RESUMEN
The basal structure of the bacterial flagellum includes a membrane embedded MS-ring (formed by multiple copies of FliF) and a cytoplasmic C-ring (composed of proteins FliG, FliM and FliN). The SRP-type GTPase FlhF is required for directing the initial flagellar protein FliF to the cell pole, but the mechanisms are unclear. Here, we show that FlhF anchors developing flagellar structures to the polar landmark protein HubP/FimV, thereby restricting their formation to the cell pole. Specifically, the GTPase domain of FlhF interacts with HubP, while a structured domain at the N-terminus of FlhF binds to FliG. FlhF-bound FliG subsequently engages with the MS-ring protein FliF. Thus, the interaction of FlhF with HubP and FliG recruits a FliF-FliG complex to the cell pole. In addition, the modulation of FlhF activity by the MinD-type ATPase FlhG controls the interaction of FliG with FliM-FliN, thereby regulating the progression of flagellar assembly at the pole.
Asunto(s)
Proteínas Bacterianas , Flagelos , Flagelos/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Unión Proteica , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , Partícula de Reconocimiento de Señal/metabolismo , Partícula de Reconocimiento de Señal/química , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de la MembranaRESUMEN
The intrinsically disordered, lipid-modified membrane anchor of small GTPases is emerging as a critical modulator of function through its ability to sort lipids in a conformation-dependent manner. We reviewed recent computational and experimental studies that have begun to shed light on the sequence-ensemble-function relationship in this unique class of lipidated intrinsically disordered regions (LIDRs).
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas Intrínsecamente Desordenadas/química , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas de Unión al GTP Monoméricas/química , Humanos , Membrana Celular/metabolismo , AnimalesRESUMEN
Protein structure has been well established to play a key role in determining function; however, intrinsically disordered proteins and regions (IDPs and IDRs) defy this paradigm. IDPs and IDRs exist as an ensemble of structures rather than a stable 3D structure yet play essential roles in many cell-signaling processes. Nearly all Ras superfamily GTPases are tethered to membranes by a lipid tail at the end of a flexible IDR. The sequence of the IDR is a key determinant of membrane localization, and interaction between the IDR and the membrane has been shown to affect signaling in RAS proteins through the modulation of dynamic membrane organization. Here, we utilized atomistic molecular dynamics simulations to study the membrane interaction, conformational dynamics, and lipid sorting of three IDRs from small GTPases Rheb, RhoA, and DiRas3 in model membranes representing their physiological target membranes. We found that complementarity between the lipidated IDR sequence and target membrane lipid composition is a determinant of conformational plasticity. We also show that electrostatic interactions between anionic lipids and basic residues on IDRs are correlated with sampling of semistable conformational substates, and lack of these interactions is associated with greater conformational diversity. Finally, we show that small GTPase IDRs with a polybasic domain alter local lipid composition by segregating anionic lipids and, in some cases, excluding other lipids from their immediate vicinity in favor of anionic lipids.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Proteína Homóloga de Ras Enriquecida en el Cerebro , Proteína de Unión al GTP rhoA , Humanos , Membrana Celular/metabolismo , Membrana Celular/química , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Simulación de Dinámica Molecular , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas de Unión al GTP Monoméricas/química , Proteína Homóloga de Ras Enriquecida en el Cerebro/metabolismo , Proteína Homóloga de Ras Enriquecida en el Cerebro/química , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoA/químicaRESUMEN
The sterile alpha motif and histidine-aspartate domain-containing protein 1 (or SAMHD1), a human dNTP-triphosphohydrolase, contributes to HIV-1 restriction in select terminally differentiated cells of the immune system. While the prevailing hypothesis is that the catalytically active form of the protein is an allosterically triggered tetramer, whose HIV-1 restriction properties are attributed to its dNTP - triphosphohydrolase activity, it is also known to bind to ssRNA and ssDNA oligomers. A complete picture of the structure-function relationship of the enzyme is still elusive and the function corresponding to its nucleic acid binding ability is debated. In this in silico study, we investigate the stability, preference and allosteric effects of DNA oligomers bound to SAMHD1. In particular, we compare the binding of DNA and RNA oligomers of the same sequence and also consider the binding of DNA fragments with phosphorothioate bonds in the backbone. The results are compared with the canonical form with the monomers connected by GTP/dATP crossbridges. The simulations indicate that SAMHD1 dimers preferably bind to DNA and RNA oligomers compared to GTP/dATP. However, allosteric communication channels are altered in the nucleic acid acid bound complexes compared to the canonical form. All results are consistent with the hypothesis that the DNA bound form of the protein correspond to an unproductive off-pathway state where the protein is sequestered and not available for dNTP hydrolysis.
Asunto(s)
Simulación de Dinámica Molecular , Proteínas de Unión al GTP Monoméricas , Humanos , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Nucleótidos/metabolismo , ADN , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Comunicación , ARNRESUMEN
Membrane-enclosed organelles are defining features of eukaryotes in distinguishing these organisms from prokaryotes. Specification of distinct membranes is critical to assemble and maintain discrete compartments. Small GTPases and their regulators are the signaling molecules that drive membrane-modifying machineries to the desired location. These signaling molecules include Rab and Rag GTPases, roadblock and longin domain proteins, and TRAPPC3-like proteins. Here, we take a structural approach to assess the relatedness of these eukaryotic-like proteins in Asgard archaea, the closest known prokaryotic relatives to eukaryotes. We find that the Asgard archaea GTPase core domains closely resemble eukaryotic Rabs and Rags. Asgard archaea roadblock, longin and TRAPPC3 domain-containing proteins form dimers similar to those found in the eukaryotic TRAPP and Ragulator complexes. We conclude that the emergence of these protein architectures predated eukaryogenesis, however further adaptations occurred in proto-eukaryotes to allow these proteins to regulate distinct internal membranes.
Asunto(s)
Proteínas de Unión al GTP Monoméricas , Proteínas de Unión al GTP Monoméricas/química , Archaea/metabolismo , Transporte de ProteínasRESUMEN
The protein kinase Mechanistic Target of Rapamycin (mTOR) in Complex 1 (mTORC1) is regulated in part by the Ras-related GTP-binding proteins (Rag GTPases). Rag GTPases form a heterodimeric complex consisting of either RagA or RagB associated with either RagC or RagD and act to localize mTORC1 to the lysosomal membrane. Until recently, RagA and RagB were thought to be functionally redundant, as were RagC and RagD. However, recent research suggests that the various isoforms differentially activate mTORC1. Here, the mRNA expression and protein abundance of the Rag GTPases was compared across male rat skeletal muscle, heart, liver, kidney, and brain. Whereas mRNA expression of RagA was higher than RagB in nearly all tissues studied, RagB protein abundance was higher than RagA in all tissues besides skeletal muscle. RagC mRNA expression was more abundant or equal to RagD mRNA, and RagD protein was more abundant than RagC protein in all tissues. Moreover, the proportion of RagB in the short isoform was greater than the long in liver, whereas the opposite was true in brain. These results serve to further elucidate Rag GTPase expression and offer potential explanations for the differential responses to amino acids that are observed in different tissues.
Asunto(s)
Proteínas de Unión al GTP Monoméricas , Transducción de Señal , Masculino , Ratas , Animales , Transducción de Señal/fisiología , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Aminoácidos/metabolismo , ARN Mensajero/genéticaRESUMEN
FlhF and FlhG control the location and number of flagella, respectively, in many polar-flagellated bacteria. The roles of FlhF and FlhG are not well characterized in bacteria that have multiple polar flagella, such as Helicobacter pylori. Deleting flhG in H. pylori shifted the flagellation pattern where most cells had approximately four flagella to a wider and more even distribution in flagellar number. As reported in other bacteria, deleting flhF in H. pylori resulted in reduced motility, hypoflagellation, and the improper localization of flagella to nonpolar sites. Motile variants of H. pylori ∆flhF mutants that had a higher proportion of flagella localizing correctly to the cell pole were isolated, but we were unable to identify the genetic determinants responsible for the increased localization of flagella to the cell pole. One motile variant though produced more flagella than the ΔflhF parental strain, which apparently resulted from a missense mutation in fliF (encodes the MS ring protein), which changed Asn-255 to aspartate. Recombinant FliFN255D, but not recombinant wild-type FliF, formed ordered ring-like assemblies in vitro that were ~50 nm wide and displayed the MS ring architecture. We infer from these findings that the FliFN225D variant forms the MS ring more effectively in vivo in the absence of FlhF than wild-type FliF. IMPORTANCE Helicobacter pylori colonizes the human stomach where it can cause a variety of diseases, including peptic ulcer disease and gastric cancer. H. pylori uses flagella for motility, which is required for host colonization. FlhG and FlhF control the flagellation patterns in many bacteria. We found that in H. pylori, FlhG ensures that cells have approximately equal number of flagella and FlhF is needed for flagellum assembly and localization. FlhF is proposed to facilitate the assembly of FliF into the MS ring, which is one of the earliest structures formed in flagellum assembly. We identified a FliF variant that assembles the MS ring in the absence of FlhF, which supports the proposed role of FlhF in facilitating MS ring assembly.
Asunto(s)
Helicobacter pylori , Proteínas de Unión al GTP Monoméricas , Humanos , Proteínas Bacterianas/metabolismo , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Flagelos/genética , Flagelos/metabolismoRESUMEN
mTORC1 is a protein kinase complex that controls cellular growth in response to nutrient availability. Amino acid signals are transmitted toward mTORC1 via the Rag/Gtr GTPases and their upstream regulators. An important regulator is LAMTOR, which localizes Rag/Gtr on the lysosomal/vacuole membrane. In human cells, LAMTOR consists of five subunits, but in yeast, only three or four. Currently, it is not known how variation of the subunit stoichiometry may affect its structural organization and biochemical properties. Here, we report a 3.1 Å-resolution structural model of the Gtr-Lam complex in Schizosaccharomyces pombe. We found that SpGtr shares conserved architecture as HsRag, but the intersubunit communication that coordinates nucleotide loading on the two subunits differs. In contrast, SpLam contains distinctive structural features, but its GTP-specific GEF activity toward SpGtr is evolutionarily conserved. Our results revealed unique evolutionary paths of the protein components of the mTORC1 pathway.
Asunto(s)
Proteínas de Unión al GTP Monoméricas , Schizosaccharomyces , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Aminoácidos/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Unión al GTP Monoméricas/químicaRESUMEN
Sterile alpha motif histidine-aspartate domain protein 1 (SAMHD1) is a deoxynucleotide triphosphohydrolase that exists in monomeric, dimeric, and tetrameric forms. It is activated by GTP binding to an A1 allosteric site on each monomer subunit, which induces dimerization, a prerequisite for dNTP-induced tetramerization. SAMHD1 is a validated drug target stemming from its inactivation of many anticancer nucleoside drugs leading to drug resistance. The enzyme also possesses a single-strand nucleic acid binding function that promotes RNA and DNA homeostasis by several mechanisms. To discover small molecule inhibitors of SAMHD1, we screened a custom â¼69â¯000-compound library for dNTPase inhibitors. Surprisingly, this effort yielded no viable hits and indicated that exceptional barriers for discovery of small molecule inhibitors existed. We then took a rational fragment-based inhibitor design approach using a deoxyguanosine (dG) A1 site targeting fragment. A targeted chemical library was synthesized by coupling a 5'-phosphoryl propylamine dG fragment (dGpC3NH2) to 376 carboxylic acids (RCOOH). Direct screening of the products (dGpC3NHCO-R) yielded nine initial hits, one of which (R = 3-(3'-bromo-[1,1'-biphenyl]), 5a) was investigated extensively. Amide 5a is a competitive inhibitor against GTP binding to the A1 site and induces inactive dimers that are deficient in tetramerization. Surprisingly, 5a also prevented ssDNA and ssRNA binding, demonstrating that the dNTPase and nucleic acid binding functions of SAMHD1 can be disrupted by a single small molecule. A structure of the SAMHD1-5a complex indicates that the biphenyl fragment impedes a conformational change in the C-terminal lobe that is required for tetramerization.
Asunto(s)
Proteínas de Unión al GTP Monoméricas , Ácidos Nucleicos , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Ácido Aspártico , Histidina , Motivo alfa Estéril , Guanosina Trifosfato/química , Desoxiguanosina , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismoRESUMEN
BACKGROUND: SAMHD1 is a deoxynucleotide triphosphohydrolase that restricts replication of HIV-1 in differentiated leucocytes. HIV-1 is not restricted in cycling cells and it has been proposed that this is due to phosphorylation of SAMHD1 at T592 in these cells inactivating the enzymatic activity. To distinguish between theories for how SAMHD1 restricts HIV-1 in differentiated but not cycling cells, we analysed the effects of substitutions at T592 on restriction and dNTP levels in both cycling and differentiated cells as well as tetramer stability and enzymatic activity in vitro. RESULTS: We first showed that HIV-1 restriction was not due to SAMHD1 nuclease activity. We then characterised a panel of SAMHD1 T592 mutants and divided them into three classes. We found that a subset of mutants lost their ability to restrict HIV-1 in differentiated cells which generally corresponded with a decrease in triphosphohydrolase activity and/or tetramer stability in vitro. Interestingly, no T592 mutants were able to restrict WT HIV-1 in cycling cells, despite not being regulated by phosphorylation and retaining their ability to hydrolyse dNTPs. Lowering dNTP levels by addition of hydroxyurea did not give rise to restriction. Compellingly however, HIV-1 RT mutants with reduced affinity for dNTPs were significantly restricted by wild-type and T592 mutant SAMHD1 in both cycling U937 cells and Jurkat T-cells. Restriction correlated with reverse transcription levels. CONCLUSIONS: Altogether, we found that the amino acid at residue 592 has a strong effect on tetramer formation and, although this is not a simple "on/off" switch, this does correlate with the ability of SAMHD1 to restrict HIV-1 replication in differentiated cells. However, preventing phosphorylation of SAMHD1 and/or lowering dNTP levels by adding hydroxyurea was not enough to restore restriction in cycling cells. Nonetheless, lowering the affinity of HIV-1 RT for dNTPs, showed that restriction is mediated by dNTP levels and we were able to observe for the first time that SAMHD1 is active and capable of inhibiting HIV-1 replication in cycling cells, if the affinity of RT for dNTPs is reduced. This suggests that the very high affinity of HIV-1 RT for dNTPs prevents HIV-1 restriction by SAMHD1 in cycling cells.
Asunto(s)
VIH-1 , Proteínas de Unión al GTP Monoméricas , Humanos , VIH-1/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Fosforilación , Células U937 , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/metabolismoRESUMEN
Different protein purification methods exist. Yet, they need to be adapted for specific downstream applications to maintain functional integrity of the recombinant proteins. This study established a purification protocol for lentiviral Vpx (viral protein X) and test its ability to degrade sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) ex vivo in resting CD4+ T cells. For this purpose, we cloned a novel eukaryotic expression plasmid for Vpx including C-terminal 10x His- and HA-tags and confirmed that those tags did not alter the ability to degrade SAMHD1. We optimized purification conditions for Vpx produced in HEK293T cells with CHAPS as detergent and Co-NTA resins yielding the highest solubility and protein amounts. Size exclusion chromatography (SEC) further enhanced the purity of recombinant Vpx proteins. Importantly, nucleofection of resting CD4+ T cells demonstrated that purified recombinant Vpx protein efficiently degraded SAMHD1 in a proteasome-dependent manner. In conclusion, this protocol is suitable for functional downstream applications of recombinant Vpx and might be transferrable to other recombinant proteins with similar functions/properties as lentiviral Vpx.
Asunto(s)
Proteínas de Unión al GTP Monoméricas , Linfocitos T , Humanos , Proteína 1 que Contiene Dominios SAM y HD/genética , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Células HEK293 , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Linfocitos T CD4-Positivos , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/metabolismoRESUMEN
The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of mammalian cell growth that is dysregulated in a number of human diseases, including metabolic syndromes, aging, and cancer. Structural, biochemical, and pharmacological studies that have increased our understanding of how mTORC1 executes growth control often relied upon purified mTORC1 protein. However, current immunoaffinity-based purification methods are expensive, inefficient, and do not necessarily isolate endogenous mTORC1, hampering their overall utility in research. Here we present a simple tool to isolate endogenous mTORC1 from various cellular sources. By recombinantly expressing and isolating mTORC1-binding Rag GTPases from Escherichia coli and using them as affinity probes, we demonstrate that mTORC1 can be isolated from mouse, bovine, and human sources. Our results indicate that mTORC1 isolated by this relatively inexpensive method is catalytically active and amenable to scaling. Collectively, this tool may be utilized to isolate mTORC1 from various cellular sources, organs, and disease contexts, aiding mTORC1-related research.
Asunto(s)
Biotecnología , Diana Mecanicista del Complejo 1 de la Rapamicina , Proteínas de Unión al GTP Monoméricas , Proteínas Recombinantes , Animales , Bovinos , Humanos , Ratones , Mamíferos/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/química , Diana Mecanicista del Complejo 1 de la Rapamicina/aislamiento & purificación , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Escherichia coli/genética , Biotecnología/métodos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos MolecularesRESUMEN
Transport of newly synthesized proteins from endoplasmic reticulum (ER) to Golgi is mediated by coat protein complex II (COPII). The assembly and disassembly of COPII vesicles is regulated by the molecular switch Sar1, which is a small GTPase and a component of COPII. Usually a small GTPase binds GDP (inactive form) or GTP (active form). Mammals have two Sar1 isoforms, Sar1a and Sar1b, that have approximately 90% sequence identity. Some experiments demonstrated that these two isoforms had distinct but overlapping functions. Here we found another instance of differing behavior: the alarmone ppGpp could bind to and inhibit the GTPase activity of human Sar1a but could not inhibit the GTPase activity of human Sar1b. The crystal structures of Sar1aâ ppGpp and Sar1bâ GDP have been determined. Superposition of the structures shows that ppGpp binds to the nucleotide-binding pocket, its guanosine base, ribose ring and 5'-diphosphate occupying nearly the same positions as for GDP. However, its 3'-diphosphate points away from the active site and, hence, away from the surface of the protein. The overall structure of Sar1aâ ppGpp is more similar to Sar1bâ GDP than to Sar1bâ GTP. We also find that the Asp140-Arg138-water-ligand interaction net is important for the binding of ppGpp to Sar1a. This study provides further evidence showing that there are biochemical differences between the Sar1a and Sar1b isoforms of Sar1.
Asunto(s)
Proteínas de Unión al GTP Monoméricas , Animales , Humanos , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/metabolismo , Guanosina Tetrafosfato , Proteínas de Transporte Vesicular/metabolismo , Difosfatos/metabolismo , Isoformas de Proteínas/metabolismo , Mamíferos/metabolismoRESUMEN
KRas is a small GTPase and membrane-bound signaling protein. Newly synthesized KRas is post-translationally modified with a membrane-anchoring prenyl group. KRas chaperones are therapeutic targets in cancer due to their participation in trafficking oncogenic KRas to membranes. SmgGDS splice variants are chaperones for small GTPases with basic residues in their hypervariable domain (HVR), including KRas. SmgGDS-607 escorts pre-prenylated small GTPases, while SmgGDS-558 escorts prenylated small GTPases. We provide a structural description of farnesylated and fully processed KRas (KRas-FMe) in complex with SmgGDS-558 and define biophysical properties of this interaction. Surface plasmon resonance measurements on biomimetic model membranes quantified the thermodynamics of the interaction of SmgGDS with KRas, and small-angle x-ray scattering was used to characterize complexes of SmgGDS-558 and KRas-FMe structurally. Structural models were refined using Monte Carlo and molecular dynamics simulations. Our results indicate that SmgGDS-558 interacts with the HVR and the farnesylated C-terminus of KRas-FMe, but not its G-domain. Therefore, SmgGDS-558 interacts differently with prenylated KRas than prenylated RhoA, whose G-domain was found in close contact with SmgGDS-558 in a recent crystal structure. Using immunoprecipitation assays, we show that SmgGDS-558 binds the GTP-bound, GDP-bound, and nucleotide-free forms of farnesylated and fully processed KRas in cells, consistent with SmgGDS-558 not engaging the G-domain of KRas. We found that the dissociation constant, Kd, for KRas-FMe binding to SmgGDS-558 is comparable with that for the KRas complex with PDEδ, a well-characterized KRas chaperone that also does not interact with the KRas G-domain. These results suggest that KRas interacts in similar ways with the two chaperones SmgGDS-558 and PDEδ. Therapeutic targeting of the SmgGDS-558/KRas complex might prove as useful as targeting the PDEδ/KRas complex in KRas-driven cancers.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido , Proteínas de Unión al GTP Monoméricas , Genes ras , Factores de Intercambio de Guanina Nucleótido/metabolismo , Guanosina Trifosfato/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismoRESUMEN
The p47 immunity-related GTPase (IRG) Irgb6 plays a pioneering role in host defense against Toxoplasma gondii infection. Irgb6 is recruited to the parasitophorous vacuole membrane (PVM) formed by T. gondii and disrupts it. Despite the importance of this process, the molecular mechanisms accounting for PVM recognition by Irgb6 remain elusive because of lack of structural information on Irgb6. Here we report the crystal structures of mouse Irgb6 in the GTP-bound and nucleotide-free forms. Irgb6 exhibits a similar overall architecture to other IRGs in which GTP binding induces conformational changes in both the dimerization interface and the membrane-binding interface. The membrane-binding interface of Irgb6 assumes a unique conformation, composed of N- and C-terminal helical regions forming a phospholipid binding site. In silico docking of phospholipids further revealed membrane-binding residues that were validated through mutagenesis and cell-based assays. Collectively, these data demonstrate a novel structural basis for Irgb6 to recognize T. gondii PVM in a manner distinct from other IRGs.
Asunto(s)
Interacciones Huésped-Parásitos , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/metabolismo , Toxoplasma , Toxoplasmosis/metabolismo , Toxoplasmosis/parasitología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Ratones , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Relación Estructura-Actividad , VacuolasRESUMEN
The mTOR complex 1 (mTORC1) controls cell growth in response to amino acid levels1. Here we report SAR1B as a leucine sensor that regulates mTORC1 signalling in response to intracellular levels of leucine. Under conditions of leucine deficiency, SAR1B inhibits mTORC1 by physically targeting its activator GATOR2. In conditions of leucine sufficiency, SAR1B binds to leucine, undergoes a conformational change and dissociates from GATOR2, which results in mTORC1 activation. SAR1B-GATOR2-mTORC1 signalling is conserved in nematodes and has a role in the regulation of lifespan. Bioinformatic analysis reveals that SAR1B deficiency correlates with the development of lung cancer. The silencing of SAR1B and its paralogue SAR1A promotes mTORC1-dependent growth of lung tumours in mice. Our results reveal that SAR1B is a conserved leucine sensor that has a potential role in the development of lung cancer.
Asunto(s)
Leucina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Transducción de Señal , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Secuencia Conservada , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Células HEK293 , Humanos , Leucina/deficiencia , Longevidad/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/agonistas , Ratones , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/deficiencia , Proteínas de Unión al GTP Monoméricas/genética , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Unión Proteica , Proteínas Supresoras de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mitochondrial ribosomes (mitoribosomes) synthesize a critical set of proteins essential for oxidative phosphorylation. Therefore, mitoribosomal function is vital to the cellular energy supply. Mitoribosome biogenesis follows distinct molecular pathways that remain poorly understood. Here, we determine the cryo-EM structures of mitoribosomes isolated from human cell lines with either depleted or overexpressed mitoribosome assembly factor GTPBP5, allowing us to capture consecutive steps during mitoribosomal large subunit (mt-LSU) biogenesis. Our structures provide essential insights into the last steps of 16S rRNA folding, methylation and peptidyl transferase centre (PTC) completion, which require the coordinated action of nine assembly factors. We show that mammalian-specific MTERF4 contributes to the folding of 16S rRNA, allowing 16 S rRNA methylation by MRM2, while GTPBP5 and NSUN4 promote fine-tuning rRNA rearrangements leading to PTC formation. Moreover, our data reveal an unexpected involvement of the elongation factor mtEF-Tu in mt-LSU assembly, where mtEF-Tu interacts with GTPBP5, similar to its interaction with tRNA during translational elongation.
Asunto(s)
Ribosomas Mitocondriales/química , Subunidades Ribosómicas Grandes/química , Línea Celular , Microscopía por Crioelectrón , Humanos , Metiltransferasas/química , Metiltransferasas/metabolismo , Ribosomas Mitocondriales/metabolismo , Modelos Moleculares , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/metabolismo , Complejos Multiproteicos , Factor Tu de Elongación Peptídica/química , Factor Tu de Elongación Peptídica/metabolismo , Peptidil Transferasas/química , Peptidil Transferasas/metabolismo , Unión Proteica , Pliegue del ARN , ARN Ribosómico 16S/química , ARN Ribosómico 16S/metabolismo , Subunidades Ribosómicas Grandes/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Ribosome biogenesis requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. Particularly, maturation of the peptidyl transferase center (PTC) is mediated by conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial large ribosomal subunit (mtLSU) using endogenous complex purification, in vitro reconstitution and cryo-EM. Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch and progression to a near-mature PTC state. Additionally, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results provide a framework for understanding step-wise PTC folding as a critical conserved quality control checkpoint.
Asunto(s)
Proteínas de Unión al GTP/química , Ribosomas Mitocondriales/química , Proteínas de Unión al GTP Monoméricas/química , Microscopía por Crioelectrón , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Humanos , Metiltransferasas/química , Metiltransferasas/metabolismo , Ribosomas Mitocondriales/metabolismo , Modelos Moleculares , Proteínas de Unión al GTP Monoméricas/metabolismo , Complejos Multiproteicos , Biogénesis de Organelos , Peptidil Transferasas/química , Peptidil Transferasas/metabolismo , Pliegue de Proteína , ARN Ribosómico/química , ARN Ribosómico/metabolismo , Subunidades Ribosómicas Grandes/química , Subunidades Ribosómicas Grandes/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Mitochondrial ribosomes are specialized for the synthesis of membrane proteins responsible for oxidative phosphorylation. Mammalian mitoribosomes have diverged considerably from the ancestral bacterial ribosomes and feature dramatically reduced ribosomal RNAs. The structural basis of the mammalian mitochondrial ribosome assembly is currently not well understood. Here we present eight distinct assembly intermediates of the human large mitoribosomal subunit involving seven assembly factors. We discover that the NSUN4-MTERF4 dimer plays a critical role in the process by stabilizing the 16S rRNA in a conformation that exposes the functionally important regions of rRNA for modification by the MRM2 methyltransferase and quality control interactions with the conserved mitochondrial GTPase MTG2 that contacts the sarcin-ricin loop and the immature active site. The successive action of these factors leads to the formation of the peptidyl transferase active site of the mitoribosome and the folding of the surrounding rRNA regions responsible for interactions with tRNAs and the small ribosomal subunit.
Asunto(s)
Ribosomas Mitocondriales/química , Peptidil Transferasas/química , Dominio Catalítico , Microscopía por Crioelectrón , Humanos , Metiltransferasas/química , Metiltransferasas/metabolismo , Ribosomas Mitocondriales/metabolismo , Modelos Moleculares , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/metabolismo , Conformación de Ácido Nucleico , Peptidil Transferasas/metabolismo , Multimerización de Proteína , ARN Ribosómico/química , ARN Ribosómico/metabolismo , Subunidades Ribosómicas Grandes/química , Subunidades Ribosómicas Grandes/metabolismo , Factores de Transcripción/metabolismoRESUMEN
SAMHD1 is a fundamental regulator of cellular dNTPs that catalyzes their hydrolysis into 2'-deoxynucleoside and triphosphate, restricting the replication of viruses, including HIV-1, in CD4+ myeloid lineage and resting T-cells. SAMHD1 mutations are associated with the autoimmune disease Aicardi-Goutières syndrome (AGS) and certain cancers. More recently, SAMHD1 has been linked to anticancer drug resistance and the suppression of the interferon response to cytosolic nucleic acids after DNA damage. Here, we probe dNTP hydrolysis and inhibition of SAMHD1 using the Rp and Sp diastereomers of dNTPαS nucleotides. Our biochemical and enzymological data show that the α-phosphorothioate substitution in Sp-dNTPαS but not Rp-dNTPαS diastereomers prevents Mg2+ ion coordination at both the allosteric and catalytic sites, rendering SAMHD1 unable to form stable, catalytically active homotetramers or hydrolyze substrate dNTPs at the catalytic site. Furthermore, we find that Sp-dNTPαS diastereomers competitively inhibit dNTP hydrolysis, while Rp-dNTPαS nucleotides stabilize tetramerization and are hydrolyzed with similar kinetic parameters to cognate dNTPs. For the first time, we present a cocrystal structure of SAMHD1 with a substrate, Rp-dGTPαS, in which an Fe-Mg-bridging water species is poised for nucleophilic attack on the Pα. We conclude that it is the incompatibility of Mg2+, a hard Lewis acid, and the α-phosphorothioate thiol, a soft Lewis base, that prevents the Sp-dNTPαS nucleotides coordinating in a catalytically productive conformation. On the basis of these data, we present a model for SAMHD1 stereospecific hydrolysis of Rp-dNTPαS nucleotides and for a mode of competitive inhibition by Sp-dNTPαS nucleotides that competes with formation of the enzyme-substrate complex.