Takashi Kuzuhara

1H nuclear magnetic resonance study of the solution conformation of an antibacterial protein, sapecin.

The solution conformation of an antibacterial protein sapecin has been determined by 1H nuclear magnetic resonance (NMR) and dynamical simulated annealing calculations. It has been shown that the polypeptide fold consists of one flexible loop (residues 4–12), one helix (residues 15–23), and two extended strands (residues 24–31 and 34–40). It was found that the tertiary structure of sapecin is completely different from that of rabbit neutrophil defensin NP‐5, which is homologous to sapecin in the amino acid sequences and also has the antibacterial activity. The three‐dimensional structure determination has revealed that a basic‐residue rich region and the hydrophobic surface face each other on the surface of sapecin.

A nuclear FK506-binding protein is a histone chaperone regulating rDNA silencing

We report a novel chromatin-modulating factor, nuclear FK506-binding protein (FKBP). It is a member of the peptidyl prolyl cis-trans isomerase (PPIase) family, whose members were originally identified as enzymes that assist in the proper folding of polypeptides. The endogenous FKBP gene is required for the in vivo silencing of gene expression at the rDNA locus and FKBP has histone chaperone activity in vitro. Both of these properties depend on the N-terminal non-PPIase domain of the protein. The C-terminal PPIase domain is not essential for the histone chaperone activity in vitro, but it regulates rDNA silencing in vivo. Chromatin immunoprecipitation showed that nuclear FKBP associates with chromatin at rDNA loci in vivo. These in vivo and in vitro findings in nuclear FKBPs reveal a hitherto unsuspected link between PPIases and the alteration of chromatin structure.

Identification and characterization of CIA/ASF1 as an interactor of bromodomains associated with TFIID

General transcription initiation factor IID (TFIID) plays a central and critical role in transcription initiation from both naked and chromatin templates. Although interaction between several DNA-binding proteins and TFIID were identified and well characterized, functional linkage between TFIID and chromatin factors has remained to be elucidated. Here we show the identification and characterization of human CIA/hASF1 (identified previously as a histone chaperone) as an interactor of two tandem bromodomain modules of human (h)TAFII250/CCG1, the largest subunit of TFIID. Although yeast (y)TAFII145, a homologue of hTAFII250/CCG1 in Saccharomyces cerevisiae, lacks bromodomains, glutathione S-transferase pull-down and immunoprecipitation assays revealed that Asf1p (antisilencing function 1), the counterpart of CIA in S. cerevisiae, interacts with Bdf1p (bromodomain factor 1), which is reported to serve as the missing bromodomain in yTAFII145. Furthermore, yeast strain lacking the BDF1 gene shows the Spt phenotype that is shown also by the ASF1 gene disruptant, and a double-knockout strain of both genes shows synthetic lethality, indicating that ASF1 genetically interacts with bromodomains associated with yTFIID. We also found that Asf1p coprecipitates with yTFIID subunits from yeast whole-cell extract, and overexpression of yTFIID subunits suppress the Spt phenotype caused by gene disruption of the ASF1. This study describes the functional linkage between TFIID and a histone chaperone.

Functional interaction of general transcription initiation factor TFIIE with general chromatin factor SPT16/CDC68.

Transcriptional initiation of class II genes is one of the major targets for the regulation of gene expression and is carried out by RNA polymerase II and many auxiliary factors, which include general transcription initiation factors (GTFs). TFIIE, one of the GTFs, functions at the later stage of transcription initiation. As recent studies indicated the possibility that TFIIE may have a role in chromatin transcriptional regulation, we isolated TFIIE‐interacting factors which have chromatin‐related functions.

The Crystal Structure of CCG1/TAFII250-interacting Factor B (CIB)

The general transcription initiation factor TFIID and its interactors play critical roles in regulating the transcription from both naked and chromatin DNA. We have isolated a novel TFIID interactor that we denoted as CCG1/TAFII250-interacting factor B (CIB). We show here that CIB activates transcription. To further understand the function of this protein, we determined its crystal structure at 2.2-Å resolution. The tertiary structure of CIB reveals an α/β-hydrolase fold that resembles structures in the prokaryotic α/β-hydrolase family proteins. It is not similar in structure or primary sequence to any eukaryotic transcription or chromatin factors that have been reported to date. CIB possesses a conserved catalytic triad that is found in other α/β-hydrolases, and our in vitro studies confirmed that it bears hydrolase activity. However, CIB differs from other α/β-hydrolases in that it lacks a binding site excursion, which facilitates the substrate selectivity of the other α/β-hydrolases. Further functional characterization of CIB based on its tertiary structure and through biochemical studies may provide novel insights into the mechanisms that regulate eukaryotic transcription.

Purification, crystallization and preliminary X-ray crystallographic analysis of human CCG1-interacting factor B.

A novel human factor CIB (CCG1-interacting factor B) has been isolated using the yeast two-hybrid system. The 22 kDa CIB protein has been expressed in Escherichia coli, purified to homogeneity and crystallized in a form suitable for crystallographic studies. The protein was crystallized in the orthogonal space group P2(1)2(1)2(1), with unit-cell parameters a = 43.60 (2), b = 44.45 (1), c = 110.70 (5) A and one molecule in the asymmetric unit. The crystal diffracted beyond 2.2 A resolution using synchrotron radiation.

The sequence of a Xenopus laevis TFIID subunit reveals remarkable conservation among vertebrates

A cDNA clone encoding a Xenopus laevis (Xl) homologue of human transcription factor IID (TFIID) subunit p80 was isolated and sequenced. The deduced 618-amino-acid (aa) sequence was compared to the homologous from human, mouse, rat and Drosophila melanogaster (Dm). A highly conserved region exists in the central region among these species. In contrast, the C-terminal region has significant homology among vertebrates, whereas the corresponding region of the Dm homologue shows poor homology.

Crystallization of human CCG1-interacting factor B (CIB)

A novel human protein factor CIB (CCG1-interacting factor B) was isolated using the yeast two-hybrid system. CIB was purified and subsequently crystallized using the vapor-diffusion method. Dynamic light-scattering studies of CIB revealed this protein to be monodispersive and present as a monomer in solution. Thin plate-like crystals were grown by the hanging-drop method in the presence of ammonium sulfate as a prime precipitant. The crystals belong to the space group P2 1 2 1 2 1 , with unit cell dimensions of a = 43.88 A, b = 44.69 A, and c = 111.50 A and contain one molecule in the asymmetric unit.