfamily of small Ras-like GDP/GTP binding proteins. Rab proteins cycle between inactive and active states as well as between cytosolic and membrane bound forms. Membrane extraction/delivery and cytosolic distribution of Rabs is mediated by interaction with the protein GDP dissociation inhibitor (GDI) that binds to prenylated inactive (GDP-bound) Rab proteins. Because the Rab:GDP:GDI complex is of high affinity, the question arises of how GDI can be displaced efficiently from Rab protein in order to allow the necessary recruitment of the Rab to its specific target membrane. While there is strong evidence that DrrA, as a bacterially encoded GDP/GTP exchange factor, contributes to this event, we show here that posttranslational modifications of Rabs can also modulate the affinity for GDI and thus cause effective displacement of GDI from Rab:GDI complexes. These activities have been found associated with the phosphocholination and adenylylation activities of the enzymes AnkX and DrrA/SidM, respectively, from the pathogenic bacterium Legionella pneumophila. Both modifications occur after spontaneous dissociation of Rab:GDI complexes within their natural equilibrium. Therefore, the effective GDI displacement that is observed is caused by inhibition of reformation of Rab:GDI complexes. Interestingly, in contrast to adenylylation by DrrA, AnkX can covalently modify inactive Rabs with high catalytic efficiency even when GDP is bound to the GTPase and hence can inhibit binding of GDI to Rab:GDP complexes. We therefore speculate that human cells could employ similar mechanisms in the absence of infection to effectively displace Rabs from GDI.
Asunto(s)
Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Unión al GTP rab/metabolismo , Adenina/metabolismo , Unión Competitiva , Humanos , Modelos Biológicos , Modelos Moleculares , Fosforilcolina/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas de Unión al GTP rab/química , Inhibidores de la Disociación del Nucleótido Guanina rho-EspecíficoRESUMEN
Rab GTPases, key regulators of vesicular transport, hydrolyze GTP very slowly unless assisted by Rab GTPase-activating proteins (RabGAPs). Dysfunction of RabGAPs is involved in many diseases. By combining X-ray structure analysis and time-resolved FTIR spectroscopy we reveal here the detailed molecular reaction mechanism of a complex between human Rab and RabGAP at the highest possible spatiotemporal resolution and in atomic detail. A glutamine residue of Rab proteins (cis-glutamine) that is essential for intrinsic activity is less important in the GAP-activated reaction. During generation of the RabGAP·Rab:GTP complex, there is a rapid conformational change in which the cis-glutamine is replaced by a glutamine from RabGAP (trans-glutamine); this differs from the RasGAP mechanism, where the cis-glutamine is also important for GAP catalysis. However, as in the case of Ras, a trans-arginine is also recruited to complete the active center during this conformational change. In contrast to the RasGAP mechanism, an accumulation of a state in which phosphate is bound is not observed, and bond breakage is the rate-limiting step. The movement of trans-glutamine and trans-arginine into the catalytic site and bond breakage during hydrolysis are monitored in real time. The combination of X-ray structure analysis and time-resolved FTIR spectroscopy provides detailed insight in the catalysis of human Rab GTPases.
Asunto(s)
Biocatálisis , Proteínas Activadoras de GTPasa/metabolismo , Mamíferos/metabolismo , Modelos Moleculares , Proteínas de Unión al GTP rab1/metabolismo , Animales , Dominio Catalítico , Análisis Mutacional de ADN , Proteínas Activadoras de GTPasa/química , Glutamina/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Hidrólisis , Cinética , Espectroscopía Infrarroja por Transformada de Fourier , Proteínas de Unión al GTP rab1/químicaRESUMEN
The Gram-negative bacterium Legionella pneumophila is the causative agent of Legionnaires' disease. During infection of eukaryotic cells, the bacterium releases about 300 different bacterial effector molecules that aid in the establishment of the Legionella-containing vacuole (LCV) among which SidC is one of these secreted proteins. However, apart from membrane lipid binding the function of SidC remains elusive. In order to characterize SidC further, we have determined the crystal structure of the N-terminal domain of SidC (amino acids 1-609, referred to as SidC-N) at 2.4Å resolution. SidC-N reveals a novel fold in which 4 potential subdomains (A-D) are arranged in a crescent-like structure. None of these subdomains currently has any known structural homologues, raising the question of how this fold has evolved. These domains are highly interconnected, with a low degree of flexibility towards each other. Due to the extended arrangement of the subdomains, SidC-N may contain multiple binding sites for potential interaction partners.
Asunto(s)
Proteínas Bacterianas/química , Legionella pneumophila , Sitios de Unión , Cristalografía por Rayos X , Modelos Moleculares , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Dispersión del Ángulo PequeñoRESUMEN
Small G-proteins of the Ras superfamily control the temporal and spatial coordination of intracellular signaling networks by acting as molecular on/off switches. Guanine nucleotide exchange factors (GEFs) regulate the activation of these G-proteins through catalytic replacement of GDP by GTP. During nucleotide exchange, three distinct substrate·enzyme complexes occur: a ternary complex with GDP at the start of the reaction (G-protein·GEF·GDP), an intermediary nucleotide-free binary complex (G-protein·GEF), and a ternary GTP complex after productive G-protein activation (G-protein·GEF·GTP). Here, we show structural snapshots of the full nucleotide exchange reaction sequence together with the G-protein substrates and products using Rabin8/GRAB (GEF) and Rab8 (G-protein) as a model system. Together with a thorough enzymatic characterization, our data provide a detailed view into the mechanism of Rabin8/GRAB-mediated nucleotide exchange.
Asunto(s)
Guanosina Trifosfato/química , Complejos Multienzimáticos/química , Proteínas Serina-Treonina Quinasas/química , Proteínas de Unión al GTP rab/química , Proteína de Unión al GTP rab3A/química , Catálisis , Quinasas del Centro Germinal , Guanosina Trifosfato/genética , Guanosina Trifosfato/metabolismo , Humanos , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteína de Unión al GTP rab3A/genética , Proteína de Unión al GTP rab3A/metabolismoRESUMEN
Modulation of the function of small GTPases that regulate vesicular trafficking is a strategy employed by several human pathogens. Legionella pneumophila infects lung macrophages and injects a plethora of different proteins into its host cell. Among these is DrrA/SidM, which catalyzes stable adenylylation of Rab1b, a regulator of endoplasmatic reticulum to Golgi trafficking, and thereby alters the function and interactions of this small GTPase. We employed time-resolved FTIR-spectroscopy to monitor the DrrA-catalyzed AMP-transfer to Tyr77 of Rab1b. A transient complex between DrrA, adenylylated Rab1b, and the pyrophosphate byproduct was resolved, allowing us to analyze the interactions at the active site. Combination of isotopic labeling and site-directed mutagenesis allowed us to derive the catalytic mechanism of DrrA from the FTIR difference spectra. DrrA shares crucial residues in the ATP-binding pocket with similar AMP-transferring enzymes such as glutamine synthetase adenylyltransferase or kanamycin nucleotidyltransferase, but provides the complete active site on a single subunit. We determined that Asp112 of DrrA functions as the catalytic base for deprotonation of Tyr77 of Rab1b to enable nucleophilic attack on the ATP. The study provides detailed understanding of the Legionella pneumophila protein DrrA and of AMP-transfer reactions in general.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/genética , Sitios de Unión , Dominio Catalítico , Factores de Intercambio de Guanina Nucleótido/genética , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fosfatos/química , Conformación Proteica , Espectroscopía Infrarroja por Transformada de Fourier , Tirosina/metabolismo , Proteínas de Unión al GTP rab1/metabolismoRESUMEN
Alpha-synuclein (αS) misfolding is associated with Parkinson's disease (PD) but little is known about the mechanisms underlying αS toxicity. Increasing evidence suggests that defects in membrane transport play an important role in neuronal dysfunction. Here we demonstrate that the GTPase Rab8a interacts with αS in rodent brain. NMR spectroscopy reveals that the C-terminus of αS binds to the functionally important switch region as well as the C-terminal tail of Rab8a. In line with a direct Rab8a/αS interaction, Rab8a enhanced αS aggregation and reduced αS-induced cellular toxicity. In addition, Rab8 - the Drosophila ortholog of Rab8a - ameliorated αS-oligomer specific locomotor impairment and neuron loss in fruit flies. In support of the pathogenic relevance of the αS-Rab8a interaction, phosphorylation of αS at S129 enhanced binding to Rab8a, increased formation of insoluble αS aggregates and reduced cellular toxicity. Our study provides novel mechanistic insights into the interplay of the GTPase Rab8a and αS cytotoxicity, and underscores the therapeutic potential of targeting this interaction.