A class-A GPCR solubilized under high hydrostatic pressure retains its ligand binding ability.

The effect of high hydrostatic pressure (HHP) on the solubilization of a class-A G protein-coupled receptor, the silkmoth pheromone biosynthesis-activating neuropeptide receptor (PBANR), was investigated. PBANR was expressed in expresSF+ insect cells as a C-terminal fusion protein with EGFP. The membrane fraction was subjected to HHP treatment (200MPa) at room temperature for 1-16h in the presence of 0-2.0% (w/v) n-dodecyl-ß-D-maltopyranoside (DDM). The solubilization yield of PBANR-EGFP in the presence of 0.6% (w/v) DDM increased to ~1.5-fold after 1h HHP treatment. Fluorescence-detection size-exclusion chromatography demonstrated that the PBANR-EGFP ligand binding ability was retained after HHP-mediated solubilization. The PBANR-EGFP solubilized with 1.0% DDM under HHP at room temperature for 6h retained ligand binding ability, whereas solubilization in the absence of HHP treatment resulted in denaturation.

Structure and function of the histone chaperone CIA/ASF1 complexed with histones H3 and H4.

CIA (CCG1-interacting factor A)/ASF1, which is the most conserved histone chaperone among the eukaryotes, was genetically identified as a factor for an anti-silencing function (Asf1) by yeast genetic screening. Shortly after that, the CIA-histone-H3-H4 complex was isolated from Drosophila as a histone chaperone CAF-1 stimulator. Human CIA-I/II (ASF1a/b) was identified as a histone chaperone that interacts with the bromodomain-an acetylated-histone-recognizing domain-of CCG1, in the general transcription initiation factor TFIID. Intensive studies have revealed that CIA/ASF1 mediates nucleosome assembly by forming a complex with another histone chaperone in human cells and yeast, and is involved in DNA replication, transcription, DNA repair and silencing/anti-silencing in yeast. CIA/ASF1 was shown as a major storage chaperone for soluble histones in proliferating human cells. Despite all these biochemical and biological functional analyses, the structure-function relationship of the nucleosome assembly/disassembly activity of CIA/ASF1 has remained elusive. Here we report the crystal structure, at 2.7 A resolution, of CIA-I in complex with histones H3 and H4. The structure shows the histone H3-H4 dimer's mutually exclusive interactions with another histone H3-H4 dimer and CIA-I. The carboxy-terminal beta-strand of histone H4 changes its partner from the beta-strand in histone H2A to that of CIA-I through large conformational change. In vitro functional analysis demonstrated that CIA-I has a histone H3-H4 tetramer-disrupting activity. Mutants with weak histone H3-H4 dimer binding activity showed critical functional effects on cellular processes related to transcription. The histone H3-H4 tetramer-disrupting activity of CIA/ASF1 and the crystal structure of the CIA/ASF1-histone-H3-H4 dimer complex should give insights into mechanisms of both nucleosome assembly/disassembly and nucleosome semi-conservative replication.

Structure of the histone chaperone CIA/ASF1-double bromodomain complex linking histone modifications and site-specific histone eviction.

Nucleosomes around the promoter region are disassembled for transcription in response to various signals, such as acetylation and methylation of histones. Although the interactions between histone-acetylation-recognizing bromodomains and factors involved in nucleosome disassembly have been reported, no structural basis connecting histone modifications and nucleosome disassembly has been obtained. Here, we determined at 3.3 A resolution the crystal structure of histone chaperone cell cycle gene 1 (CCG1) interacting factor A/antisilencing function 1 (CIA/ASF1) in complex with the double bromodomain in the CCG1/TAF1/TAF(II)250 subunit of transcription factor IID. Structural, biochemical, and biological studies suggested that interaction between double bromodomain and CIA/ASF1 is required for their colocalization, histone eviction, and pol II entry at active promoter regions. Furthermore, the present crystal structure has characteristics that can connect histone acetylation and CIA/ASF1-mediated histone eviction. These findings suggest that the molecular complex between CIA/ASF1 and the double bromodomain plays a key role in site-specific histone eviction at active promoter regions. The model we propose here is the initial structure-based model of the biological signaling from histone modifications to structural change of the nucleosome (hi-MOST model).

Expression, purification and crystallization of N-acetyl-(R)-ß-phenylalanine acylases derived from Burkholderia sp. AJ110349 and Variovorax sp. AJ110348 and structure determination of the Burkholderia enzyme.

N-Acetyl-(R)-ß-phenylalanine acylase is an enzyme that hydrolyzes the amide bond of N-acetyl-(R)-ß-phenylalanine to produce enantiopure (R)-ß-phenylalanine. In previous studies, Burkholderia sp. AJ110349 and Variovorax sp. AJ110348 were isolated as (R)-enantiomer-specific N-acetyl-(R)-ß-phenylalanine acylase-producing organisms and the properties of the native enzyme from Burkholderia sp. AJ110349 were characterized. In this study, structural analyses were carried out in order to investigate the structure-function relationships of the enzymes derived from both organisms. The recombinant N-acetyl-(R)-ß-phenylalanine acylases were crystallized by the hanging-drop vapor-diffusion method under multiple crystallization solution conditions. The crystals of the Burkholderia enzyme belonged to space group P41212, with unit-cell parameters a = b = 112.70-112.97, c = 341.50-343.32 Å, and were likely to contain two subunits in the asymmetric unit. The crystal structure was solved by the Se-SAD method, suggesting that two subunits in the asymmetric unit form a dimer. Each subunit was composed of three domains, and they showed structural similarity to the corresponding domains of the large subunit of N,N-dimethylformamidase from Paracoccus sp. strain DMF. The crystals of the Variovorax enzyme grew as twinned crystals and were not suitable for structure determination. Using size-exclusion chromatography with online static light-scattering analysis, the N-acetyl-(R)-ß-phenylalanine acylases were clarified to be dimeric in solution.

Roles of histone chaperone CIA/Asf1 in nascent DNA elongation during nucleosome replication.

The nucleosome, which is composed of DNA wrapped around a histone octamer, is a fundamental unit of chromatin and is duplicated during the eukaryotic DNA replication process. The evolutionarily conserved histone chaperone cell cycle gene 1 (CCG1) interacting factor A/anti-silencing function 1 (CIA/Asf1) is involved in histone transfer and nucleosome reassembly during DNA replication. CIA/Asf1 has been reported to split the histone (H3-H4)(2) tetramer into histone H3-H4 dimer(s) in vitro, raising a possibility that, in DNA replication, CIA/Asf1 is involved in nucleosome disassembly and the promotion of semi-conservative histone H3-H4 dimer deposition onto each daughter strand in vivo. Despite numerous studies on the functional roles of CIA/Asf1, its mechanistic role(s) remains elusive because of lack of biochemical analyses. The biochemical studies described here show that a V94R CIA/Asf1 mutant, which lacks histone (H3-H4)(2) tetramer splitting activity, does not form efficiently a quaternary complex with histones H3-H4 and the minichromosome maintenance 2 (Mcm2) subunit of the Mcm2-7 replicative DNA helicase. Interestingly, the mutant enhances nascent DNA strand synthesis in a cell-free chromosomal DNA replication system using Xenopus egg extracts. These results suggest that CIA/Asf1 in the CIA/Asf1-H3-H4-Mcm2 complex, which is considered to be an intermediate in histone transfer during DNA replication, negatively regulates the progression of the replication fork.

Regulatory system of the protocatechuate 4,5-cleavage pathway genes essential for lignin downstream catabolism.

Sphingobium sp. strain SYK-6 converts various lignin-derived biaryls with guaiacyl (4-hydroxy-3-methoxyphenyl) and syringyl (4-hydroxy-3,5-dimethoxyphenyl) moieties to vanillate and syringate. These compounds are further catabolized through the protocatechuate (PCA) 4,5-cleavage (PCA45) pathway. In this article, the regulatory system of the PCA45 pathway is described. A LysR-type transcriptional regulator (LTTR), LigR, activated the transcription of the ligK-orf1-ligI-lsdA and ligJABC operons in the presence of PCA or gallate (GA), which is an intermediate metabolite of vanillate or syringate, respectively, and repressed transcription of its own gene. LigR bound to the positions -77 to -51 and -80 to -48 of the ligK and ligJ promoters, respectively, and induced DNA bending. In the presence of PCA or GA, DNA bending on both promoters was enhanced. The LigR-binding regions of the ligK and ligJ promoters in the presence of inducer molecules were extended and shortened, respectively. The LTTR consensus sequences (Box-K and Box-J) in the ligK and ligJ promoters were essential for the binding of LigR and transcriptional activation of both operons. In addition, the regions between the LigR binding boxes and the -35 regions were required for the enhancement of DNA bending, although the binding of LigR to the -35 region of the ligJ promoter was not observed in DNase I footprinting experiments. This study shows the binding features of LigR on the ligK and ligJ promoters and explains how the PCA45 pathway genes are expressed during degradation of lignin-derived biaryls by this bacterium.

Crystal structure of Methanococcus jannaschii TATA box-binding protein.

As the archaeal transcription system consists of a eukaryotic-type transcription apparatus and bacterial-type regulatory transcription factors, analyses of the molecular interface between the transcription apparatus and regulatory transcription factors are critical to reveal the evolutionary change of the transcription system. TATA box-binding protein (TBP), the central components of the transcription apparatus are classified into three groups: eukaryotic, archaeal-I and archaeal-II TBPs. Thus, comparative functional analysis of these three groups of TBP is important for the study of the evolution of the transcription system. Here, we present the first crystal structure of an archaeal-II TBP from Methanococcus jannaschii. The highly conserved and group-specific conserved surfaces of TBP bind to DNA and TFIIB/TFB, respectively. The phylogenetic trees of TBP and TFIIB/TFB revealed that they evolved in a coupled manner. The diversified surface of TBP is negatively charged in the archaeal-II TBP, which is completely different from the case of eukaryotic and archaeal-I TBPs, which are positively charged and biphasic, respectively. This difference is responsible for the diversification of the regulatory functions of TBP during evolution.

Crystallization and preliminary crystallographic analysis of DtsR1, a carboxyltransferase subunit of acetyl-CoA carboxylase from Corynebacterium glutamicum.

DtsR1, a carboxyltransferase subunit of acetyl-CoA carboxylase derived from Corynebacterium glutamicum, was crystallized by the sitting-drop vapour-diffusion method using polyethylene glycol 6000 as a precipitant. The crystal belongs to the trigonal system with space group R32 and contains three subunits in the asymmetric unit. A molecular-replacement solution was found using the structure of transcarboxylase 12S from Propionibacterium shermanii as a search model.

Crystallization and preliminary crystallographic analysis of a haloalkane dehalogenase, DbjA, from Bradyrhizobium japonicum USDA110.

Haloalkane dehalogenases are key enzymes for the degradation of halogenated aliphatic pollutants. The haloalkane dehalogenase DbjA constitutes a novel substrate-specificity class with high catalytic activity for beta-methylated haloalkanes. In order to reveal the mechanism of its substrate specificity, DbjA has been crystallized using the hanging-drop vapour-diffusion method. The best crystals were obtained using the microseeding technique with a reservoir solution consisting of 17-19.5%(w/v) PEG 4000, 0.2 M calcium acetate and 0.1 M Tris-HCl pH 7.7-8.0. The space group of the DbjA crystal is P2(1)2(1)2, with unit-cell parameters a = 212.9, b = 117.8, c = 55.8 A. The crystal diffracts to 1.75 A resolution.

Crystal structure of a gamma-butyrolactone autoregulator receptor protein in Streptomyces coelicolor A3(2).

The gamma-butyrolactone-type autoregulator/receptor systems in the Gram-positive bacterial genus Streptomyces regulate morphological differentiation or antibiotic production, or both. The autoregulator receptors act as DNA-binding proteins, and on binding their cognate ligands (gamma-butyrolactones) they are released from the DNA, thus serving as repressors. The crystal structure of CprB in Streptomyces coelicolor A3(2), a homologue of the A-factor-receptor protein, ArpA, in Streptomyces griseus, was determined. The overall structure of CprB shows that the gamma-butyrolactone receptors belong to the TetR family. CprB is composed of two domains, a DNA-binding domain and a regulatory domain. The regulatory domain contains a hydrophobic cavity, which probably serves as a ligand-binding pocket. On the basis of the crystal structure of CprB and on the analogy of the characteristics of ligand-TetR binding, the binding of gamma-butyrolactones to the regulatory domain of the receptors is supposed to induce the relocation of the DNA-binding domain through conformational changes of residues located between the ligand-binding site and the DNA-binding domain, which would result in the dissociation of the receptors from their target DNA.

Purification, crystallization and preliminary X-ray analysis of Methanococcus jannaschii TATA box-binding protein (TBP).

TATA box-binding protein (TBP) from Methanococcus jannaschii has been crystallized by the hanging-drop vapour-diffusion method using PEG MME 2000 as a precipitant. The crystal belongs to space group P21, with unit-cell parameters a = 53.2, b = 55.5, c = 123.4 A,a = 90.0, fi = 91.0, y = 90.0 degrees, and contains four molecules in the asymmetric unit. A data set was collected to 1.9 A resolution using synchrotron radiation. A molecular-replacement solution was found using the structure of TBP from Sulfolobus acidocaldarius as a model. Crystallographic refinement is in progress.

Crystallization of CprB, an autoregulator-receptor protein from Streptomyces coelicolor A3(2).

CprB, an autoregulator-receptor protein from Streptomyces coelicolor A3(2), was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 6000 as a precipitant. Three crystal forms (I, II and III) were obtained; crystal forms I and II were useful for structure determination. Form I crystals belong to an orthorhombic system with space group P2(1)2(1)2(1) and diffracted to better than 2.4 A. Form II crystals belong to a tetragonal system with space group P4(1(3))2(1)2.