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1.
EMBO J ; 40(14): e107294, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34031912

RESUMO

Potassium-coupled chloride transporters (KCCs) play crucial roles in regulating cell volume and intracellular chloride concentration. They are characteristically inhibited under isotonic conditions via phospho-regulatory sites located within the cytoplasmic termini. Decreased inhibitory phosphorylation in response to hypotonic cell swelling stimulates transport activity, and dysfunction of this regulatory process has been associated with various human diseases. Here, we present cryo-EM structures of human KCC3b and KCC1, revealing structural determinants for phospho-regulation in both N- and C-termini. We show that phospho-mimetic KCC3b is arrested in an inward-facing state in which intracellular ion access is blocked by extensive contacts with the N-terminus. In another mutant with increased isotonic transport activity, KCC1Δ19, this interdomain interaction is absent, likely due to a unique phospho-regulatory site in the KCC1 N-terminus. Furthermore, we map additional phosphorylation sites as well as a previously unknown ATP/ADP-binding pocket in the large C-terminal domain and show enhanced thermal stabilization of other CCCs by adenine nucleotides. These findings provide fundamentally new insights into the complex regulation of KCCs and may unlock innovative strategies for drug development.


Assuntos
Cloretos/metabolismo , Nucleotídeos/metabolismo , Potássio/metabolismo , Simportadores/metabolismo , Animais , Linhagem Celular , Tamanho Celular , Humanos , Fosforilação/fisiologia , Células Sf9 , Transdução de Sinais/fisiologia , Cotransportadores de K e Cl-
2.
Genes Dev ; 28(20): 2291-303, 2014 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-25319829

RESUMO

Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2-7 (minichromosome maintenance proteins 2-7) double hexamer. During S phase, each Mcm2-7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood. The helicase loaders ORC-Cdc6 function to recruit a single Cdt1-Mcm2-7 heptamer to replication origins prior to Cdt1 release and ORC-Cdc6-Mcm2-7 complex formation, but how the second Mcm2-7 hexamer is recruited to promote double-hexamer formation is not well understood. Here, structural evidence for intermediates consisting of an ORC-Cdc6-Mcm2-7 complex and an ORC-Cdc6-Mcm2-7-Mcm2-7 complex are reported, which together provide new insights into DNA licensing. Detailed structural analysis of the loaded Mcm2-7 double-hexamer complex demonstrates that the two hexamers are interlocked and misaligned along the DNA axis and lack ATP hydrolysis activity that is essential for DNA helicase activity. Moreover, we show that the head-to-head juxtaposition of the Mcm2-7 double hexamer generates a new protein interaction surface that creates a multisubunit-binding site for an S-phase protein kinase that is known to activate DNA replication. The data suggest how the double hexamer is assembled and how helicase activity is regulated during DNA licensing, with implications for cell cycle control of DNA replication and genome stability.


Assuntos
Proteínas de Manutenção de Minicromossomo/química , Proteínas de Manutenção de Minicromossomo/metabolismo , Saccharomyces cerevisiae/enzimologia , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Ativação Enzimática , Hidrólise , Microscopia Eletrônica , Proteínas de Manutenção de Minicromossomo/isolamento & purificação , Conformação Molecular , Ligação Proteica
3.
Genes Dev ; 28(15): 1653-66, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-25085418

RESUMO

The regulated loading of the replicative helicase minichromosome maintenance proteins 2-7 (MCM2-7) onto replication origins is a prerequisite for replication fork establishment and genomic stability. Origin recognition complex (ORC), Cdc6, and Cdt1 assemble two MCM2-7 hexamers into one double hexamer around dsDNA. Although the MCM2-7 hexamer can adopt a ring shape with a gap between Mcm2 and Mcm5, it is unknown which Mcm interface functions as the DNA entry gate during regulated helicase loading. Here, we establish that the Saccharomyces cerevisiae MCM2-7 hexamer assumes a closed ring structure, suggesting that helicase loading requires active ring opening. Using a chemical biology approach, we show that ORC-Cdc6-Cdt1-dependent helicase loading occurs through a unique DNA entry gate comprised of the Mcm2 and Mcm5 subunits. Controlled inhibition of DNA insertion triggers ATPase-driven complex disassembly in vitro, while in vivo analysis establishes that Mcm2/Mcm5 gate opening is essential for both helicase loading onto chromatin and cell cycle progression. Importantly, we demonstrate that the MCM2-7 helicase becomes loaded onto DNA as a single hexamer during ORC/Cdc6/Cdt1/MCM2-7 complex formation prior to MCM2-7 double hexamer formation. Our study establishes the existence of a unique DNA entry gate for regulated helicase loading, revealing key mechanisms in helicase loading, which has important implications for helicase activation.


Assuntos
DNA Fúngico/metabolismo , Proteínas de Manutenção de Minicromossomo/metabolismo , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/enzimologia , Trifosfato de Adenosina/metabolismo , Ciclo Celular , Cromossomos Fúngicos/metabolismo , Ativação Enzimática , Hidrólise , Proteínas de Manutenção de Minicromossomo/química , Proteínas de Manutenção de Minicromossomo/genética , Proteínas de Manutenção de Minicromossomo/ultraestrutura , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestrutura , Subunidades Proteicas/química , Subunidades Proteicas/genética , Origem de Replicação/fisiologia , Saccharomyces cerevisiae/genética
4.
Mol Cell ; 50(4): 577-88, 2013 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-23603117

RESUMO

In Saccharomyces cerevisiae and higher eukaryotes, the loading of the replicative helicase MCM2-7 onto DNA requires the combined activities of ORC, Cdc6, and Cdt1. These proteins load MCM2-7 in an unknown way into a double hexamer around DNA. Here we show that MCM2-7 recruitment by ORC/Cdc6 is blocked by an autoinhibitory domain in the C terminus of Mcm6. Interestingly, Cdt1 can overcome this inhibitory activity, and consequently the Cdt1-MCM2-7 complex activates ORC/Cdc6 ATP-hydrolysis to promote helicase loading. While Cdc6 ATPase activity is known to facilitate Cdt1 release and MCM2-7 loading, we discovered that Orc1 ATP-hydrolysis is equally important in this process. Moreover, we found that Orc1/Cdc6 ATP-hydrolysis promotes the formation of the ORC/Cdc6/MCM2-7 (OCM) complex, which functions in MCM2-7 double-hexamer assembly. Importantly, CDK-dependent phosphorylation of ORC inhibits OCM establishment to ensure once per cell cycle replication. In summary, this work reveals multiple critical mechanisms that redefine our understanding of DNA licensing.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Complexos Multiproteicos/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação/genética , Western Blotting , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Quinases Ciclina-Dependentes/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Hidrólise , Modelos Biológicos , Complexos Multiproteicos/genética , Mutação , Complexo de Reconhecimento de Origem/genética , Fosforilação , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
5.
J Biol Chem ; 291(14): 7267-85, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26865637

RESUMO

Hexokinase 2 (Hxk2) fromSaccharomyces cerevisiaeis a bi-functional enzyme, being both a catalyst in the cytosol and an important regulator of the glucose repression signal in the nucleus. Despite considerable recent progress, little is known about the regulatory mechanism that controls nuclear Hxk2 association with theSUC2promoter chromatin and how this association is necessary forSUC2gene repression. Our data indicate that in theSUC2promoter context, Hxk2 functions through a variety of structurally unrelated factors, mainly the DNA-binding Mig1 and Mig2 repressors and the regulatory Snf1 and Reg1 factors. Hxk2 sustains the repressor complex architecture maintaining transcriptional repression at theSUC2gene. Using chromatin immunoprecipitation assays, we discovered that the Hxk2 in its open configuration, at low glucose conditions, leaves the repressor complex that induces its dissociation and promotesSUC2gene expression. In high glucose conditions, Hxk2 adopts a close conformation that promotes Hxk2 binding to the Mig1 protein and the reassembly of theSUC2repressor complex. Additional findings highlight the possibility that Hxk2 constitutes an intracellular glucose sensor that operates by changing its conformation in response to cytoplasmic glucose levels that regulate its interaction with Mig1 and thus its recruitment to the repressor complex of theSUC2promoter. Thus, our data indicate that Hxk2 is more intimately involved in gene regulation than previously thought.


Assuntos
Glucose/metabolismo , Hexoquinase/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Glucose/genética , Hexoquinase/genética , Complexos Multiproteicos/genética , Ligação Proteica , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , beta-Frutofuranosidase/biossíntese , beta-Frutofuranosidase/genética
6.
Nucleic Acids Res ; 43(21): 10238-50, 2015 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-26338774

RESUMO

During G1-phase of the cell-cycle the replicative MCM2-7 helicase becomes loaded onto DNA into pre-replicative complexes (pre-RCs), resulting in MCM2-7 double-hexamers on DNA. In S-phase, Dbf4-dependent kinase (DDK) and cyclin-dependent-kinase (CDK) direct with the help of a large number of helicase-activation factors the assembly of a Cdc45-MCM2-7-GINS (CMG) complex. However, in the absence of S-phase kinases complex assembly is inhibited, which is unexpected, as the MCM2-7 double-hexamer represents a very large interaction surface. Currently it is unclear what mechanisms restricts complex assembly and how DDK can overcome this inhibition to promote CMG-assembly. We developed an advanced reconstituted-system to study helicase activation in-solution and discovered that individual factors like Sld3 and Sld2 can bind directly to the pre-RC, while Cdc45 cannot. When Sld3 and Sld2 were incubated together with the pre-RC, we observed that competitive interactions restrict complex assembly. DDK stabilizes the Sld3/Sld2-pre-RC complex, but the complex is only short-lived, indicating an anti-cooperative mechanism. Yet, a Sld3/Cdc45-pre-RC can form in the presence of DDK and the addition of Sld2 enhances complex stability. Our results indicate that helicase activation is regulated by competitive and cooperative interactions, which restrict illegitimate complex formation and direct limiting helicase-activation factors into pre-initiation complexes.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Manutenção de Minicromossomo/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Origem de Replicação , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte/metabolismo , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Manutenção de Minicromossomo/química , Proteínas Nucleares/metabolismo
7.
Nucleic Acids Res ; 42(4): 2257-69, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24234446

RESUMO

The replicative mini-chromosome-maintenance 2-7 (MCM2-7) helicase is loaded in Saccharomyces cerevisiae and other eukaryotes as a head-to-head double-hexamer around origin DNA. At first, ORC/Cdc6 recruits with the help of Cdt1 a single MCM2-7 hexamer to form an 'initial' ORC/Cdc6/Cdt1/MCM2-7 complex. Then, on ATP hydrolysis and Cdt1 release, the 'initial' complex is transformed into an ORC/Cdc6/MCM2-7 (OCM) complex. However, it remains unclear how the OCM is subsequently converted into a MCM2-7 double-hexamer. Through analysis of MCM2-7 hexamer-interface mutants we discovered a complex competent for MCM2-7 dimerization. We demonstrate that these MCM2-7 mutants arrest during prereplicative complex (pre-RC) assembly after OCM formation, but before MCM2-7 double-hexamer assembly. Remarkably, only the OCM complex, but not the 'initial' ORC/Cdc6/Cdt1/MCM2-7 complex, is competent for MCM2-7 dimerization. The MCM2-7 dimer, in contrast to the MCM2-7 double-hexamer, interacts with ORC/Cdc6 and is salt-sensitive, classifying the arrested complex as a helicase-loading intermediate. Accordingly, we found that overexpression of the mutants cause cell-cycle arrest and dominant lethality. Our work identifies the OCM complex as competent for MCM2-7 dimerization, reveals MCM2-7 dimerization as a limiting step during pre-RC formation and defines critical mechanisms that explain how origins are licensed.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Pontos de Checagem do Ciclo Celular , Proteínas de Ligação a DNA/metabolismo , Componente 7 do Complexo de Manutenção de Minicromossomo/química , Componente 7 do Complexo de Manutenção de Minicromossomo/genética , Proteínas de Manutenção de Minicromossomo/química , Proteínas de Manutenção de Minicromossomo/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Dados de Sequência Molecular , Mutação , Multimerização Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
8.
Nucleic Acids Res ; 41(5): 3162-72, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23376927

RESUMO

The origin recognition complex (ORC) of Saccharomyces cerevisiae binds origin DNA and cooperates with Cdc6 and Cdt1 to load the replicative helicase MCM2-7 onto DNA. Helicase loading involves two MCM2-7 hexamers that assemble into a double hexamer around double-stranded DNA. This reaction requires ORC and Cdc6 ATPase activity, but it is unknown how these proteins control MCM2-7 double hexamer formation. We demonstrate that mutations in Cdc6 sensor-2 and Walker A motifs, which are predicted to affect ATP binding, influence the ORC-Cdc6 interaction and MCM2-7 recruitment. In contrast, a Cdc6 sensor-1 mutant affects MCM2-7 loading and Cdt1 release, similar as a Cdc6 Walker B ATPase mutant. Moreover, we show that Orc1 ATP hydrolysis is not involved in helicase loading or in releasing ORC from loaded MCM2-7. To determine whether Cdc6 regulates MCM2-7 double hexamer formation, we analysed complex assembly. We discovered that inhibition of Cdc6 ATPase restricts MCM2-7 association with origin DNA to a single hexamer, while active Cdc6 ATPase promotes recruitment of two MCM2-7 hexamer to origin DNA. Our findings illustrate how conserved Cdc6 AAA+ motifs modulate MCM2-7 recruitment, show that ATPase activity is required for MCM2-7 hexamer dimerization and demonstrate that MCM2-7 hexamers are recruited to origins in a consecutive process.


Assuntos
Proteínas de Ciclo Celular/química , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae/química , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Substituição de Aminoácidos , Proteínas de Ciclo Celular/genética , Linhagem Celular , Proteínas Cromossômicas não Histona/química , Replicação do DNA , DNA Fúngico/química , Proteínas de Ligação a DNA/química , Hidrólise , Componente 7 do Complexo de Manutenção de Minicromossomo , Mutagênese Sítio-Dirigida , Proteínas Nucleares/química , Complexo de Reconhecimento de Origem/antagonistas & inibidores , Complexo de Reconhecimento de Origem/química , Complexo de Reconhecimento de Origem/genética , Ligação Proteica , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética
9.
Structure ; 32(8): 1137-1149.e4, 2024 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-38815576

RESUMO

Two pore channels are lysosomal cation channels with crucial roles in tumor angiogenesis and viral release from endosomes. Inhibition of the two-pore channel 2 (TPC2) has emerged as potential therapeutic strategy for the treatment of cancers and viral infections, including Ebola and COVID-19. Here, we demonstrate that antagonist SG-094, a synthetic analog of the Chinese alkaloid medicine tetrandrine with increased potency and reduced toxicity, induces asymmetrical structural changes leading to a single binding pocket at only one intersubunit interface within the asymmetrical dimer. Supported by functional characterization of mutants by Ca2+ imaging and patch clamp experiments, we identify key residues in S1 and S4 involved in compound binding to the voltage sensing domain II. SG-094 arrests IIS4 in a downward shifted state which prevents pore opening via the IIS4/S5 linker, hence resembling gating modifiers of canonical VGICs. These findings may guide the rational development of new therapeutics antagonizing TPC2 activity.


Assuntos
Canais de Cálcio , Humanos , Canais de Cálcio/metabolismo , Canais de Cálcio/química , Sítios de Ligação , Lisossomos/metabolismo , Células HEK293 , Ligação Proteica , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/metabolismo , Modelos Moleculares , Canais de Dois Poros
10.
J Vis Exp ; (199)2023 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-37843272

RESUMO

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening.


Assuntos
Proteínas de Membrana Transportadoras , Proteínas Carreadoras de Solutos , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Carreadoras de Solutos/química , Proteínas Carreadoras de Solutos/metabolismo , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala , Proteínas de Membrana/metabolismo , Preparações Farmacêuticas
11.
BMC Cell Biol ; 13: 31, 2012 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-23131016

RESUMO

BACKGROUND: Mig2 has been described as a transcriptional factor that in the absence of Mig1 protein is required for glucose repression of the SUC2 gene. Recently it has been reported that Mig2 has two different subcellular localizations. In high-glucose conditions it is a nuclear modulator of several Mig1-regulated genes, but in low-glucose most of the Mig2 protein accumulates in mitochondria. Thus, the Mig2 protein enters and leaves the nucleus in a glucose regulated manner. However, the mechanism by which Mig2 enters into the nucleus was unknown until now. RESULTS: Here, we report that the Mig2 protein is an import substrate of the carrier Kap95 (importin-ß). The Mig2 nuclear import mechanism bypasses the requirement for Kap60 (importin-α) as an adaptor protein, since Mig2 directly binds to Kap95 in the presence of Gsp1(GDP). We also show that the Mig2 nuclear import and the binding of Mig2 with Kap95 are not glucose-dependent processes and require a basic NLS motif, located between lysine-32 and arginine-37. Mig2 interaction with Kap95 was assessed in vitro using purified proteins, demonstrating that importin-ß, together with the GTP-binding protein Gsp1, is able to mediate efficient Mig2-Kap95 interaction in the absence of the importin-α (Kap60). It was also demonstrated, that the directionality of Mig2 transport is regulated by association with the small GTPase Gsp1 in the GDP- or GTP-bound forms, which promote cargo recognition and release, respectively. CONCLUSIONS: The Mig2 protein accumulates in the nucleus through a Kap95 and NLS-dependent nuclear import pathway, which is independent of importin-α in Saccharomyces cerevisiae.


Assuntos
Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Motivos de Aminoácidos , Núcleo Celular/metabolismo , Carioferinas/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Repressoras/química , Proteínas de Saccharomyces cerevisiae/química , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , beta Carioferinas/genética
12.
Nat Commun ; 13(1): 4087, 2022 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-35840580

RESUMO

Kv3 channels have distinctive gating kinetics tailored for rapid repolarization in fast-spiking neurons. Malfunction of this process due to genetic variants in the KCNC1 gene causes severe epileptic disorders, yet the structural determinants for the unusual gating properties remain elusive. Here, we present cryo-electron microscopy structures of the human Kv3.1a channel, revealing a unique arrangement of the cytoplasmic tetramerization domain T1 which facilitates interactions with C-terminal axonal targeting motif and key components of the gating machinery. Additional interactions between S1/S2 linker and turret domain strengthen the interface between voltage sensor and pore domain. Supported by molecular dynamics simulations, electrophysiological and mutational analyses, we identify several residues in the S4/S5 linker which influence the gating kinetics and an electrostatic interaction between acidic residues in α6 of T1 and R449 in the pore-flanking S6T helices. These findings provide insights into gating control and disease mechanisms and may guide strategies for the design of pharmaceutical drugs targeting Kv3 channels.


Assuntos
Ativação do Canal Iônico , Canais de Potássio Shaw , Microscopia Crioeletrônica , Humanos , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , Canais de Potássio Shaw/química , Canais de Potássio Shaw/genética , Canais de Potássio Shaw/metabolismo , Eletricidade Estática
13.
Methods Mol Biol ; 2199: 23-43, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33125643

RESUMO

Structural genomics groups have identified the need to generate multiple truncated versions of each target to improve their success in producing a well-expressed, soluble, and stable protein and one that crystallizes and diffracts to a sufficient resolution for structural determination. At the Structural Genomics Consortium, we opted for the ligation-independent cloning (LIC) method which provides the throughput we desire to produce and screen many proteins in a parallel process. Here, we describe our LIC protocol for generating constructs in 96-well format and provide a choice of vectors suitable for expressing proteins in both E. coli and the baculovirus expression vector system (BEVS).


Assuntos
Baculoviridae/genética , Clonagem Molecular , Escherichia coli , Expressão Gênica , Vetores Genéticos/genética , Proteínas Recombinantes , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
14.
Methods Mol Biol ; 2199: 45-66, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33125644

RESUMO

In Chapter 3 , we described the Structural Genomics Consortium (SGC) process for generating multiple constructs of truncated versions of each protein using LIC. In this chapter we provide a step-by-step procedure of our E. coli system for test expressing intracellular (soluble) proteins in a 96-well format that enables us to identify which proteins or truncated versions are expressed in a soluble and stable form suitable for structural studies. In addition, we detail the process for scaling up cultures for large-scale protein purification. This level of production is required to obtain sufficient quantities (i.e., milligram amounts) of protein for further characterization and/or structural studies (e.g., crystallization or cryo-EM experiments). Our standard process is purification by immobilized metal affinity chromatography (IMAC) using nickel resin followed by size exclusion chromatography (SEC), with additional procedures arising from the complexity of the protein itself.


Assuntos
Cromatografia de Afinidade , Cromatografia em Gel , Escherichia coli , Proteínas Recombinantes , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
15.
Methods Mol Biol ; 2199: 67-94, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33125645

RESUMO

This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the baculovirus expression vector system (BEVS). This eukaryotic expression system was selected and a screening process established in 2007 as a measure to tackle the more challenging kinase, RNA-DNA processing, and integral membrane protein families on our target list. Here, we discuss our platform for identifying soluble proteins from 3 mL of insect cell culture and describe the procedures involved in producing protein from liter-scale cultures.


Assuntos
Baculoviridae/genética , Vetores Genéticos/genética , Proteínas de Membrana , Animais , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Células Sf9 , Spodoptera
16.
Methods Mol Biol ; 2199: 95-115, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33125646

RESUMO

This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the BacMam system. This eukaryotic expression system was selected and a screening process established in 2016 to enable production of highly challenging human integral membrane proteins (IMPs), which are a significant component of our target list. Here, we discuss our recently developed platform for identifying expression and monodispersity of IMPs from 3 mL of HEK293 cells.


Assuntos
Expressão Gênica , Vetores Genéticos/genética , Proteínas de Membrana , Células HEK293 , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
17.
Commun Biol ; 4(1): 934, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34345007

RESUMO

We describe an analytical method for the identification, mapping and relative quantitation of glycopeptides from SARS-CoV-2 Spike protein. The method may be executed using a LC-TOF mass spectrometer, requires no specialized knowledge of glycan analysis and exploits the differential resolving power of reverse phase HPLC. While this separation technique resolves peptides with high efficiency, glycans are resolved poorly, if at all. Consequently, glycopeptides consisting of the same peptide bearing different glycan structures will all possess very similar retention times and co-elute. Rather than a disadvantage, we show that shared retention time can be used to map multiple glycan species to the same peptide and location. In combination with MSMS and pseudo MS3, we have constructed a detailed mass-retention time database for Spike glycopeptides. This database allows any accurate mass LC-MS laboratory to reliably identify and quantify Spike glycopeptides from a single overnight elastase digest in less than 90 minutes.


Assuntos
Glicopeptídeos/química , Espectrometria de Massas/métodos , Glicoproteína da Espícula de Coronavírus/química , Bases de Dados de Proteínas , Fatores de Tempo
18.
Nat Struct Mol Biol ; 20(8): 944-51, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23851460

RESUMO

In eukaryotes, the Cdt1-bound replicative helicase core MCM2-7 is loaded onto DNA by the ORC-Cdc6 ATPase to form a prereplicative complex (pre-RC) with an MCM2-7 double hexamer encircling DNA. Using purified components in the presence of ATP-γS, we have captured in vitro an intermediate in pre-RC assembly that contains a complex between the ORC-Cdc6 and Cdt1-MCM2-7 heteroheptamers called the OCCM. Cryo-EM studies of this 14-subunit complex reveal that the two separate heptameric complexes are engaged extensively, with the ORC-Cdc6 N-terminal AAA+ domains latching onto the C-terminal AAA+ motor domains of the MCM2-7 hexamer. The conformation of ORC-Cdc6 undergoes a concerted change into a right-handed spiral with helical symmetry that is identical to that of the DNA double helix. The resulting ORC-Cdc6 helicase loader shows a notable structural similarity to the replication factor C clamp loader, suggesting a conserved mechanism of action.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona/química , Proteínas de Ligação a DNA/química , Modelos Moleculares , Complexos Multiproteicos/química , Complexo de Reconhecimento de Origem/química , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Clonagem Molecular , Microscopia Crioeletrônica , Replicação do DNA/fisiologia , DNA Fúngico/química , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Processamento de Imagem Assistida por Computador , Imunoprecipitação , Mutagênese Sítio-Dirigida , Complexo de Reconhecimento de Origem/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
19.
Mitochondrion ; 12(3): 370-80, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22353369

RESUMO

Mig2 has been described as a transcriptional factor that in the absence of Mig1 protein is required for glucose repression of the SUC2 gene. Thus, until now, the main role assigned to Mig2 has been the functional redundancy to Mig1. In this study, we report that Mig2 has a double subcellular localization. As expected, in high-glucose conditions it is accumulated in the nucleus but in low-glucose conditions Mig2 has an unexpected mitochondrial localization and role in mitochondrial morphology. We describe that Mig2 physically interacts with the mitochondrial protein Ups1 in a glucose-dependent manner. We also show that Δmig2 mutant cells exhibit a fragmented network of mitochondrial tubules, a phenotype similarly observed in cells lacking Fzo1 and Ups1. Furthermore, Mig2 acts antagonistically with respect to the fission-promoting components, because mitochondrial aggregation induced by DNM1 deletion was rescued in the Δdnm1Δmig2 double mutant. Thus, our studies have revealed an additional role for Mig2 as a novel factor required for the maintenance of fusion-competent mitochondria in Saccharomyces cerevisiae and strongly suggest that Mig2 could be involved in the cross talk between the nucleus and the mitochondria through Ups1 to regulate mitochondrial morphology in a glucose dependent manner.


Assuntos
Núcleo Celular/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Mitocôndrias/metabolismo , Proteínas Repressoras/efeitos dos fármacos , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Glucose/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas Repressoras/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
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