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1.
Nucleic Acids Res ; 50(13): 7697-7720, 2022 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-35801871

RESUMO

Artemis nuclease and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are key components in nonhomologous DNA end joining (NHEJ), the major repair mechanism for double-strand DNA breaks. Artemis activation by DNA-PKcs resolves hairpin DNA ends formed during V(D)J recombination. Artemis deficiency disrupts development of adaptive immunity and leads to radiosensitive T- B- severe combined immunodeficiency (RS-SCID). An activated state of Artemis in complex with DNA-PK was solved by cryo-EM recently, which showed Artemis bound to the DNA. Here, we report that the pre-activated form (basal state) of the Artemis:DNA-PKcs complex is stable on an agarose-acrylamide gel system, and suitable for cryo-EM structural analysis. Structures show that the Artemis catalytic domain is dynamically positioned externally to DNA-PKcs prior to ABCDE autophosphorylation and show how both the catalytic and regulatory domains of Artemis interact with the N-HEAT and FAT domains of DNA-PKcs. We define a mutually exclusive binding site for Artemis and XRCC4 on DNA-PKcs and show that an XRCC4 peptide disrupts the Artemis:DNA-PKcs complex. All of the findings are useful in explaining how a hypomorphic L3062R missense mutation of DNA-PKcs could lead to insufficient Artemis activation, hence RS-SCID. Our results provide various target site candidates to design disruptors for Artemis:DNA-PKcs complex formation.


Assuntos
Proteína Quinase Ativada por DNA/química , Proteínas de Ligação a DNA/química , Endonucleases/química , Reparo do DNA , Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Imunodeficiência Combinada Severa/genética
2.
J Vis Exp ; (177)2021 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-34866621

RESUMO

Electron crystallography is a powerful tool for high-resolution structure determination. Macromolecules such as soluble or membrane proteins can be grown into highly ordered two-dimensional (2D) crystals under favorable conditions. The quality of the grown 2D crystals is crucial to the resolution of the final reconstruction via 2D image processing. Over the years, lipid monolayers have been used as a supporting layer to foster the 2D crystallization of peripheral membrane proteins as well as soluble proteins. This method can also be applied to 2D crystallization of integral membrane proteins but requires more extensive empirical investigation to determine detergent and dialysis conditions to promote partitioning to the monolayer. A lipid monolayer forms at the air-water interface such that the polar lipid head groups remain hydrated in the aqueous phase and the non-polar, acyl chains, tails partition into the air, breaking the surface tension and flattening the water surface. The charged nature or distinctive chemical moieties of the head groups provide affinity for proteins in solution, promoting binding for 2D array formation. A newly formed monolayer with the 2D array can be readily transfer into an electron microscope (EM) on a carbon-coated copper grid used to lift and support the crystalline array. In this work, we describe a lipid monolayer methodology for cryogenic electron microscopic (cryo-EM) imaging.


Assuntos
Elétrons , Diálise Renal , Microscopia Crioeletrônica/métodos , Cristalografia por Raios X , Lipídeos/química , Proteínas de Membrana/química
3.
Int J Mol Sci ; 22(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34360842

RESUMO

IBMPFD/ALS is a genetic disorder caused by a single amino acid mutation on the p97 ATPase, promoting ATPase activity and cofactor dysregulation. The disease mechanism underlying p97 ATPase malfunction remains unclear. To understand how the mutation alters the ATPase regulation, we assembled a full-length p97R155H with its p47 cofactor and first visualized their structures using single-particle cryo-EM. More than one-third of the population was the dodecameric form. Nucleotide presence dissociates the dodecamer into two hexamers for its highly elevated function. The N-domains of the p97R155H mutant all show up configurations in ADP- or ATPγS-bound states. Our functional and structural analyses showed that the p47 binding is likely to impact the p97R155H ATPase activities via changing the conformations of arginine fingers. These functional and structural analyses underline the ATPase dysregulation with the miscommunication between the functional modules of the p97R155H.


Assuntos
Demência Frontotemporal/metabolismo , Modelos Moleculares , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Mutação , Miosite de Corpos de Inclusão/metabolismo , Osteíte Deformante/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Proteína com Valosina/genética , Demência Frontotemporal/genética , Humanos , Microscopia Eletrônica de Transmissão , Distrofia Muscular do Cíngulo dos Membros/genética , Miosite de Corpos de Inclusão/genética , Osteíte Deformante/genética , Conformação Proteica , Proteína com Valosina/metabolismo
4.
J Biol Chem ; 292(45): 18392-18407, 2017 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-28939772

RESUMO

p97 is an essential ATPase associated with various cellular activities (AAA+) that functions as a segregase in diverse cellular processes, including the maintenance of proteostasis. p97 interacts with different cofactors that target it to distinct pathways; an important example is the deubiquitinase ataxin3, which collaborates with p97 in endoplasmic reticulum-associated degradation. However, the molecular details of this interaction have been unclear. Here, we characterized the binding of ataxin3 to p97, showing that ataxin3 binds with low-micromolar affinity to both wild-type p97 and mutants linked to degenerative disorders known as multisystem proteinopathy 1 (MSP1); we further showed that the stoichiometry of binding is one ataxin3 molecule per p97 hexamer. We mapped the binding determinants on each protein, demonstrating that ataxin3's p97/VCP-binding motif interacts with the inter-lobe cleft in the N-domain of p97. We also probed the nucleotide dependence of this interaction, confirming that ataxin3 and p97 associate in the presence of ATP and in the absence of nucleotide, but not in the presence of ADP. Our experiments suggest that an ADP-driven downward movement of the p97 N-terminal domain dislodges ataxin3 by inducing a steric clash between the D1-domain and ataxin3's C terminus. In contrast, MSP1 mutants of p97 bind ataxin3 irrespective of their nucleotide state, indicating a failure by these mutants to translate ADP binding into a movement of the N-terminal domain. Our model provides a mechanistic explanation for how nucleotides regulate the p97-ataxin3 interaction and why atypical cofactor binding is observed with MSP1 mutants.


Assuntos
Ataxina-3/metabolismo , Coenzimas/metabolismo , Miopatias Distais/metabolismo , Modelos Moleculares , Deficiências na Proteostase/metabolismo , Proteínas Repressoras/metabolismo , Proteína com Valosina/metabolismo , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Ataxina-3/química , Ataxina-3/genética , Sítios de Ligação , Ligação Competitiva , Coenzimas/química , Coenzimas/genética , Cristalografia por Raios X , Bases de Dados de Proteínas , Miopatias Distais/enzimologia , Miopatias Distais/genética , Humanos , Microscopia Eletrônica de Transmissão , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Multimerização Proteica , Deficiências na Proteostase/enzimologia , Deficiências na Proteostase/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteína com Valosina/química , Proteína com Valosina/genética
5.
Proc Natl Acad Sci U S A ; 113(5): 1162-7, 2016 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-26787853

RESUMO

A library of eight amphiphilic Janus glycodendrimers (GDs) with d-mannose (Man) headgroups, a known routing signal for lectin-mediated transport processes, was constructed via an iterative modular methodology. Sequence-defined variations of the Janus GD modulate the surface density and sequence of Man after self-assembly into multilamellar glycodendrimersomes (GDSs). The spatial mode of Man presentation is decisive for formation of either unilamellar or onion-like GDS vesicles. Man presentation and Janus GD concentration determine GDS size and number of bilayers. Beyond vesicle architecture, Man topological display affects kinetics and plateau level of GDS aggregation by a tetravalent model lectin: the leguminous agglutinin Con A, which is structurally related to endogenous cargo transporters. The agglutination process was rapid, efficient, and readily reversible for onion-like GDSs, demonstrating their value as versatile tools to explore the nature of physiologically relevant glycan/lectin pairing.


Assuntos
Carboidratos/química , Dendrímeros/química , Lectinas/química , Microscopia Eletrônica de Transmissão
6.
Proc Natl Acad Sci U S A ; 112(46): E6293-300, 2015 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-26578789

RESUMO

Telomeric repeat-containing RNA (TERRA) has been identified as a telomere-associated regulator of chromosome end protection. Here, we report that TERRA can also be found in extracellular fractions that stimulate innate immune signaling. We identified extracellular forms of TERRA in mouse tumor and embryonic brain tissue, as well as in human tissue culture cell lines using RNA in situ hybridization. RNA-seq analyses revealed TERRA to be among the most highly represented transcripts in extracellular fractions derived from both normal and cancer patient blood plasma. Cell-free TERRA (cfTERRA) could be isolated from the exosome fractions derived from human lymphoblastoid cell line (LCL) culture media. cfTERRA is a shorter form (∼200 nt) of cellular TERRA and copurifies with CD63- and CD83-positive exosome vesicles that could be visualized by cyro-electron microscopy. These fractions were also enriched for histone proteins that physically associate with TERRA in extracellular ChIP assays. Incubation of cfTERRA-containing exosomes with peripheral blood mononuclear cells stimulated transcription of several inflammatory cytokine genes, including TNFα, IL6, and C-X-C chemokine 10 (CXCL10) Exosomes engineered with elevated TERRA or liposomes with synthetic TERRA further stimulated inflammatory cytokines, suggesting that exosome-associated TERRA augments innate immune signaling. These findings imply a previously unidentified extrinsic function for TERRA and a mechanism of communication between telomeres and innate immune signals in tissue and tumor microenvironments.


Assuntos
Exossomos/imunologia , Imunidade Inata , Neoplasias/imunologia , RNA não Traduzido/imunologia , Transdução de Sinais/imunologia , Telômero , Animais , Antígenos CD/sangue , Antígenos CD/genética , Antígenos CD/imunologia , Linhagem Celular Tumoral , Citocinas/sangue , Citocinas/genética , Citocinas/imunologia , Exossomos/genética , Exossomos/metabolismo , Histonas/sangue , Histonas/genética , Histonas/imunologia , Humanos , Imunoglobulinas/sangue , Imunoglobulinas/genética , Imunoglobulinas/imunologia , Inflamação/sangue , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Glicoproteínas de Membrana/sangue , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Neoplasias/sangue , Neoplasias/genética , Neoplasias/patologia , RNA não Traduzido/sangue , RNA não Traduzido/genética , Transdução de Sinais/genética , Tetraspanina 30/sangue , Tetraspanina 30/genética , Tetraspanina 30/imunologia , Antígeno CD83
7.
J Virol ; 88(1): 469-76, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24155406

RESUMO

Previous human antibody studies have shown that the human VH1-46 antibody variable gene segment encodes much of the naturally occurring human B cell response to rotavirus and is directed to virus protein 6 (VP6). It is currently unknown why some of the VH1-46-encoded human VP6 monoclonal antibodies inhibit viral transcription while others do not. In part, there are affinity differences between antibodies that likely affect inhibitory activity, but we also hypothesize that there are differing modes of binding to VP6 that affect the ability to block the transcriptional pore on double-layered particles. Here, we used a hybrid method approach for antibody epitope mapping, including single-particle cryo-electron microscopy (cryo-EM) and enhanced amide hydrogen-deuterium exchange mass spectrometry (DXMS) to determine the location and mode of binding of a VH1-46-encoded antibody, RV6-25. The structure of the RV6-25 antibody-double-layered particle (DLP) complex indicated a very complex binding pattern that revealed subtle differences in accessibility of the VP6 epitope depending on its position in the type I, II, or III channels. These subtle variations in the presentation or accessibility of the RV VP6 capsid layer led to position-specific differences in occupancy for binding of the RV6-25 antibody. The studies also showed that the location of binding of the noninhibitory antibody RV6-25 on the apical surface of RV VP6 head domain does not obstruct the transcription pore upon antibody binding, in contrast to binding of an inhibitory antibody, RV6-26, deeper in the transcriptional pore.


Assuntos
Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Proteínas do Capsídeo/imunologia , Epitopos/imunologia , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação de Anticorpos , Biopolímeros/imunologia , Microscopia Crioeletrônica , Primers do DNA , Epitopos/química , Espectrometria de Massas , Dados de Sequência Molecular
8.
J Mol Biol ; 425(18): 3311-24, 2013 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-23796516

RESUMO

The circadian control of cellular processes in cyanobacteria is regulated by a posttranslational oscillator formed by three Kai proteins. During the oscillator cycle, KaiA serves to promote autophosphorylation of KaiC while KaiB counteracts this effect. Here, we present a crystallographic structure of the wild-type Synechococcus elongatus KaiB and a cryo-electron microscopy (cryoEM) structure of a KaiBC complex. The crystal structure shows the expected dimer core structure and significant conformational variations of the KaiB C-terminal region, which is functionally important in maintaining rhythmicity. The KaiBC sample was formed with a C-terminally truncated form of KaiC, KaiC-Δ489, which is persistently phosphorylated. The KaiB-KaiC-Δ489 structure reveals that the KaiC hexamer can bind six monomers of KaiB, which form a continuous ring of density in the KaiBC complex. We performed cryoEM-guided molecular dynamics flexible fitting simulations with crystal structures of KaiB and KaiC to probe the KaiBC protein-protein interface. This analysis indicated a favorable binding mode for the KaiB monomer on the CII end of KaiC, involving two adjacent KaiC subunits and spanning an ATP binding cleft. A KaiC mutation, R468C, which has been shown to affect the affinity of KaiB for KaiC and lengthen the period in a bioluminescence rhythm assay, is found within the middle of the predicted KaiBC interface. The proposed KaiB binding mode blocks access to the ATP binding cleft in the CII ring of KaiC, which provides insight into how KaiB might influence the phosphorylation status of KaiC.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/metabolismo , Synechococcus/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Sítios de Ligação/fisiologia , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/química , Microscopia Crioeletrônica , Cristalografia por Raios X , Modelos Moleculares , Simulação de Dinâmica Molecular , Complexos Multiproteicos/química , Fosforilação , Ligação Proteica , Multimerização Proteica/fisiologia , Estrutura Quaternária de Proteína , Synechococcus/genética
9.
PLoS One ; 8(5): e61101, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23671563

RESUMO

Several live attenuated rotavirus (RV) vaccines have been licensed, but the mechanisms of protective immunity are still poorly understood. The most frequent human B cell response is directed to the internal protein VP6 on the surface of double-layered particles, which is normally exposed only in the intracellular environment. Here, we show that the canonical VP6 antibodies secreted by humans bind to such particles and inhibit viral transcription. Polymeric IgA RV antibodies mediated an inhibitory effect against virus replication inside cells during IgA transcytosis. We defined the recognition site on VP6 as a quaternary epitope containing a high density of charged residues. RV human mAbs appear to bind to a negatively-charged patch on the surface of the Type I channel in the transcriptionally active particle, and they sterically block the channel. This unique mucosal mechanism of viral neutralization, which is not apparent from conventional immunoassays, may contribute significantly to human immunity to RV.


Assuntos
Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Proteínas do Capsídeo/imunologia , Infecções por Rotavirus/imunologia , Rotavirus/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/metabolismo , Antígenos Virais/genética , Antígenos Virais/metabolismo , Linfócitos B/imunologia , Linfócitos B/virologia , Células CACO-2 , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunoglobulina A/imunologia , Imunoglobulina A/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Testes de Neutralização , Ligação Proteica/imunologia , Estrutura Terciária de Proteína , Rotavirus/metabolismo , Rotavirus/fisiologia , Infecções por Rotavirus/virologia , Homologia de Sequência de Aminoácidos , Transcrição Gênica , Vírion/genética , Vírion/imunologia , Vírion/metabolismo , Replicação Viral/genética , Replicação Viral/imunologia
10.
Cell Host Microbe ; 12(6): 815-23, 2012 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-23245326

RESUMO

Plasmodium falciparum pathogenesis is affected by various cell types in the blood, including platelets, which can kill intraerythrocytic malaria parasites. Platelets could mediate these antimalarial effects through human defense peptides (HDPs), which exert antimicrobial effects by permeabilizing membranes. Therefore, we screened a panel of HDPs and determined that human platelet factor 4 (hPF4) kills malaria parasites inside erythrocytes by selectively lysing the parasite digestive vacuole (DV). PF4 rapidly accumulates only within infected erythrocytes and is required for parasite killing in infected erythrocyte-platelet cocultures. To exploit this antimalarial mechanism, we tested a library of small, nonpeptidic mimics of HDPs (smHDPs) and identified compounds that kill P. falciparum by rapidly lysing the parasite DV while sparing the erythrocyte plasma membrane. Lead smHDPs also reduced parasitemia in a murine malaria model. Thus, identifying host molecules that control parasite growth can further the development of related molecules with therapeutic potential.


Assuntos
Antimaláricos/isolamento & purificação , Antimaláricos/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Fator Plaquetário 4/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Eritrócitos/parasitologia , Malária/tratamento farmacológico , Malária/parasitologia , Camundongos , Carga Parasitária , Parasitemia/tratamento farmacológico , Parasitemia/parasitologia
11.
PLoS One ; 6(8): e23697, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21887298

RESUMO

The circadian clock in the cyanobacterium Synechococcus elongatus is composed of a post-translational oscillator (PTO) that can be reconstituted in vitro from three different proteins in the presence of ATP and a transcription-translation feedback loop (TTFL). The homo-hexameric KaiC kinase, phosphatase and ATPase alternates between hypo- and hyper-phosphorylated states over the 24-h cycle, with KaiA enhancing phosphorylation, and KaiB antagonizing KaiA and promoting KaiC subunit exchange. SasA is a His kinase that relays output signals from the PTO formed by the three Kai proteins to the TTFL. Although the crystal structures for all three Kai proteins are known, atomic resolution structures of Kai and Kai/SasA protein complexes have remained elusive. Here, we present models of the KaiAC and KaiBC complexes derived from solution small angle X-ray scattering (SAXS), which are consistent with previous EM based models. We also present a combined SAXS/EM model of the KaiC/SasA complex, which has two N-terminal SasA sensory domains occupying positions on the C-terminal KaiC ring reminiscent of the orientations adopted by KaiB dimers. Using EM we demonstrate that KaiB and SasA compete for similar binding sites on KaiC. We also propose an EM based model of the ternary KaiABC complex that is consistent with the sequestering of KaiA by KaiB on KaiC during the PTO dephosphorylation phase. This work provides the first 3D-catalogue of protein-protein interactions in the KaiABC PTO and the output pathway mediated by SasA.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/química , Fosfotransferases/metabolismo , Domínios e Motivos de Interação entre Proteínas , Synechococcus/química , Difração de Raios X , Sítios de Ligação , Relógios Circadianos , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/metabolismo , Cristalografia por Raios X , Histidina Quinase , Microscopia Eletrônica , Conformação Proteica , Proteínas Quinases , Espalhamento a Baixo Ângulo
12.
Proc Natl Acad Sci U S A ; 107(33): 14805-10, 2010 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-20679240

RESUMO

Three proteins from cyanobacteria (KaiA, KaiB, and KaiC) can reconstitute circadian oscillations in vitro. At least three molecular properties oscillate during this reaction, namely rhythmic phosphorylation of KaiC, ATP hydrolytic activity of KaiC, and assembly/disassembly of intermolecular complexes among KaiA, KaiB, and KaiC. We found that the intermolecular associations determine key dynamic properties of this in vitro oscillator. For example, mutations within KaiB that alter the rates of binding of KaiB to KaiC also predictably modulate the period of the oscillator. Moreover, we show that KaiA can bind stably to complexes of KaiB and hyperphosphorylated KaiC. Modeling simulations indicate that the function of this binding of KaiA to the KaiB*KaiC complex is to inactivate KaiA's activity, thereby promoting the dephosphorylation phase of the reaction. Therefore, we report here dynamics of interaction of KaiA and KaiB with KaiC that determine the period and amplitude of this in vitro oscillator.


Assuntos
Proteínas de Bactérias/metabolismo , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/metabolismo , Simulação de Dinâmica Molecular , Synechococcus/metabolismo , Algoritmos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Ritmo Circadiano , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/química , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/genética , Eletroforese em Gel de Poliacrilamida , Polarização de Fluorescência , Cinética , Modelos Biológicos , Modelos Químicos , Mutação , Peptídeos , Fosforilação , Ligação Proteica , Synechococcus/genética
13.
J Struct Biol ; 164(1): 166-9, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18621546

RESUMO

High throughput methods of data acquisition are advantageous for cryoelectron microscopy and single particle reconstruction as high-resolution structure determination requires thousands of particle images. We have developed a semi-automated data collection method that utilizes the scripting languages provided by FEI for their Tecnai User Interface (TUI) and by Gatan for their Digital Micrograph package. Our Script-Assisted Microscopy (SAM) method allows for the selection of multiple locations within a low magnification, search mode, micrograph and for subsequent automated imaging of these locations at a higher exposure magnification. The SAM approach permits the user to retain control over the microscope, while streamlining the most repetitive steps of collecting and evaluating micrographs. With SAM, we have found an average of 1000 micrographs can be collected per day on any grid type, either irregular homemade grids or prefabricated grids with regularly spaced holes. This rate of data collection represents a fivefold improvement over our manual collection rates. SAM provides an example of an individually tailored approach to data acquisition utilizing the scripting interfaces provided by the equipment manufacturers. The SAM method has proven valuable for determination of a subnanometer resolution cryoEM structure of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a 469kDa protein.


Assuntos
Microscopia Crioeletrônica/instrumentação , Fotomicrografia/instrumentação , Software , Proteína Quinase Ativada por DNA/química , Nanotecnologia
14.
EMBO J ; 27(12): 1767-78, 2008 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-18497745

RESUMO

The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro by the KaiA, KaiB and KaiC proteins in the presence of ATP. The principal clock component, KaiC, undergoes regular cycles between hyper- and hypo-phosphorylated states with a period of ca. 24 h that is temperature compensated. KaiA enhances KaiC phosphorylation and this enhancement is antagonized by KaiB. Throughout the cycle Kai proteins interact in a dynamic manner to form complexes of different composition. We present a three-dimensional model of the S. elongatus KaiB-KaiC complex based on X-ray crystallography, negative-stain and cryo-electron microscopy, native gel electrophoresis and modelling techniques. We provide experimental evidence that KaiB dimers interact with KaiC from the same side as KaiA and for a conformational rearrangement of the C-terminal regions of KaiC subunits. The enlarged central channel and thus KaiC subunit separation in the C-terminal ring of the hexamer is consistent with KaiC subunit exchange during the dephosphorylation phase. The proposed binding mode of KaiB explains the observation of simultaneous binding of KaiA and KaiB to KaiC, and provides insight into the mechanism of KaiB's antagonism of KaiA.


Assuntos
Proteínas de Bactérias/metabolismo , Relógios Biológicos , Ritmo Circadiano , Modelos Moleculares , Synechococcus/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano , Microscopia Crioeletrônica , Cristalografia por Raios X , Coloração Negativa , Fragmentos de Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína
15.
Structure ; 16(3): 468-77, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18334221

RESUMO

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) regulates the nonhomologous end joining pathway for repair of double-stranded DNA (dsDNA) breaks. Here, we present a 7A resolution structure of DNA-PKcs determined by cryo-electron microscopy single-particle reconstruction. This structure is composed of density rods throughout the molecule that are indicative of alpha helices and reveals structural features not observed in lower resolution EM structures. Docking of homology models into the DNA-PKcs structure demonstrates that up to eight helical HEAT repeat motifs fit well within the density. Surprisingly, models for the kinase domain can be docked into either the crown or base of the molecule at this resolution, although real space refinement suggests that the base location is the best fit. We propose a model for the interaction of DNA with DNA-PKcs in which one turn of dsDNA enters the central channel and interacts with a resolved alpha-helical protrusion.


Assuntos
Domínio Catalítico , Microscopia Crioeletrônica , Proteína Quinase Ativada por DNA/química , Proteína Quinase Ativada por DNA/metabolismo , DNA/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador/métodos , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
16.
PLoS Biol ; 5(4): e93, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17388688

RESUMO

A biochemical oscillator can be reconstituted in vitro with three purified proteins, that displays the salient properties of circadian (daily) rhythms, including self-sustained 24-h periodicity that is temperature compensated. We analyze the biochemical basis of this oscillator by quantifying the time-dependent interactions of the three proteins (KaiA, KaiB, and KaiC) by electron microscopy and native gel electrophoresis to elucidate the timing of the formation of complexes among the Kai proteins. The data are used to derive a dynamic model for the in vitro oscillator that accurately reproduces the rhythms of KaiABC complexes and of KaiC phosphorylation, and is consistent with biophysical observations of individual Kai protein interactions. We use fluorescence resonance energy transfer (FRET) to confirm that monomer exchange among KaiC hexamers occurs. The model demonstrates that the function of this monomer exchange may be to maintain synchrony among the KaiC hexamers in the reaction, thereby sustaining a high-amplitude oscillation. Finally, we apply the first perturbation analyses of an in vitro oscillator by using temperature pulses to reset the phase of the KaiABC oscillator, thereby testing the resetting characteristics of this unique circadian oscillator. This study analyzes a circadian clockwork to an unprecedented level of molecular detail.


Assuntos
Ritmo Circadiano , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação , Eletroforese em Gel de Poliacrilamida , Transferência Ressonante de Energia de Fluorescência , Técnicas In Vitro , Microscopia Eletrônica , Fosforilação , Synechococcus/metabolismo , Synechococcus/fisiologia
17.
EMBO J ; 25(9): 2017-28, 2006 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-16628225

RESUMO

The cyanobacterial circadian clock can be reconstituted in vitro by mixing recombinant KaiA, KaiB and KaiC proteins with ATP, producing KaiC phosphorylation and dephosphorylation cycles that have a regular rhythm with a ca. 24-h period and are temperature-compensated. KaiA and KaiB are modulators of KaiC phosphorylation, whereby KaiB antagonizes KaiA's action. Here, we present a complete crystallographic model of the Synechococcus elongatus KaiC hexamer that includes previously unresolved portions of the C-terminal regions, and a negative-stain electron microscopy study of S. elongatus and Thermosynechococcus elongatus BP-1 KaiA-KaiC complexes. Site-directed mutagenesis in combination with EM reveals that KaiA binds exclusively to the CII half of the KaiC hexamer. The EM-based model of the KaiA-KaiC complex reveals protein-protein interactions at two sites: the known interaction of the flexible C-terminal KaiC peptide with KaiA, and a second postulated interaction between the apical region of KaiA and the ATP binding cleft on KaiC. This model brings KaiA mutation sites that alter clock period or abolish rhythmicity into contact with KaiC and suggests how KaiA might regulate KaiC phosphorylation.


Assuntos
Proteínas de Bactérias/química , Relógios Biológicos , Ritmo Circadiano , Synechococcus/fisiologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/ultraestrutura , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano , Cristalografia por Raios X , Microscopia Eletrônica , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fosforilação , Mapeamento de Interação de Proteínas , Deleção de Sequência , Synechococcus/metabolismo
18.
Eukaryot Cell ; 4(1): 166-77, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15643072

RESUMO

Mcl1p is an essential fission yeast chromatin-binding protein that belongs to a family of highly conserved eukaryotic proteins important for sister chromatid cohesion. The essential function is believed to result from its role as a Pol1p (polymerase alpha) accessory protein, a conclusion based primarily on analogy to Ctf4p's interaction with Pol1p. In this study, we show that Mcl1p also binds to Pol1p with high affinity for the N terminus of Pol1p during S phase and DNA damage. Characterization of an inducible allele of mcl1+, (nmt41)mcl1-MH, shows that altered expression levels of Mcl1p lead to sensitivity to DNA-damaging agents and synthetic lethality with the replication checkpoint mutations rad3Delta, rqh1Delta, and hsk1-1312. Further, we find that the overexpression of the S-phase checkpoint kinase, Cds1, or the loss of Hsk1 kinase activity can disrupt Mcl1p's interaction with chromatin and Pol1p during replication arrest with hydroxyurea. We take these data to mean that Mcl1p is a dynamic component of the polymerase alpha complex during replication and is important for the replication stress response in fission yeast.


Assuntos
Proteínas Cromossômicas não Histona/fisiologia , DNA Polimerase I/metabolismo , Fase S , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/metabolismo , Alelos , Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatina/metabolismo , DNA/química , Dano ao DNA , Eletroforese em Gel Bidimensional , Genótipo , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde/química , Modelos Biológicos , Mutação , Fenótipo , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas de Schizosaccharomyces pombe/metabolismo , Sacarose/farmacologia , Temperatura
19.
Eukaryot Cell ; 1(5): 758-73, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12455694

RESUMO

The fission yeast minichromosome loss mutant mcl1-1 was identified in a screen for mutants defective in chromosome segregation. Missegregation of the chromosomes in mcl1-1 mutant cells results from decreased centromeric cohesion between sister chromatids. mcl1+ encodes a beta-transducin-like protein with similarity to a family of eukaryotic proteins that includes Ctf4p from Saccharomyces cerevisiae, sepB from Aspergillus nidulans, and AND-1 from humans. The previously identified fungal members of this protein family also have chromosome segregation defects, but they primarily affect DNA metabolism. Chromosomes from mcl1-1 cells were heterogeneous in size or structure on pulsed-field electrophoresis gels and had elongated heterogeneous telomeres. mcl1-1 was lethal in combination with the DNA checkpoint mutations rad3delta and rad26delta, demonstrating that loss of Mcl1p function leads to DNA damage. mcl1-1 showed an acute sensitivity to DNA damage that affects S-phase progression. It interacts genetically with replication components and causes an S-phase delay when overexpressed. We propose that Mcl1p, like Ctf4p, has a role in regulating DNA replication complexes.


Assuntos
Proteínas Cromossômicas não Histona/genética , Cromossomos Fúngicos/genética , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Proteínas , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Sequência de Aminoácidos , Cromátides/fisiologia , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Replicação do DNA , Proteínas de Ligação a DNA/química , Dados de Sequência Molecular , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Homologia de Sequência de Aminoácidos
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