RESUMEN
The Ser/Thr protein kinase ataxia telangiectasia mutated (ATM) plays an important role in the DNA damage response, signaling in response to redox signals, the control of metabolic processes, and mitochondrial homeostasis. ATM localizes to the nucleus and at the plasma membrane, mitochondria, peroxisomes, and other cytoplasmic vesicular structures. It has been shown that the C-terminal FATC domain of human ATM (hATMfatc) can interact with a range of membrane mimetics and may thereby act as a membrane-anchoring unit. Here, NMR structural and 15N relaxation data, NMR data using spin-labeled micelles, and MD simulations of micelle-associated hATMfatc revealed that it binds the micelle by a dynamic assembly of three helices with many residues of hATMfatc located in the headgroup region. We observed that none of the three helices penetrates the micelle deeply or makes significant tertiary contacts to the other helices. NMR-monitored interaction experiments with hATMfatc variants in which two conserved aromatic residues (Phe3049 and Trp3052) were either individually or both replaced by alanine disclosed that the double substitution does not abrogate the interaction with micelles and bicelles at the high concentrations at which these aggregates are typically used, but impairs interactions with small unilamellar vesicles, usually used at much lower lipid concentrations and considered a better mimetic for natural membranes. We conclude that the observed dynamic structure of micelle-associated hATMfatc may enable it to interact with differently composed membranes or membrane-associated interaction partners and thereby regulate ATM's kinase activity. Moreover, the FATC domain of ATM may function as a membrane-anchoring unit for other biomolecules.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/química , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Membrana Celular/metabolismo , Humanos , Micelas , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Dominios ProteicosRESUMEN
Mycobacterium tuberculosis escapes killing in human macrophages by secreting protein kinase G (PknG). PknG intercepts host signaling to prevent fusion of the phagosome engulfing the mycobacteria with the lysosome and, thus, their degradation. The N-terminal NORS (no regulatory secondary structure) region of PknG (approximately residues 1-75) has been shown to play a role in PknG regulation by (auto)phosphorylation, whereas the following rubredoxin-like metal-binding motif (RD, residues â¼74-147) has been shown to interact tightly with the subsequent catalytic domain (approximately residues 148-420) to mediate its redox regulation. Deletions or mutations in NORS or the redox-sensitive RD significantly decrease PknG survival function. Based on combined NMR spectroscopy, in vitro kinase assay, and molecular dynamics simulation data, we provide novel insights into the regulatory roles of the N-terminal regions. The NORS region is indeed natively disordered and rather dynamic. Consistent with most earlier data, autophosphorylation occurs in our assays only when the NORS region is present and, thus, in the NORS region. Phosphorylation of it results only in local conformational changes and does not induce interactions with the subsequent RD. Although the reduced, metal-bound RD makes tight interactions with the following catalytic domain in the published crystal structures, it can also fold in its absence. Our data further suggest that oxidation-induced unfolding of the RD regulates substrate access to the catalytic domain and, thereby, PknG function under different redox conditions, e.g. when exposed to increased levels of reactive oxidative species in host macrophages.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/química , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Mycobacterium tuberculosis/metabolismo , Desplegamiento Proteico , Rubredoxinas/química , Rubredoxinas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Proteínas Quinasas Dependientes de GMP Cíclico/genética , Humanos , Simulación de Dinámica Molecular , Mutación/genética , Oxidación-Reducción , Estructura Secundaria de Proteína , Rubredoxinas/genéticaRESUMEN
Mycobacterium tuberculosis protein kinase G (PknG) is secreted into host macrophages to block lysosomal degradation. The catalytic domain (â¼147-405) is C-terminally flanked by a tetratricopeptide repeat domain (TPRD). The preceding rubredoxin-like metal-binding motif (RD, â¼74-147) mediates PknG redox regulation. The N-terminal â¼75 residues were predicted to show no regulatory secondary structure (NORS) and harbor the only site (T63) phosphorylated in vivo. Deletions or mutations in the NORS or the redox-sensitive RD significantly decrease the survival function. Here, we show that the RD appears only to be present in the folded, metal-bound state if ZnCl2 is added upon induction of protein expression in minimal medium. Since factor Xa cleaves at the end of its recognition site (IEGR), a modified expression plasmid for PknG1-147 was obtained by mutating the N-terminal thrombin to a factor Xa recognition site. This allows preparing PknG1-147 with its native N-terminus. We further present a fast approach to generate expression plasmids for only the NORS or the RD by site-directed mutagenesis of the expression plasmid for His-tagged PknG1-147. An expression plasmid for PknG1-75 was obtained by introducing a stop codon at position 76 and one for PknG74-174 by introducing a factor Xa recognition site before position 74. SDS-PAGE analysis shows that all fragments are highly expressed in E. coli and can be purified to high purity. Thereby, the established preparation protocols pave the route for the NMR structural characterization of PknG regulation by its N-terminal regions, which is demonstrated by the recorded initial (1)H-(15)N-HSQC spectra.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Quinasas Dependientes de GMP Cíclico/biosíntesis , Proteínas Quinasas Dependientes de GMP Cíclico/química , Expresión Génica , Mycobacterium tuberculosis/enzimología , Proteínas Bacterianas/genética , Proteínas Quinasas Dependientes de GMP Cíclico/genética , Mycobacterium tuberculosis/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
The nematocyst walls of Hydra are formed by proteins containing small cysteine-rich domains (CRDs) of ~25 amino acids. The first CRD of nematocyst outer all antigen (NW1) and the C-terminal CRD of minicollagen-1 (Mcol1C) contain six cysteines at identical sequence positions, however adopt different disulfide bonded structures. NW1 shows the disulfide connectivities C2-C14/C6-C19/C10-C18 and Mcol1C C2-C18/C6-C14/C10-C19. To analyze if both show structural preferences in the open, non-disulfide bonded form, which explain the formation of either disulfide connectivity pattern, molecular dynamics (MD) simulations at different temperatures were performed. NW1 maintained in the 100-ns MD simulations at 283 K a rather compact fold that is stabilized by specific hydrogen bonds. The Mcol1C structure fluctuated overall more, however stayed most of the time also rather compact. The analysis of the backbone Φ/ψ angles indicated different turn propensities for NW1 and Mcol1C, which mostly can be explained based on published data about the influence of different amino acid side chains on the local backbone conformation. Whereas a folded precursor mechanism may be considered for NW1, Mcol1C may fold according to the quasi-stochastic folding model involving disulfide bond reshuffling and conformational changes, locking the native disulfide conformations. The study further demonstrates the power of MD simulations to detect local structural preferences in rather dynamic systems such as the open, non-disulfide bonded forms of NW1 and Mcol1C, which complement published information from NMR backbone residual dipolar couplings. Because the backbone structural preferences encoded by the amino acid sequence embedding the cysteines influence which disulfide connectivities are formed, the data are generally interesting for a better understanding of oxidative folding and the design of disulfide stabilized therapeutics.
Asunto(s)
Cisteína/química , Disulfuros/química , Hydra/química , Simulación de Dinámica Molecular , Proteínas/química , Animales , Enlace de Hidrógeno , Nematocisto/química , Oxidación-Reducción , Pliegue de Proteína , Temperatura , TermodinámicaRESUMEN
The FATC domain is shared by all members of the family of phosphatidylinositol-3 kinase-related kinases (PIKKs). It has been shown that the FATC domain plays an important role for the regulation of each PIKK. However, other than an involvement in protein-protein interactions, a common principle for the action of the FATC domain has not been detected. A detailed characterization of the structure and lipid binding properties of the FATC domain of the Ser/Thr kinase target of rapamycin (TOR) revealed that it contains a redox-sensitive membrane anchor in its C terminus. Because the C-terminal regions of the FATC domains of all known PIKKs are rather hydrophobic and especially rich in aromatic residues, we examined whether the ability to interact with lipids and membranes might be a general property. Here, we present the characterization of the interactions with lipids and different membrane mimetics for the FATC domains of human DNA-PKcs, human ATM, human ATR, human SMG-1, and human TRRAP by NMR and CD spectroscopy. The data indicate that all of these can interact with different membrane mimetics and may have different preferences only for membrane properties such as surface charge, curvature, and lipid packing. The oxidized form of the TOR FATC domain is well structured overall and forms an α-helix that is followed by a disulfide-bonded loop. In contrast, the FATC domains of the other PIKKs are rather unstructured in the isolated form and only significantly populate α-helical secondary structure upon interaction with membrane mimetics.
Asunto(s)
Lípidos/química , Membranas Artificiales , Fosfatidilinositol 3-Quinasas/química , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Humanos , Resonancia Magnética Nuclear Biomolecular , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
Antimicrobial peptides are a promising class of potential antibiotics that interact selectively with negatively charged lipid bilayers. This paper presents the structural characterization of the antimicrobial peptides myxinidin and WMR associated with bacterial membrane mimetic micelles and bicelles by NMR, CD spectroscopy, and molecular dynamics simulations. Both peptides adopt a different conformation in the lipidic environment than in aqueous solution. The location of the peptides in micelles and bicelles has been studied by paramagnetic relaxation enhancement experiments with paramagnetic tagged 5- and 16-doxyl stearic acid (5-/16-SASL). Molecular dynamics simulations of multiple copies of the peptides were used to obtain an atomic level of detail on membrane-peptide and peptide-peptide interactions. Our results highlight an essential role of the negatively charged membrane mimetic in the structural stability of both myxinidin and WMR. The peptides localize predominantly in the membrane's headgroup region and have a noticeable membrane thinning effect on the overall bilayer structure. Myxinidin and WMR show a different tendency to self-aggregate, which is also influenced by the membrane composition (DOPE/DOPG versus DOPE/DOPG/CL) and can be related to the previously observed difference in the ability of the peptides to disrupt different types of model membranes.
Asunto(s)
Péptidos Antimicrobianos , Micelas , Péptidos/química , Membrana Dobles de Lípidos/química , MembranasRESUMEN
The Ser/Thr kinase target of rapamycin (TOR) is a central controller of cellular growth and metabolism. Misregulation of TOR signaling is involved in metabolic and neurological disorders and tumor formation. TOR can be inhibited by association of a complex of rapamycin and FKBP12 to the FKBP12-rapamycin binding (FRB) domain. This domain was further proposed to interact with phosphatidic acid (PA), a lipid second messenger present in cellular membranes. Because mammalian TOR has been localized at various cellular membranes and in the nucleus, the output of TOR signaling may depend on its localization, which is expected to be influenced by the interaction with complex partners and regulators in response to cellular signals. Here, we present a detailed characterization of the interaction of the FRB domain with PA and how it is influenced by the surrounding membrane environment. On the basis of nuclear magnetic resonance- and circular dichroism-monitored binding studies using different neutral and negatively charged lipids as well as different membrane mimetics (micelles, bicelles, and liposomes), the FRB domain may function as a conditional peripheral membrane protein. However, the data for the isolated domain just indicate an increased affinity for negatively charged lipids and membrane patches but no specific preference for PA or PA-enriched regions. The membrane-mimetic environment induces strong conformational changes that largely maintain the α-helical secondary structure content but presumably disperse the helices in the lipidic environment. Consistent with overlapping binding surfaces for different lipids and the FKBP12-rapamycin complex, binding of the inhibitor complex protects the FRB domain from interactions with membrane mimetics at lower lipid concentrations.
Asunto(s)
Membrana Celular/química , Liposomas/farmacología , Micelas , Ácidos Fosfatidicos/farmacología , Sirolimus/metabolismo , Serina-Treonina Quinasas TOR/química , Proteína 1A de Unión a Tacrolimus/metabolismo , Humanos , Modelos Moleculares , Estructura Secundaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Dictyostelium Formin C (ForC) is involved in the regulation of local actin cytoskeleton reorganization (e.g. during cellular adhesion or migration). ForC contains formin homology 2 and 3 (FH2 and -3) domains and an N-terminal putative GTPase-binding domain (GBD) but lacks a canonical FH1 region. To better understand the role of the GBD, its structure, dynamics, lipid-binding properties, and cellular functions were analyzed by NMR and CD spectroscopy and by in vivo fluorescence microscopy. Moreover, the program CS-Rosetta was tested for the structure prediction based on chemical shift data only. The ForC GBD adopts an ubiquitin-like α/ß-roll fold with an unusually long loop between ß-strands 1 and 2. Based on the lipid-binding data, the presence of DPC micelles induces the formation of α-helical secondary structure and a rearrangement of the tertiary structure. Lipid-binding studies with a mutant protein and a peptide suggest that the ß1-ß2 loop is not relevant for these conformational changes. Whereas small amounts of negatively charged phosphoinositides (1,2-dioctanoyl-sn-glycero-3-(phosphoinositol 4,5-bisphosphate) and 1,2-dihexanoyl-sn-glycero-3-(phosphoinositol 3,4,5-trisphosphate)) lower the micelle concentration necessary to induce the observed spectral changes, other negatively charged phospholipids (1,2-dihexanoyl-sn-glycero-3-(phospho-L-serine) and 1,2-dihexanoyl-sn-glycero-3-phospho-(1'-rac-glycerol)) had no such effect. Interestingly, bicelles and micelles composed of diacylphosphocholines had no effect on the GBD structure. Our data suggest a model in which part of the large positively charged surface area of the GBD mediates localization to specific membrane patches, thereby regulating interactions with signaling proteins. Our cellular localization studies show that both the GBD and the FH3 domain are required for ForC targeting to cell-cell contacts and early phagocytic cups and macropinosomes.
Asunto(s)
Proteínas del Citoesqueleto/química , Dictyostelium/química , Lípidos/química , Micelas , Modelos Moleculares , Proteínas Protozoarias/química , Dicroismo Circular , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Dictyostelium/citología , Dictyostelium/genética , Dictyostelium/metabolismo , Lípidos/genética , Microscopía Fluorescente , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Relación Estructura-ActividadRESUMEN
Many ß-peptides fold in a 14-helical secondary structure in organic solvents, but similar 14-helix formation in water requires additional stabilizing elements. Especially the 14-helix stabilization of short ß-peptides in aqueous solution is critical, due to the limited freedom for incorporating stabilizing elements. Here we show how a single lactam bridge, connecting two ß-amino acid side-chains, can lead to high 14-helix character in short ß(3)-peptides in water. A comparative study, using CD and NMR spectroscopy and structure calculations, revealed the strong 14-helix inducing power of a side-chain-to-side-chain cyclization and its optimal position on the ß(3)-peptide scaffold with respect to pH and ionic strength effects. The lactam bridge is ideally incorporated in the N-terminal region of the ß(3)-peptide, where it limits the conformational flexibility of the peptide backbone. The lactam bridge induces a 14-helical conformation in methanol and water to a similar extent. Based on the presented first high resolution NMR 3D structure of a lactam bridged ß(3)-peptide, the fold shows a large degree of high order, both in the backbone and in the side-chains, leading to a highly compact and stable folded structure.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular , Oligopéptidos/química , Secuencia de Aminoácidos , Concentración de Iones de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Secundaria de ProteínaRESUMEN
The target of rapamycin (TOR) is a conserved eukaryotic Ser/Thr kinase that regulates cellular growth in response to the nutrient and energy state. TOR signaling plays an important role in the development of diseases such as cancer, obesity, and diabetes and in different redox-sensitive processes (hypoxia, apoptosis, and aging). Because TOR has been detected at different cellular membranes and in the nucleus, its localization may influence the specific signaling readout. To better understand how TOR can associate with different membranes, the lipid-binding properties of the redox-sensitive yeast TOR1 FATC domain (y1fatc) have been characterized by solution NMR spectroscopy. Binding studies with different lipids indicate that y1fatc interacts specifically with a membrane-mimetic environment but appears not to recognize a specific lipid headgroup. In both, the structures of oxidized and reduced micelle-bound y1fatc, residues Ile-2456 to Trp-2470 of the lipid-binding motif form a hydrophobic bulb that has a rim of charged residues. The diffusion constants for both micelle-bound states are consistent with the rotational correlation times from the analysis of the (15)N relaxation data. Based on the K(d) values, the oxidized form (K(d) approximately 0.31 mm) binds dodecyl phosphocholine micelles slightly tighter than the reduced form (K(d) approximately 1.86 mM). Binding studies with y1fatc in which one or both tryptophans (Trp-2466 and Trp-2470) were replaced by alanine suggest that these residues are important for the exact positioning in the membrane and that the other aromatic (His-2462, Tyr-2463, and Phe-2469) and aliphatic residues (Ile-2456, Leu-2459, Ile-2464, and Pro-2468) in the lipid-binding motif contribute significantly to the affinity.
Asunto(s)
Micelas , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Oxidación-Reducción , Fosfatidilinositol 3-Quinasas/genética , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/genética , Alineación de SecuenciaRESUMEN
Transcription elongation is regulated by the cellular protein Hexim1, which inhibits phosphorylation of RNA polymerase II by interacting with the positive transcription elongation factor P-TEFb. Hexim1 binds directly to Cyclin T1 of P-TEFb with its coiled coil domain that is subdivided into a highly polar N-terminal segment containing nonconservative residues in the dimer interface and a C-terminal segment with an evolutionarily conserved sequence composition. Here we show that the noncanonical sequence composition of the first coiled coil segment is required for the interaction with Cyclin T1 while the second segment keeps the Cyclin T-binding domain dimeric upon binding. Both coiled coil segments exhibit distinct melting points as shown by heat denaturation experiments using circular dichroism spectroscopy. Deletion of the central stammer motif (Delta316-318) leads to a single denaturation reaction, suggesting formation of a continuous coiled coil. Mutation of noncanonical coiled coil residues K284 and Y291 to valines in the dimer interface of the first segment only slightly increases its stability. Concomitantly, deletion of the stammer but not the double point mutation led to a reduced affinity for Cyclin T1 as shown by isothermal titration calorimetry. Moreover, Cyclin T1 bound Hexim1 with a 1:2 stoichiometry, whereas truncation of the C-terminal coiled coil led to formation of an equimolar complex. These observations suggest that binding to Cyclin T1 induces an asymmetry or sterical hindrance in the first coiled coil segment of dimeric Hexim1 that disallows formation of a 2:2 complex as further supported by analytical ultracentrifugation and cross-linking experiments.
Asunto(s)
Ciclina T/química , Factor B de Elongación Transcripcional Positiva/química , Proteínas de Unión al ARN/química , Sitios de Unión , Dicroismo Circular , Reactivos de Enlaces Cruzados/química , Humanos , Modelos Moleculares , Complejos Multiproteicos , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Termodinámica , Factores de Transcripción , Temperatura de Transición , UltracentrifugaciónRESUMEN
Mammalian target of rapamycin (TOR) controls cell growth and metabolism in response to the availability of nutrients, growth factors, and the cellular energy status. Misregulation of TOR can result in a pathogenic increase or decrease in organ size and in cancer. TOR can be inhibited by binding of a complex of rapamycin and FKBP to the FKBP-rapamycin binding (FRB) domain. Rapamycin and derivatives of it have been used as immunosuppressive drugs. Because TOR is further an interesting drug target in cancer research, we established an expression, purification, and refolding protocol for the FRB domain of human TOR (hFRB). hFRB is extracted from inclusion bodies, purified by reversed phase HPLC, and refolded by drop-wise dilution of the denatured protein into a native buffer. The procedure is very simple and can easily be scaled up to prepare large amounts of functional protein for high-throughput cancer drug screening assays by NMR and other techniques.
Asunto(s)
Proteínas Portadoras/aislamiento & purificación , Fosfotransferasas (Aceptor de Grupo Alcohol)/aislamiento & purificación , Proteínas Quinasas/aislamiento & purificación , Factores de Transcripción/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos , Resonancia Magnética Nuclear Biomolecular , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas , Serina-Treonina Quinasas TORRESUMEN
Ras homolog enriched in brain (Rheb) is a small GTPase that regulates mammalian/mechanistic target of rapamycin complex 1 (mTORC1) and, thereby, cell growth and metabolism. Here we show that cycling between the inactive GDP- and the active GTP-bound state modulates the backbone dynamics of a C-terminal truncated form, RhebΔCT, which is suggested to influence its interactions. We further investigated the interactions between RhebΔCT and the proposed Rheb-binding domain of the regulatory protein FKBP38. The observed weak interactions with the GTP-analogue- (GppNHp-) but not the GDP-bound state, appear to accelerate the GDP to GTP exchange, but only very weakly compared to a genuine GEF. Thus, FKBP38 is most likely not a GEF but a Rheb effector that may function in membrane targeting of Rheb.
Asunto(s)
Proteína Homóloga de Ras Enriquecida en el Cerebro/química , Proteínas de Unión a Tacrolimus/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Simulación de Dinámica Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteína Homóloga de Ras Enriquecida en el Cerebro/genética , Proteína Homóloga de Ras Enriquecida en el Cerebro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/metabolismoRESUMEN
Increased efforts have been undertaken to better understand the formation of signaling complexes at cellular membranes. Since the preparation of proteins containing a transmembrane domain or a prenylation motif is generally challenging an alternative membrane anchoring unit that is easy to attach, water-soluble and binds to different membrane mimetics would find broad application. The 33-residue long FATC domain of yeast TOR1 (y1fatc) fulfills these criteria and binds to neutral and negatively charged micelles, bicelles, and liposomes. As a case study, we fused it to the FKBP506-binding region of the protein FKBP38 (FKBP38-BD) and used 1 H-15 N NMR spectroscopy to characterize localization of the chimeric protein to micelles, bicelles, and liposomes. Based on these and published data for y1fatc, its use as a C-terminally attachable membrane anchor for other proteins is compatible with a wide range of buffer conditions (pH circa 6-8.5, NaCl 0 to >150 mM, presence of reducing agents, different salts such as MgCl2 and CaCl2 ). The high water-solubility of y1fatc enables its use for titration experiments against a membrane-localized interaction partner of the fused target protein. Results from studies with peptides corresponding to the C-terminal 17-11 residues of the 33-residue long domain by 1D 1 H NMR and CD spectroscopy indicate that they still can interact with membrane mimetics. Thus, they may be used as membrane anchors if the full y1fatc sequence is disturbing or if a chemically synthesized y1fatc peptide shall be attached by native chemical ligation, for example, unlabeled peptide to 15 N-labeled target protein for NMR studies.
Asunto(s)
Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Unión a Tacrolimus/química , Proteínas de Unión a Tacrolimus/metabolismo , Membrana Celular/metabolismo , Dicroismo Circular , Humanos , Liposomas/metabolismo , Micelas , Modelos Moleculares , Simulación de Dinámica Molecular , Mutación , Resonancia Magnética Nuclear Biomolecular , Fosfatidilinositol 3-Quinasas/genética , Conformación Proteica , Dominios Proteicos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Unión a Tacrolimus/genéticaRESUMEN
FAT C-terminal (FATC) is a circa 33 residue-long domain. It controls the kinase functionality in phosphatidylinositol-3 kinase-related kinases (PIKKs). Recent NMR- and CD-monitored interaction studies indicated that the FATC domains of all PIKKs can interact with membrane mimetics albeit with different preferences for membrane properties such as surface charge and curvature. Thus they may generally act as membrane anchoring unit. Here, we present the 1H, 15N, and 13C chemical shift assignments of the DPC micelle immersed FATC domains of the human PIKKs ataxia-telangiectasia mutated (ATM, residues 3024-3056) and DNA protein kinase catalytic subunit (DNA-PKcs, residues 4096-4128), both fused to the 56 residue long B1 domain of Streptococcal protein G (GB1). Each fusion protein is 100 amino acids long and contains in the linking region between the GB1 tag and the FATC region a thrombin (LVPRGS) and an enterokinase (DDDDK) protease site. The assignments pave the route for the detailed structural characterization of the membrane mimetic bound states, which will help to better understand the role of the proper cellular localization at membranes for the function and regulation of PIKKs. The chemical shift assignment of the GB1 tag is useful for NMR spectroscopists developing new experiments or using GB1 otherwise for case studies in the field of in-cell NMR spectroscopy or protein folding. Moreover it is often used as purification tag. Earlier we showed already that GB1 does not interact with membrane mimetics and thus does not disturb the NMR monitoring of membrane mimetic interactions of attached proteins.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/química , Proteínas Bacterianas/química , Micelas , Resonancia Magnética Nuclear Biomolecular , Proteínas Quinasas/química , Proteínas Recombinantes de Fusión/química , Humanos , Dominios ProteicosRESUMEN
Mycobacterium tuberculosis protein kinase G (PknG) is a 82 kDa multidomain eukaryotic-like serine/threonine kinase mediating the survival of pathogenic mycobacteria within host macrophages. The N-terminal sequence preceding the catalytic kinase domain contains an approximately 75 residues long tail, which was predicted to show no regulatory secondary structure (1-75 = NORS) but harbors the major in vivo phosphorylation site (T63), and a rubredoxin-like metal binding motif (74-147 = RD). In the reduced rubredoxin motif, four conserved cysteine residues that are present as two C-X-X-C-G motifs coordinate a metal ion. The cysteines are further involved in sensing the redox environment to regulate PknG catalytic activity. Here, we report the (1)H, (13)C, and (15)N resonance assignments of the highly dynamic unstructured N-terminal region NORS and the RD in the reduced, metal bound, presumably folded and the oxidized, presumably unfolded state. Chemical shifts have been deposited at the BioMagResBank under the BMRB accession numbers 26,028 for the His-PknG1-147 with the RD in reduced, metal bound state, 26,027 for His-PknG1-75, and 26,030 and 26,029 for PknG74-147 either in the reduced, metal bound or oxidized state, respectively. The presented chemical shift assignments pave the route for the structural characterization of the regulation of PknG by redox changes and posttranslational modifications (phosphorylation).
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/química , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Metales/metabolismo , Mycobacterium tuberculosis/enzimología , Resonancia Magnética Nuclear Biomolecular , Desplegamiento Proteico , Rubredoxinas/metabolismo , Oxidación-Reducción , Dominios ProteicosRESUMEN
Flexible polypeptides such as unfolded proteins may access an astronomical number of conformations. The most advanced simulations of such states usually comprise tens of thousands of individual structures. In principle, a comparison of parameters predicted from such ensembles to experimental data provides a measure of their quality. In practice, analyses that go beyond the comparison of unbiased average data have been impossible to carry out on the entirety of such very large ensembles and have, therefore, been restricted to much smaller subensembles and/or nondeterministic algorithms. Here, we show that such very large ensembles, on the order of 10(4) to 10(5) conformations, can be analyzed in full by a maximum entropy fit to experimental average data. Maximizing the entropy of the population weights of individual conformations under experimental χ(2) constraints is a convex optimization problem, which can be solved in a very efficient and robust manner to a unique global solution even for very large ensembles. Since the population weights can be determined reliably, the reweighted full ensemble presents the best model of the combined information from simulation and experiment. Furthermore, since the reduction of entropy due to the experimental constraints is well-defined, its value provides a robust measure of the information content of the experimental data relative to the simulated ensemble and an indication for the density of the sampling of conformational space. The method is applied to the reweighting of a 35,000 frame molecular dynamics trajectory of the nonapeptide EGAAWAASS by extensive NMR (3)J coupling and RDC data. The analysis shows that RDCs provide significantly more information than (3)J couplings and that a discontinuity in the RDC pattern at the central tryptophan is caused by a cluster of helical conformations. Reweighting factors are moderate and consistent with errors in MD force fields of less than 3kT. The required reweighting is larger for an ensemble derived from a statistical coil model, consistent with its coarser nature. We call the method COPER, for convex optimization for ensemble reweighting. Similar advantages of large-scale efficiency and robustness can be obtained for other ensemble analysis methods with convex targets and constraints, such as constrained χ(2) minimization and the maximum occurrence method.
Asunto(s)
Algoritmos , Péptidos/química , Secuencia de Aminoácidos , Análisis por Conglomerados , Entropía , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Oligopéptidos/química , Oligopéptidos/metabolismo , Péptidos/metabolismoRESUMEN
Phosphatidylinositol 3-kinase-related kinases (PIKKs) play vital roles in the regulation of cell growth, proliferation, survival, and consequently metabolism, as well as in the cellular response to stresses such as ionizing radiation or redox changes. In humans six family members are known to date, namely mammalian/mechanistic target of rapamycin (mTOR), ataxia-telangiectasia mutated (ATM), ataxia- and Rad3-related (ATR), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), suppressor of morphogenesis in genitalia-1 (SMG-1), and transformation/transcription domain-associated protein (TRRAP). All fulfill rather diverse functions and most of them have been detected in different cellular compartments including various cellular membranes. It has been suggested that the regulation of the localization of signaling proteins allows for generating a locally specific output. Moreover, spatial partitioning is expected to improve the reliability of biochemical signaling. Since these assumptions may also be true for the regulation of PIKK function, the current knowledge about the regulation of the localization of PIKKs at different cellular (membrane) compartments by a network of interactions is reviewed. Membrane targeting can involve direct lipid-/membrane interactions as well as interactions with membrane-anchored regulatory proteins, such as, for example, small GTPases, or a combination of both.
RESUMEN
The estrogen receptor (ER) is the number one target for the treatment of endocrine responsive breast cancer and remains a highly attractive target for new drug development. Despite considerable efforts to understand the role of ER post-translational modifications (PTMs), the complexity of these modifications and their impact, at the molecular level, are poorly understood. Using a chemical biology approach, fundamentally rooted in an efficient protein semisynthesis of tyrosine phosphorylated ER constructs, the complex role of the ER tyrosine phosphorylation is addressed here for the first time on a molecular level. The semisynthetic approach allows for the site-specific introduction of PTMs as well as biophysical probes. A combination of biophysical techniques, including NMR, with molecular dynamics studies reveals the role of the phosphorylation of the clinically relevant tyrosine 537 (Y537) in ERα and the analogous tyrosine (Y488) in ERß. Phosphorylation has important effects on the dynamics of the ER Helix 12, which is centrally involved in receptor activity regulation, and on its interplay with ligand and cofactor binding, but with differential regulatory effects of the analogous PTMs on the two ER subtypes. Combined, the results bring forward a novel molecular model of a phosphorylation-induced subtype specific ER modulatory mechanism, alternative to the widely accepted ligand-induced activation mechanism.
Asunto(s)
Coactivadores de Receptor Nuclear/metabolismo , Receptores de Estrógenos/metabolismo , Sitios de Unión , Modelos Moleculares , Coactivadores de Receptor Nuclear/química , Fosforilación , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Receptores de Estrógenos/químicaRESUMEN
The conserved C-terminal FATC domain of the kinase 'target of rapamycin' is important for its regulation and was suggested to contain a peripheral membrane anchor. Here, we present the characterization of the interactions of the yeast TOR1 FATC domain (2438-2470=y1fatc) and 15 mutants with membrane mimetic micelles, bicelles, and small unilamellar vesicles (SUVs) by NMR and CD spectroscopy. Replacement of up to 6-7 residues did not result in a significant abrogation of the association with micelles or bicelles. However, replacement of only one residue could result in an impairment of the interaction with SUVs that are usually used at low concentrations. Some mutants not binding liposomes may be introduced in full-length TOR for future functional and localization studies in vivo.