RESUMEN
High-throughput protein production systems have become an important issue, because protein production is one of the bottleneck steps in large-scale structural and functional analyses of proteins. We have developed a dialysis reactor and a fully automated system for protein production using the dialysis cell-free synthesis method, which we previously established to produce protein samples on a milligram scale in a high-throughput manner. The dialysis reactor was designed to be suitable for an automated system and has six dialysis cups attached to a flat dialysis membrane. The automated system is based on a Tecan Freedom EVO 200 workstation in a three-arm configuration, and is equipped with shaking incubators, a vacuum module, a robotic centrifuge, a plate heat sealer, and a custom-made tilting carrier for collection of reaction solutions from the flat-bottom cups with dialysis membranes. The consecutive process, from the dialysis cell-free protein synthesis to the partial purification by immobilized metal affinity chromatography on a 96-well filtration plate, was performed within ca. 14h, including 8h of cell-free protein synthesis. The proteins were eluted stepwise in a high concentration using EDTA by centrifugation, while the resin in the filtration plate was washed on the vacuum manifold. The system was validated to be able to simultaneously and automatically produce up to 96 proteins in yields of several milligrams with high well-to-well reliability, sufficient for structural and functional analyses of proteins. The protein samples produced by the automated system have been utilized for NMR screening to judge the protein foldedness and for structure determinations using heteronuclear multi-dimensional NMR spectroscopy. The automated high-throughput protein production system represents an important breakthrough in the structural and functional studies of proteins and has already contributed a massive amount of results in the structural genomics project at the RIKEN Structural Genomics/Proteomics Initiative (RSGI).
Asunto(s)
Automatización de Laboratorios/instrumentación , Sistema Libre de Células , Diálisis/instrumentación , Biosíntesis de Proteínas , Proteínas/aislamiento & purificación , Cromatografía de Afinidad/instrumentación , Cromatografía de Afinidad/métodos , Diálisis/métodos , Electroforesis en Gel de Poliacrilamida , Diseño de Equipo , Histidina , Oligopéptidos , Proteínas/genéticaRESUMEN
SWIRM is a conserved domain found in several chromatin-associated proteins. Based on their sequences, the SWIRM family members can be classified into three subfamilies, which are represented by Swi3, LSD1, and Ada2. Here we report the SWIRM structure of human MYb-like, Swirm and Mpn domain-containing protein-1 (MYSM1). The MYSM1 SWIRM structure forms a compact HTH-related fold comprising five alpha-helices, which best resembles the Swi3 SWIRM structure, among the known SWIRM structures. The MYSM1 and Swi3 SWIRM structures are more similar to the LSD1 structure than the Ada2alpha structure. The SWIRM domains of MYSM1 and LSD1 lacked DNA binding activity, while those of Ada2alpha and the human Swi3 counterpart, SMARCC2, bound DNA. The dissimilarity in the DNA-binding ability of the MYSM1 and SMARCC2 SWIRM domains might be due to a couple of amino acid differences in the last helix. These results indicate that the SWIRM family has indeed diverged into three structural subfamilies (Swi3/MYSM1, LSD1, and Ada2 types), and that the Swi3/MYSM1-type subfamily has further diverged into two functionally distinct groups. We also solved the structure of the SANT domain of MYSM1, and demonstrated that it bound DNA with a similar mode to that of the c-Myb DNA-binding domain.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , ADN/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Soluciones , Relación Estructura-Actividad , Transactivadores , Proteasas Ubiquitina-EspecíficasRESUMEN
Microtubule-associated protein/microtubule affinity-regulating kinases (MARKs)/PAR-1 are common regulators of cell polarity that are conserved from nematode to human. All of these kinases have a highly conserved C-terminal domain, which is termed the kinase-associated domain 1 (KA1), although its function is unknown. In this study, we determined the solution structure of the KA1 domain of mouse MARK3 by NMR spectroscopy. We found that approximately 50 additional residues preceding the previously defined KA1 domain are required for its proper folding. The newly defined KA1 domain adopts a compact alpha+beta structure with a betaalphabetabetabetabetaalpha topology. We also found a characteristic hydrophobic, concave surface surrounded by positively charged residues. This concave surface includes the highly conserved Glu-Leu-Lys-Leu motif at the C terminus, indicating that it is important for the function of the KA1 domain.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas Serina-Treonina Quinasas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Proteínas Asociadas a Microtúbulos/química , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia/métodos , Homología de Secuencia de Aminoácido , Solventes , Homología Estructural de ProteínaRESUMEN
SWIRM is an evolutionarily conserved domain involved in several chromatin-modifying complexes. Recently, the LSD1 protein, which bears a SWIRM domain, was found to be a demethylase for Lys4-methylated histone H3. Here, we report a solution structure of the SWIRM domain of human LSD1. It forms a compact fold composed of 6 alpha helices, in which a 20 amino acid long helix (alpha4) is surrounded by 5 other short helices. The SWIRM domain structure could be divided into the N-terminal part (alpha1-alpha3) and the C-terminal part (alpha4-alpha6), which are connected to each other by a salt bridge. While the N-terminal part forms a SWIRM-specific structure, the C-terminal part adopts a helix-turn-helix (HTH)-related fold. We discuss a model in which the SWIRM domain acts as an anchor site for a histone tail.
Asunto(s)
Ensamble y Desensamble de Cromatina , Secuencias Hélice-Giro-Hélice , Modelos Moleculares , Oxidorreductasas N-Desmetilantes/genética , Factores de Transcripción/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas de Unión al ADN , Histona Demetilasas , Histonas/análisis , Histonas/metabolismo , Datos de Secuencia Molecular , Oxidorreductasas N-Desmetilantes/química , Conformación Proteica , Estructura Terciaria de Proteína , Especificidad por Sustrato , Factores de Tiempo , Factores de Transcripción/análisisRESUMEN
SQUAMOSA promoter-binding proteins (SBPs) form a major family of plant-specific transcription factors, mainly related to flower development. SBPs share a highly conserved DNA-binding domain of approximately 80 amino acids (SBP domain), which contains two non-interleaved zinc-binding sites formed by eight conserved Cys or His residues. In the present study, an Arabidopsis SPL12 SBP-domain fragment that lacks a Cys residue involved in the C-terminal zinc-binding pocket was found to retain a folded structure, even though only a single Zn2+ ion binds to the fragment. Solution structure of this fragment determined by NMR is very similar to the previously determined structures of the full SBP domains of Arabidopsis SPL4 and SPL7. Considering the previous observations that chelating all the Zn2+ ions of SBPs resulted in the complete unfolding of the structure and that a mutation of the Cys residue equivalent to that described above impaired the DNA-binding activity, we propose that the Zn2+ ion at the N-terminal site is necessary to maintain the overall tertiary structure, while the Zn2+ ion at the C-terminal site is necessary for the DNA binding, mainly by guiding the basic C-terminal loop to correctly fit into the DNA groove.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/química , Arabidopsis/metabolismo , Fragmentos de Péptidos/química , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Zinc/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Dicroismo Circular , ADN/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Resonancia por Plasmón de SuperficieRESUMEN
Ethylene-insensitive3 (EIN3) and EIN3-like (EIL) proteins are essential transcription factors in the ethylene signaling of higher plants. The EIN3/EIL proteins bind to the promoter regions of the downstream genes and regulate their expression. The location of the DNA-binding domain (DBD) in the primary structure was unclear, since the proteins show no sequence similarity to other known DBDs. Here, we identify the major DBD of an EIN3/EIL protein, Arabidopsis thaliana EIL3, containing a key mutational site for DNA binding and signaling (ein3-3 site), and determine its solution structure by NMR spectroscopy. The structure consists of five alpha-helices, possessing a novel fold dissimilar to known DBD structures. By a chemical-shift perturbation analysis, a region including the ein3-3 site is suggested to be involved in DNA binding.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/química , Proteínas de Unión al ADN/química , ADN/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , ADN/química , ADN/genética , Proteínas de Unión al ADN/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Prolina/metabolismo , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología Estructural de Proteína , Resonancia por Plasmón de SuperficieRESUMEN
The WRKY proteins comprise a major family of transcription factors that are essential in pathogen and salicylic acid responses of higher plants as well as a variety of plant-specific reactions. They share a DNA binding domain, designated as the WRKY domain, which contains an invariant WRKYGQK sequence and a CX4-5CX22-23HXH zinc binding motif. Herein, we report the NMR solution structure of the C-terminal WRKY domain of the Arabidopsis thaliana WRKY4 protein. The structure consists of a four-stranded beta-sheet, with a zinc binding pocket formed by the conserved Cys/His residues located at one end of the beta-sheet, revealing a novel zinc and DNA binding structure. The WRKYGQK residues correspond to the most N-terminal beta-strand, kinked in the middle of the sequence by the Gly residue, which enables extensive hydrophobic interactions involving the Trp residue and contributes to the structural stability of the beta-sheet. Based on a profile of NMR chemical shift perturbations, we propose that the same strand enters the DNA groove and forms contacts with the DNA bases.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Unión al ADN/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Secuencia de Bases , Sitios de Unión , ADN de Plantas/genética , ADN de Plantas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Soluciones , Electricidad Estática , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The B3 DNA binding domain is shared amongst various plant-specific transcription factors, including factors involved in auxin-regulated and abscisic acid-regulated transcription. Herein, we report the NMR solution structure of the B3 domain of the Arabidopsis thaliana cold-responsive transcription factor RAV1. The structure consists of a seven-stranded open beta-barrel and two alpha-helices located at the ends of the barrel and is significantly similar to the structure of the noncatalytic DNA binding domain of the restriction enzyme EcoRII. An NMR titration experiment revealed a DNA recognition interface that enabled us to propose a structural model of the protein-DNA complex. The locations of the DNA-contacting residues are also likely to be similar to those of the EcoRII DNA binding domain.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Aclimatación/fisiología , Secuencia de Aminoácidos/fisiología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sitios de Unión/genética , Frío , Proteínas de Unión al ADN/genética , Desoxirribonucleasas de Localización Especificada Tipo II/química , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Evolución Molecular , Modelos Moleculares , Conformación Molecular , Resonancia Magnética Nuclear Biomolecular , Filogenia , Estructura Secundaria de Proteína/fisiología , Estructura Terciaria de Proteína/genética , Homología de Secuencia de AminoácidoRESUMEN
GCN2 is the alpha-subunit of the only translation initiation factor (eIF2alpha) kinase that appears in all eukaryotes. Its function requires an interaction with GCN1 via the domain at its N-terminus, which is termed the RWD domain after three major RWD-containing proteins: RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases. In this study, we determined the solution structure of the mouse GCN2 RWD domain using NMR spectroscopy. The structure forms an alpha + beta sandwich fold consisting of two layers: a four-stranded antiparallel beta-sheet, and three side-by-side alpha-helices, with an alphabetabetabetabetaalphaalpha topology. A characteristic YPXXXP motif, which always occurs in RWD domains, forms a stable loop including three consecutive beta-turns that overlap with each other by two residues (triple beta-turn). As putative binding sites with GCN1, a structure-based alignment allowed the identification of several surface residues in alpha-helix 3 that are characteristic of the GCN2 RWD domains. Despite the apparent absence of sequence similarity, the RWD structure significantly resembles that of ubiquitin-conjugating enzymes (E2s), with most of the structural differences in the region connecting beta-strand 4 and alpha-helix 3. The structural architecture, including the triple beta-turn, is fundamentally common among various RWD domains and E2s, but most of the surface residues on the structure vary. Thus, it appears that the RWD domain is a novel structural domain for protein-binding that plays specific roles in individual RWD-containing proteins.
Asunto(s)
Proteínas Quinasas/química , Alineación de Secuencia , Homología Estructural de Proteína , Secuencia de Aminoácidos , Animales , Ratones , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Proteínas Serina-Treonina Quinasas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , eIF-2 Quinasa/químicaRESUMEN
The BolA-like proteins are widely conserved from prokaryotes to eukaryotes. The BolA-like proteins seem to be involved in cell proliferation or cell-cycle regulation, but the molecular function is still unknown. Here we determined the structure of a mouse BolA-like protein. The overall topology is alphabetabetaalphaalphabetaalpha, in which beta(1) and beta(2) are antiparallel, and beta(3) is parallel to beta(2). This fold is similar to the class II KH fold, except for the absence of the GXXG loop, which is well conserved in the KH fold. The conserved residues in the BolA-like proteins are assembled on the one side of the protein.