RESUMO
The RNA genome of SARS-CoV-2 contains a 5' cap that facilitates the translation of viral proteins, protection from exonucleases and evasion of the host immune response1-4. How this cap is made in SARS-CoV-2 is not completely understood. Here we reconstitute the N7- and 2'-O-methylated SARS-CoV-2 RNA cap (7MeGpppA2'-O-Me) using virally encoded non-structural proteins (nsps). We show that the kinase-like nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain5 of nsp12 transfers the RNA to the amino terminus of nsp9, forming a covalent RNA-protein intermediate (a process termed RNAylation). Subsequently, the NiRAN domain transfers the RNA to GDP, forming the core cap structure GpppA-RNA. The nsp146 and nsp167 methyltransferases then add methyl groups to form functional cap structures. Structural analyses of the replication-transcription complex bound to nsp9 identified key interactions that mediate the capping reaction. Furthermore, we demonstrate in a reverse genetics system8 that the N terminus of nsp9 and the kinase-like active-site residues in the NiRAN domain are required for successful SARS-CoV-2 replication. Collectively, our results reveal an unconventional mechanism by which SARS-CoV-2 caps its RNA genome, thus exposing a new target in the development of antivirals to treat COVID-19.
Assuntos
Capuzes de RNA , RNA Viral , SARS-CoV-2 , Proteínas Virais , Antivirais , COVID-19/virologia , Domínio Catalítico , Guanosina Difosfato/metabolismo , Humanos , Metiltransferases/metabolismo , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Domínios Proteicos , Capuzes de RNA/química , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Tratamento Farmacológico da COVID-19RESUMO
Daptomycin is a cyclic lipodepsipeptide antibiotic used to treat infections caused by Gram-positive pathogens, including multi-drug resistant strains such as methicillin-resistant Staphylococcus au-reus (MRSA) and vancomycin-resistant enterococci (VRE). The emergence of daptomycin-resistant bacterial strains has renewed interest in generating daptomycin analogs. Previous studies have shown that replacing the tryptophan of daptomycin with aromatic groups can generate analogs with enhanced potency. Additionally, we have demonstrated that aromatic prenyltransferases can attach diverse groups to the tryptophan of daptomycin. Here, we report the use of the prenyltransferase CdpNPT to derivatize the tryptophan of daptomycin with a library of benzylic and heterocyclic pyrophosphates. An analytical-scale study revealed that CdpNPT can transfer various aromatic groups onto daptomycin. Subsequent scaled-up and purified reactions indicated that the enzyme can attach aromatic groups to N1, C2, C5 and C6 positions of Trp1 of daptomycin. In vitro antibacterial activity assays using six of these purified compounds identified aromatic substituted daptomycin analogs show potency against both daptomycin-susceptible and resistant strains of Gram-positive bacteria. These findings suggest that installing aromatic groups on the Trp1 of daptomycin can lead to the generation of potent daptomycin analogs.
RESUMO
Ykt6 is a soluble N-ethylmaleimide sensitive factor activating protein receptor (SNARE) critically involved in diverse vesicular fusion pathways. While most SNAREs rely on transmembrane domains for their activity, Ykt6 dynamically cycles between the cytosol and membrane-bound compartments where it is active. The mechanism that regulates these transitions and allows Ykt6 to achieve specificity toward vesicular pathways is unknown. Using a Parkinson's disease (PD) model, we found that Ykt6 is phosphorylated at an evolutionarily conserved site which is regulated by Ca2+ signaling. Through a multidisciplinary approach, we show that phosphorylation triggers a conformational change that allows Ykt6 to switch from a closed cytosolic to an open membrane-bound form. In the phosphorylated open form, the spectrum of protein interactions changes, leading to defects in both the secretory and autophagy pathways, enhancing toxicity in PD models. Our studies reveal a mechanism by which Ykt6 conformation and activity are regulated with potential implications for PD.
Assuntos
Sequência Conservada , Modelos Moleculares , Conformação Proteica , Proteínas R-SNARE/química , Proteínas R-SNARE/metabolismo , Aminoácidos , Autofagia , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Evolução Molecular , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas R-SNARE/genética , Relação Estrutura-AtividadeRESUMO
Chemically labile ester linkages can be introduced into lignin by incorporation of monolignol conjugates, which are synthesized in planta by acyltransferases that use a coenzymeâ A (CoA) thioester donor and a nucleophilic monolignol alcohol acceptor. The presence of these esters facilitates processing and aids in the valorization of renewable biomass feedstocks. However, the effectiveness of this strategy is potentially limited by the low steady-state levels of aromatic acid thioester donors in plants. As part of an effort to overcome this, aromatic acid CoA ligases involved in microbial aromatic degradation were identified and screened against a broad panel of substituted cinnamic and benzoic acids involved in plant lignification. Functional fingerprinting of this ligase library identified four robust, highly active enzymes capable of facile, rapid, and high-yield synthesis of aromatic acid CoA thioesters under mild aqueous reaction conditions mimicking in planta activity.
Assuntos
Coenzima A Ligases , Ligases , Coenzima A Ligases/metabolismo , Lignina/metabolismo , Plantas/metabolismo , ÉsteresRESUMO
The small GTPase KRAS is localized at the plasma membrane where it functions as a molecular switch, coupling extracellular growth factor stimulation to intracellular signaling networks. In this process, KRAS recruits effectors, such as RAF kinase, to the plasma membrane where they are activated by a series of complex molecular steps. Defining the membrane-bound state of KRAS is fundamental to understanding the activation of RAF kinase and in evaluating novel therapeutic opportunities for the inhibition of oncogenic KRAS-mediated signaling. We combined multiple biophysical measurements and computational methodologies to generate a consensus model for authentically processed, membrane-anchored KRAS. In contrast to the two membrane-proximal conformations previously reported, we identify a third significantly populated state using a combination of neutron reflectivity, fast photochemical oxidation of proteins (FPOP), and NMR. In this highly populated state, which we refer to as "membrane-distal" and estimate to comprise â¼90% of the ensemble, the G-domain does not directly contact the membrane but is tethered via its C-terminal hypervariable region and carboxymethylated farnesyl moiety, as shown by FPOP. Subsequent interaction of the RAF1 RAS binding domain with KRAS does not significantly change G-domain configurations on the membrane but affects their relative populations. Overall, our results are consistent with a directional fly-casting mechanism for KRAS, in which the membrane-distal state of the G-domain can effectively recruit RAF kinase from the cytoplasm for activation at the membrane.
Assuntos
Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Quinases raf/metabolismo , Membrana Celular/metabolismo , Simulação de Dinâmica MolecularRESUMO
NMR spectroscopy is well known for its superb resolution, especially at high applied magnetic field. However, the sensitivity of this technique is very low. Liquid-state low-concentration photo-chemically-induced dynamic nuclear polarization (LC-photo-CIDNP) is a promising emerging methodology capable of enhancing NMR sensitivity in solution. LC-photo-CIDNP works well on solvent-exposed Trp and Tyr residues, either in isolation or within proteins. This study explores the magnetic-field dependence of the LC-photo-CIDNP experienced by two tryptophan isotopologs in solution upon in situ LED-mediated optical irradiation. Out of the two uniformly 13C,15N-labeled Trp (Trp-U-13C,15N) and Trp-α-13C-ß,ß,2,4,5,6,7-d7 species employed here, only the latter bears a quasi-isolated 1Hα-13Cα spin pair. Computer simulations of the predicted polarization due to geminate recombination of both species display a roughly bell-shaped field dependence. However, while Trp-U-13C,15N is predicted to show a maximum at ca. 500 MHz (11.7 T) and a fairly weak field dependence, Trp-α-13C-ß,ß,2,4,5,6,7-d7 is expected to display a much sharper field dependence accompanied by a dramatic polarization increase at lower field (ca. 200 MHz, 4.7 T). Experimental LC-photo-CIDNP studies on both Trp isotopologs at 1µM concentration, performed at selected fields, are consistent with the theoretical predictions. In summary, this study highlights the prominent field-dependence of LC-photo-CIDNP enhancements (ε) experienced by Trp isotopologs bearing a quasi-isolated spin pair.
RESUMO
During the activation of mitogen-activated protein kinase (MAPK) signaling, the RAS-binding domain (RBD) and cysteine-rich domain (CRD) of RAF bind to active RAS at the plasma membrane. The orientation of RAS at the membrane may be critical for formation of the RAS-RBDCRD complex and subsequent signaling. To explore how RAS membrane orientation relates to the protein dynamics within the RAS-RBDCRD complex, we perform multiscale coarse-grained and all-atom molecular dynamics (MD) simulations of KRAS4b bound to the RBD and CRD domains of RAF-1, both in solution and anchored to a model plasma membrane. Solution MD simulations describe dynamic KRAS4b-CRD conformations, suggesting that the CRD has sufficient flexibility in this environment to substantially change its binding interface with KRAS4b. In contrast, when the ternary complex is anchored to the membrane, the mobility of the CRD relative to KRAS4b is restricted, resulting in fewer distinct KRAS4b-CRD conformations. These simulations implicate membrane orientations of the ternary complex that are consistent with NMR measurements. While a crystal structure-like conformation is observed in both solution and membrane simulations, a particular intermolecular rearrangement of the ternary complex is observed only when it is anchored to the membrane. This configuration emerges when the CRD hydrophobic loops are inserted into the membrane and helices α3-5 of KRAS4b are solvent exposed. This membrane-specific configuration is stabilized by KRAS4b-CRD contacts that are not observed in the crystal structure. These results suggest modulatory interplay between the CRD and plasma membrane that correlate with RAS/RAF complex structure and dynamics, and potentially influence subsequent steps in the activation of MAPK signaling.
Assuntos
Cisteína , Proteínas Proto-Oncogênicas c-raf , Sítios de Ligação , Membrana Celular/metabolismo , Cisteína/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Solventes/metabolismoRESUMO
ATP7A and ATP7B are structurally similar but functionally distinct active copper transporters that regulate copper levels in the human cells and deliver copper to the biosynthetic pathways. Both proteins have a chain of six cytosolic metal-binding domains (MBDs) believed to be involved in the copper-dependent regulation of the activity and intracellular localization of these enzymes. Although all the MBDs are quite similar in structure, their spacing differs markedly between ATP7A and ATP7B. We show by NMR that the long polypeptide between MBD1 and MBD2 of ATP7A forms an additional seventh metastable domain, which we called HMA1A (heavy metal associated domain 1A). The structure of HMA1A resembles the MBDs but contains no copper-binding site. The HMA1A domain, which is unique to ATP7A, may modulate regulatory interactions between MBD1-3, contributing to the distinct functional properties of ATP7A and ATP7B.
Assuntos
ATPases Transportadoras de Cobre , Cobre , Sítios de Ligação , Cobre/metabolismo , ATPases Transportadoras de Cobre/genética , Humanos , Domínios ProteicosRESUMO
Using atomic force microscopy (AFM) and nuclear magnetic resonance (NMR), we describe small Aß40 oligomers, termed nanodroplet oligomers (NanDOs), which form rapidly and at Aß40 concentrations too low for fibril formation. NanDOs were observed in putatively monomeric solutions of Aß40 (e.g., by size exclusion chromatography). Video-rate scanning AFM shows rapid fusion and dissolution of small oligomer-sized particles, of which the median size increases with peptide concentration. In NMR (13C HSQC), a small number of chemical shifts changed with a change in peptide concentration. Paramagnetic relaxation enhancement NMR experiments also support the formation of NanDOs and suggest prominent interactions in hydrophobic domains of Aß40. Addition of Zn2+ to Aß40 solutions caused flocculation of NanDO-containing solutions, and selective loss of signal intensity in NMR spectra from residues in the N-terminal domain of Aß40. NanDOs may represent the earliest aggregated form of Aß40 in the aggregation pathway and are akin to premicelles in solutions of amphiphilies.
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Espectroscopia de Ressonância Magnética/métodos , Microscopia de Força Atômica/métodos , Nanopartículas/química , Agregados Proteicos/fisiologia , Doença de Alzheimer/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica/métodos , HumanosRESUMO
Calorie restriction (CR) extends life span in diverse species. Mitochondria play a key role in CR adaptation; however, the molecular details remain elusive. We developed and applied a quantitative mass spectrometry method to probe the liver mitochondrial acetyl-proteome during CR versus control diet in mice that were wild-type or lacked the protein deacetylase SIRT3. Quantification of 3,285 acetylation sites-2,193 from mitochondrial proteins-rendered a comprehensive atlas of the acetyl-proteome and enabled global site-specific, relative acetyl occupancy measurements between all four experimental conditions. Bioinformatic and biochemical analyses provided additional support for the effects of specific acetylation on mitochondrial protein function. Our results (1) reveal widespread reprogramming of mitochondrial protein acetylation in response to CR and SIRT3, (2) identify three biochemically distinct classes of acetylation sites, and (3) provide evidence that SIRT3 is a prominent regulator in CR adaptation by coordinately deacetylating proteins involved in diverse pathways of metabolism and mitochondrial maintenance.
Assuntos
Restrição Calórica , Proteínas Mitocondriais/metabolismo , Proteoma/metabolismo , Sirtuína 3/fisiologia , Acetilcoenzima A/metabolismo , Acetilação , Adaptação Fisiológica , Motivos de Aminoácidos , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Metabolismo dos Carboidratos , Células Cultivadas , Cromatografia por Troca Iônica , Análise por Conglomerados , Sequência Consenso , Expressão Gênica , Genes Mitocondriais , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/isolamento & purificação , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Processamento de Proteína Pós-Traducional , Proteoma/química , Proteoma/isolamento & purificação , Sirtuína 3/química , Sirtuína 3/isolamento & purificação , Sirtuína 3/metabolismo , Coloração e Rotulagem , Espectrometria de Massas em TandemRESUMO
The ATPase Family, AAA domain-containing protein 2 (ATAD2) bromodomain (BRD) has a canonical bromodomain structure consisting of four α-helices. ATAD2 functions as a co-activator of the androgen and estrogen receptors as well as the MYC and E2F transcription factors. ATAD2 also functions during DNA replication, recognizing newly synthesized histones. In addition, ATAD2 is shown to be up-regulated in multiple forms of cancer including breast, lung, gastric, endometrial, renal, and prostate. Furthermore, up-regulation of ATAD2 is strongly correlated with poor prognosis in many types of cancer, making the ATAD2 bromodomain an innovative target for cancer therapeutics. In this study, we describe the recognition of histone acetyllysine modifications by the ATAD2 bromodomain. Residue-specific information on the complex formed between the histone tail and the ATAD2 bromodomain, obtained through nuclear magnetic resonance spectroscopy (NMR) and X-ray crystallography, illustrates key residues lining the binding pocket, which are involved in coordination of di-acetylated histone tails. Analytical ultracentrifugation, NMR relaxation data, and isothermal titration calorimetry further confirm the monomeric state of the functionally active ATAD2 bromodomain in complex with di-acetylated histone ligands. Overall, we describe histone tail recognition by ATAD2 BRD and illustrate that one acetyllysine group is primarily engaged by the conserved asparagine (N1064), the "RVF" shelf residues, and the flexible ZA loop. Coordination of a second acetyllysine group also occurs within the same binding pocket but is essentially governed by unique hydrophobic and electrostatic interactions making the di-acetyllysine histone coordination more specific than previously presumed.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/química , Proteínas de Ligação a DNA/química , Histonas/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Acetilação , Proteínas de Ligação a DNA/metabolismo , Código das Histonas , Histonas/química , Humanos , Ligação Proteica , Domínios ProteicosRESUMO
There are few methods available for the rapid discovery of multitarget drugs. Herein, we describe the template-assisted, target-guided discovery of small molecules that recognize d(CTG) in the expanded d(CTG·CAG) sequence and its r(CUG) transcript that cause myotonic dystrophy type 1. A positive cross-selection was performed using a small library of 30 monomeric alkyne- and azide-containing ligands capable of producing >5000 possible di- and trimeric click products. The monomers were incubated with d(CTG)16 or r(CUG)16 under physiological conditions, and both sequences showed selectivity in the proximity-accelerated azide-alkyne [3+2] cycloaddition click reaction. The limited number of click products formed in both selections and the even smaller number of common products suggests that this method is a useful tool for the discovery of single-target and multitarget lead therapeutic agents.
Assuntos
DNA/antagonistas & inibidores , Distrofia Miotônica/tratamento farmacológico , Distrofia Miotônica/genética , RNA/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Expansão das Repetições de Trinucleotídeos/efeitos dos fármacos , Células Cultivadas , DNA/genética , DNA/metabolismo , Humanos , Distrofia Miotônica/patologia , RNA/genética , RNA/metabolismo , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
We report the solution-phase structures of native signal peptides and related analogs capable of either strongly agonizing or antagonizing the AgrC quorum sensing (QS) receptor in the emerging pathogen Staphylococcus epidermidis. Chronic S. epidermidis infections are often recalcitrant to traditional therapies due to antibiotic resistance and formation of robust biofilms. The accessory gene regulator (agr) QS system plays an important role in biofilm formation in this opportunistic pathogen, and the binding of an autoinducing peptide (AIP) signal to its cognate transmembrane receptor (AgrC) is responsible for controlling agr. Small molecules or peptides capable of modulating this binding event are of significant interest as probes to investigate both the agr system and QS as a potential antivirulence target. We used NMR spectroscopy to characterize the structures of the three native S. epidermidis AIP signals and five non-native analogs with distinct activity profiles in the AgrC-I receptor from S. epidermidis. These studies revealed a suite of structural motifs critical for ligand activity. Interestingly, a unique ß-turn was present in the macrocycles of the two most potent AgrC-I modulators, in both an agonist and an antagonist, which was distinct from the macrocycle conformation in the less-potent AgrC-I modulators and in the native AIP-I itself. This previously unknown ß-turn provides a structural rationale for these ligands' respective biological activity profiles. Development of analogs to reinforce the ß-turn resulted in our first antagonist with subnanomolar potency in AgrC-I, while analogs designed to contain a disrupted ß-turn were dramatically less potent relative to their parent compounds. Collectively, these studies provide new insights into the AIP:AgrC interactions crucial for QS activation in S. epidermidis and advance the understanding of QS at the molecular level.
Assuntos
Sinais Direcionadores de Proteínas/fisiologia , Percepção de Quorum , Staphylococcus epidermidis/fisiologiaRESUMO
Objectives: A milder clinical course of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been anecdotally reported over the latest phase of COVID-19 pandemic in Italy. Several factors may contribute to this observation, including the effect of lockdown, social distancing, lower humidity, lower air pollution, and potential changes in the intrinsic pathogenicity of the virus. In this regard, the clinical severity of COVID-19 could be attenuated by mutations in SARS-CoV-2 genome that decrease its virulence, as well as by lower virus inocula. Methods: In this pilot study, we compared the reverse transcription polymerase chain reaction (RT-PCR) amplification profile of 100 nasopharyngeal swabs consecutively collected in April, during the peak of SARS-CoV-2 epidemic, to that of 100 swabs collected using the same procedure in May. Results: The mean Ct value of positive samples collected in May was significantly higher than that of samples collected in the previous period (ORF 1a/b gene: 31.85 ± 0.32 vs. 28.37 ± 0.5, p<0.001; E gene: 33.76 ± 0.38 vs. 29.79 ± 0.63, p<0.001), suggesting a lower viral load at the time of sampling. No significant differences were observed between male and females in the two periods, whilst higher viral loads were found in (i) patients over 60-years old, and (ii) patients that experienced severe COVID-19 during the early stages of the pandemic. Conclusions: This pilot study prompts further investigation on the correlation between SARS-CoV-2 load and different clinical manifestation of COVID-19 during different phases of the pandemic. Laboratories should consider reporting quantitative viral load data in the molecular diagnosis of SARS-CoV-2 infection.
Assuntos
Betacoronavirus , Infecções por Coronavirus/virologia , Nasofaringe/virologia , Pneumonia Viral/virologia , Carga Viral , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , COVID-19 , Teste para COVID-19 , Criança , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Feminino , Hospitais Universitários , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Pandemias , Projetos Piloto , Pneumonia Viral/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2 , Adulto JovemRESUMO
Daptomycin is a last resort antibiotic for the treatment of infections caused by many Gram-positive bacterial strains, including vancomycin-resistant Enterococcus (VRE) and methicillin- and vancomycin-resistant Staphylococcus aureus (MRSA and VRSA). However, the emergence of daptomycin-resistant strains of S. aureus and Enterococcus in recent years has renewed interest in synthesizing daptomycin analogs to overcome resistance mechanisms. Within this context, three aromatic prenyltransferases have been shown to accept daptomycin as a substrate, and the resulting prenylated analog was shown to be more potent against Gram-positive strains than the parent compound. Consequently, utilizing prenyltransferases to derivatize daptomycin offered an attractive alternative to traditional synthetic approaches, especially given the molecule's structural complexity. Herein, we report exploiting the ability of prenyltransferase CdpNPT to synthesize alkyl-diversified daptomycin analogs in combination with a library of synthetic non-native alkyl-pyrophosphates. The results revealed that CdpNPT can transfer a variety of alkyl groups onto daptomycin's tryptophan residue using the corresponding alkyl-pyrophosphates, while subsequent scaled-up reactions suggested that the enzyme can alkylate the N1, C2, C5, and C6 positions of the indole ring. In vitro antibacterial activity assays using 16 daptomycin analogs revealed that some of the analogs displayed 2-80-fold improvements in potency against MRSA, VRE, and daptomycin-resistant strains of S. aureus and Enterococcus faecalis. Thus, along with the new potent analogs, these findings have established that the regio-chemistry of alkyl substitution on the tryptophan residue can modulate daptomycin's potency. With additional protein engineering to improve the regio-selectivity, the described method has the potential to become a powerful tool for diversifying complex indole-containing molecules. KEY POINTS: ⢠CdpNPT displays impressive donor promiscuity with daptomycin as the acceptor. ⢠CdpNPT catalyzes N1-, C2-, C5-, and C6-alkylation on daptomycin's tryptophan residue. ⢠Differential alkylation of daptomycin's tryptophan residue modulates its activity.
Assuntos
Daptomicina , Staphylococcus aureus Resistente à Meticilina , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Daptomicina/farmacologia , Testes de Sensibilidade Microbiana , Staphylococcus aureus , VancomicinaRESUMO
By binding to a multitude of polypeptide substrates, Hsp70-based molecular chaperone systems perform a range of cellular functions. All J-protein co-chaperones play the essential role, via action of their J-domains, of stimulating the ATPase activity of Hsp70, thereby stabilizing its interaction with substrate. In addition, J-proteins drive the functional diversity of Hsp70 chaperone systems through action of regions outside their J-domains. Targeting to specific locations within a cellular compartment and binding of specific substrates for delivery to Hsp70 have been identified as modes of J-protein specialization. To better understand J-protein specialization, we concentrated on Saccharomyces cerevisiae SIS1, which encodes an essential J-protein of the cytosol/nucleus. We selected suppressors that allowed cells lacking SIS1 to form colonies. Substitutions changing single residues in Ydj1, a J-protein, which, like Sis1, partners with Hsp70 Ssa1, were isolated. These gain-of-function substitutions were located at the end of the J-domain, suggesting that suppression was connected to interaction with its partner Hsp70, rather than substrate binding or subcellular localization. Reasoning that, if YDJ1 suppressors affect Ssa1 function, substitutions in Hsp70 itself might also be able to overcome the cellular requirement for Sis1, we carried out a selection for SSA1 suppressor mutations. Suppressing substitutions were isolated that altered sites in Ssa1 affecting the cycle of substrate interaction. Together, our results point to a third, additional means by which J-proteins can drive Hsp70's ability to function in a wide range of cellular processes-modulating the Hsp70-substrate interaction cycle.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/metabolismo , Núcleo Celular/metabolismo , Citosol/metabolismo , Ligação Proteica/fisiologia , Domínios ProteicosRESUMO
Deregulation of KRAS4b signaling pathway has been implicated in 30% of all cancers. Membrane localization of KRAS4b is an essential step for the initiation of the downstream signaling cascades that guide various cellular mechanisms. KRAS4b plasma membrane (PM) binding is mediated by the insertion of a prenylated moiety that is attached to the terminal carboxy-methylated cysteine, in addition to electrostatic interactions of its positively charged hypervariable region with anionic lipids. Calmodulin (CaM) has been suggested to selectively bind KRAS4b to act as a negative regulator of the RAS/mitogen-activated protein kinase (MAPK) signaling pathway by displacing KRAS4b from the membrane. However, the mechanism by which CaM can recognize and displace KRAS4b from the membrane is not well understood. In this study, we employed biophysical and structural techniques to characterize this mechanism in detail. We show that KRAS4b prenylation is required for binding to CaM and that the hydrophobic pockets of CaM can accommodate the prenylated region of KRAS4b, which might represent a novel CaM-binding motif. Remarkably, prenylated KRAS4b forms a 2:1 stoichiometric complex with CaM in a nucleotide-independent manner. The interaction between prenylated KRAS4b and CaM is enthalpically driven, and electrostatic interactions also contribute to the formation of the complex. The prenylated KRAS4b terminal KSKTKC-farnesylation and carboxy-methylation is sufficient for binding and defines the minimal CaM-binding motif. This is the same region implicated in membrane and phosphodiesterase6-δ binding. Finally, we provide a structure-based docking model by which CaM binds to prenylated KRAS4b. Our data provide new insights into the KRAS4b-CaM interaction and suggest a possible mechanism whereby CaM can regulate KRAS4b membrane localization.
Assuntos
Calmodulina/metabolismo , Prenilação de Proteína , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Calmodulina/química , Humanos , Modelos Moleculares , Nucleotídeos/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas p21(ras)/químicaRESUMO
Various methods for understanding the structural and dynamic properties of proteins rely on the analysis of their NMR chemical shifts. These methods require the initial assignment of NMR signals to particular atoms in the sequence of the protein, a step that can be very time-consuming. The probabilistic interaction network of evidence (PINE) algorithm for automated assignment of backbone and side chain chemical shifts utilizes a Bayesian probabilistic network model that analyzes sequence data and peak lists from multiple NMR experiments. PINE, which is one of the most popular and reliable automated chemical shift assignment algorithms, has been available to the protein NMR community for longer than a decade. We announce here a new web server version of PINE, called Integrative PINE (I-PINE), which supports more types of NMR experiments than PINE (including three-dimensional nuclear Overhauser enhancement and four-dimensional J-coupling experiments) along with more comprehensive visualization of chemical shift based analysis of protein structure and dynamics. The I-PINE server is freely accessible at http://i-pine.nmrfam.wisc.edu . Help pages and tutorial including browser capability are available at: http://i-pine.nmrfam.wisc.edu/instruction.html . Sample data that can be used for testing the web server are available at: http://i-pine.nmrfam.wisc.edu/examples.html .
Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Algoritmos , Proteínas/análiseRESUMO
Whereas iron-sulfur (Fe-S) cluster assembly on the wild-type scaffold protein ISCU, as catalyzed by the human cysteine desulfurase complex (NIA)2, exhibits a requirement for frataxin (FXN), in yeast, ISCU variant M108I has been shown to bypass the FXN requirement. Wild-type ISCU populates two interconverting conformational states: one structured and one dynamically disordered. We show here that variants ISCU(M108I) and ISCU(D39V) of human ISCU populate only the structured state. We have compared the properties of ISCU, ISCU(M108I), and ISCU(D39V), with and without FXN, in both the cysteine desulfurase step of Fe-S cluster assembly and the overall Fe-S cluster assembly reaction catalyzed by (NIA)2. In the cysteine desulfurase step with dithiothreitol (DTT) as the reductant, FXN was found to stimulate cysteine desulfurase activity with both the wild-type and structured variants, although the effect was less prominent with ISCU(D39V) than with the wild-type or ISCU(M108I). In overall Fe-S cluster assembly, frataxin was found to stimulate cluster assembly with both the wild-type and structured variants when the reductant was DTT; however, with the physiological reductant, reduced ferredoxin 2 (rdFDX2), FXN stimulated the reaction with wild-type ISCU but not with either ISCU(M108I) or ISCU(D39V). Nuclear magnetic resonance titration experiments revealed that wild-type ISCU, FXN, and rdFDX2 all bind to (NIA)2. However, when ISCU was replaced by the fully structured variant ISCU(M108I), the addition of rdFDX2 to the [NIA-ISCU(M108I)-FXN]2 complex led to the release of FXN. Thus, the displacement of FXN by rdFDX2 explains the failure of FXN to stimulate Fe-S cluster assembly on ISCU(M108I).
Assuntos
Liases de Carbono-Enxofre/metabolismo , Ferredoxinas/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Proteínas Ferro-Enxofre/genética , Camundongos , Modelos Moleculares , FrataxinaRESUMO
The human transporter ATP7B delivers copper to the biosynthetic pathways and maintains copper homeostasis in the liver. Mutations in ATP7B cause the potentially fatal hepatoneurological disorder Wilson disease. The activity and intracellular localization of ATP7B are regulated by copper, but the molecular mechanism of this regulation is largely unknown. We show that the copper chaperone Atox1, which delivers copper to ATP7B, and the group of the first three metal-binding domains (MBD1-3) are central to the activity regulation of ATP7B. Atox1-Cu binding to ATP7B changes domain dynamics and interactions within the MBD1-3 group and activates ATP hydrolysis. To understand the mechanism linking Atox1-MBD interactions and enzyme activity, we have determined the MBD1-3 conformational space using small angle X-ray scattering and identified changes in MBD dynamics caused by apo-Atox1 and Atox1-Cu by solution NMR. The results show that copper transfer from Atox1 decreases domain interactions within the MBD1-3 group and increases the mobility of the individual domains. The N-terminal segment of MBD1-3 was found to interact with the nucleotide-binding domain of ATP7B, thus physically coupling the domains involved in copper binding and those involved in ATP hydrolysis. Taken together, the data suggest a regulatory mechanism in which Atox1-mediated copper transfer activates ATP7B by releasing inhibitory constraints through increased freedom of MBD1-3 motions.