Toshiya Senda

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 Å resolution, of CIA-I in complex with histones H3 and H4. The structure shows the histone H3–H4 dimers mutually exclusive interactions with another histone H3–H4 dimer and CIA-I. The carboxy-terminal β-strand of histone H4 changes its partner from the β-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.

Histone chaperones: 30 years from isolation to elucidation of the mechanisms of nucleosome assembly and disassembly

Abstract.Some three decades have passed since the discovery of nucleosomes in 1974 and the first isolation of a histone chaperone in 1978. While various types of histone chaperones have been isolated and functionally analyzed, the elementary processes of nucleosome assembly and disassembly have been less well characterized. Recently, the tertiary structure of a hetero-trimeric complex composed of the histone chaperone CIA/ASF1 and the histone H3-H4 dimer was determined, and this complex was proposed to be an intermediate in nucleosome assembly and disassembly reactions. In addition, CIA alone was biochemically shown to dissociate the histone (H3-H4)2 tetramer into two histone H3-H4 dimers. This activity suggested that CIA regulates the semi-conservative replication of nucleosomes. Here, we provide an overview of prominent histone chaperones with the goal of elucidating the mechanisms that preserve and modify epigenetic information. We also discuss the reactions involved in nucleosome assembly and disassembly.

Relationship between the structure of SET/TAF-Iβ/INHAT and its histone chaperone activity

Histone chaperones assemble and disassemble nucleosomes in an ATP-independent manner and thus regulate the most fundamental step in the alteration of chromatin structure. The molecular mechanisms underlying histone chaperone activity remain unclear. To gain insights into these mechanisms, we solved the crystal structure of the functional domain of SET/TAF-Iβ/INHAT at a resolution of 2.3 Å. We found that SET/TAF-Iβ/INHAT formed a dimer that assumed a “headphone”-like structure. Each subunit of the SET/TAF-Iβ/INHAT dimer consisted of an N terminus, a backbone helix, and an “earmuff” domain. It resembles the structure of the related protein NAP-1. Comparison of the crystal structures of SET/TAF-Iβ/INHAT and NAP-1 revealed that the two proteins were folded similarly except for an inserted helix. However, their backbone helices were shaped differently, and the relative dispositions of the backbone helix and the earmuff domain between the two proteins differed by ≈40°. Our biochemical analyses of mutants revealed that the region of SET/TAF-Iβ/INHAT that is engaged in histone chaperone activity is the bottom surface of the earmuff domain, because this surface bound both core histones and double-stranded DNA. This overlap or closeness of the activity surface and the binding surfaces suggests that the specific association among SET/TAF-Iβ/INHAT, core histones, and double-stranded DNA is requisite for histone chaperone activity. These findings provide insights into the possible mechanisms by which histone chaperones assemble and disassemble nucleosome structures.

Enantioselectivity of Haloalkane Dehalogenases and its Modulation by Surface Loop Engineering

Engineering of the surface loop in haloalkane dehalogenases affects their enantiodiscrimination behavior. The temperature dependence of the enantioselectivity (lnE versus 1/T) of -bromoalkanes by haloalkane dehalogenases is reversed (red data points) by deletion of the surface loop; the selectivity switches back when an additional single-point mutation is made. This behavior is not observed for -bromoesters.

Crystal Structure of Recombinant Native SDF-1α with Additional Mutagenesis Studies: An Attempt at a More Comprehensive Interpretation of Accumulated Structure-Activity Relationship Data

Crystal structures, forms 1 and 2, of recombinant native stromal cell-derived factor-1alpha (SDF-1alpha), expressed using the Sendai virus expression vector system, have been determined by x-ray crystallography at 2.0 A resolution. The crystal of form 1 is almost isomorphous with that used in the previous crystal structure analysis of the synthetic [N33A] mutant of SDF-1alpha (Dealwis, C., et al. Proc. Natl. Acad. Sci. USA 1998;95, 6941-6946). However, the present structure analysis led to considerably better refinement statistics, revealing an error in the structural assignment of N-terminal residues in the previous report. Comparison of the solution structure, as previously determined by nuclear magnetic resonance (NMR) spectroscopy, and the present structure, with two monomers in the asymmetric unit, reveals several local conformational differences. Alanine scan mutagenesis studies for each residue in the so-called RFFESH motif revealed that only the first residue, Arg12, is effective in enhancing receptor binding (and successive activation). A new notion that steric restraint between Arg8 and Arg12 is favorable (if not vital) for retaining SDF activities appears to explain more consistently the structure-activity relationship data accumulated to date. Four guiding principles are presented that may be useful for designing potent therapeutic compounds interfering with HIV-1 infection through competition at the CXCR4 coreceptor.

Structural basis for recognition of cognate tRNA by tyrosyl-tRNA synthetase from three kingdoms

The specific aminoacylation of tRNA by tyrosyl-tRNA synthetases (TyrRSs) relies on the identity determinants in the cognate tRNATyrs. We have determined the crystal structure of Saccharomyces cerevisiae TyrRS (SceTyrRS) complexed with a Tyr-AMP analog and the native tRNATyr(GΨA). Structural information for TyrRS–tRNATyr complexes is now full-line for three kingdoms. Because the archaeal/eukaryotic TyrRSs–tRNATyrs pairs do not cross-react with their bacterial counterparts, the recognition modes of the identity determinants by the archaeal/eukaryotic TyrRSs were expected to be similar to each other but different from that by the bacterial TyrRSs. Interestingly, however, the tRNATyr recognition modes of SceTyrRS have both similarities and differences compared with those in the archaeal TyrRS: the recognition of the C1-G72 base pair by SceTyrRS is similar to that by the archaeal TyrRS, whereas the recognition of the A73 by SceTyrRS is different from that by the archaeal TyrRS but similar to that by the bacterial TyrRS. Thus, the lack of cross-reactivity between archaeal/eukaryotic and bacterial TyrRS-tRNATyr pairs most probably lies in the different sequence of the last base pair of the acceptor stem (C1-G72 vs G1-C72) of tRNATyr. On the other hand, the recognition mode of Tyr-AMP is conserved among the TyrRSs from the three kingdoms.

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.

Dramatic increase in SHP2 binding activity of Helicobacter pylori Western CagA by EPIYA-C duplication: its implications in gastric carcinogenesis

Infection with cagA-positive Helicobacter pylori is critically associated with the development of gastric cancer. The cagA-encoded CagA is delivered into gastric epithelial cells via type IV secretion, where it interacts with and thereby deregulates the pro-oncogenic phosphatase SHP2. East Asian CagA and Western CagA are two major CagA species produced by H. pylori circulating in East Asian countries and in the rest of the world, respectively. The SHP2 binding site of Western CagA, termed the EPIYA-C segment, variably duplicates and infection with H. pylori carrying Western CagA with multiple EPIYA-C segments is a distinct risk factor of gastric cancer. Here we show that duplication of EPIYA-C from one to two or more increases SHP2 binding of Western CagA by more than one hundredfold. Based on the decisive difference in SHP2 binding, Western CagA can be divided into two types: type I CagA carrying a single EPIYA-C segment and type II CagA carrying multiple EPIYA-C segments. Gastric epithelial cells expressing type II CagA acquire the ability to invade extracellular matrices, a malignant cellular trait associated with deregulated SHP2. A big leap in SHP2 binding activity may therefore provide molecular basis that makes type II Western CagA a distinct gastric cancer risk.

Crystal structure of a zinc-dependent D-serine dehydratase from chicken kidney

d-Serine is a physiological co-agonist of the N-methyl-d-aspartate receptor. It regulates excitatory neurotransmission, which is important for higher brain functions in vertebrates. In mammalian brains, d-amino acid oxidase degrades d-serine. However, we have found recently that in chicken brains the oxidase is not expressed and instead a d-serine dehydratase degrades d-serine. The primary structure of the enzyme shows significant similarities to those of metal-activated d-threonine aldolases, which are fold-type III pyridoxal 5′-phosphate (PLP)-dependent enzymes, suggesting that it is a novel class of d-serine dehydratase. In the present study, we characterized the chicken enzyme biochemically and also by x-ray crystallography. The enzyme activity on d-serine decreased 20-fold by EDTA treatment and recovered nearly completely by the addition of Zn2+. None of the reaction products that would be expected from side reactions of the PLP-d-serine Schiff base were detected during the >6000 catalytic cycles of dehydration, indicating high reaction specificity. We have determined the first crystal structure of the d-serine dehydratase at 1.9 Å resolution. In the active site pocket, a zinc ion that coordinates His347 and Cys349 is located near the PLP-Lys45 Schiff base. A theoretical model of the enzyme-d-serine complex suggested that the hydroxyl group of d-serine directly coordinates the zinc ion, and that the ϵ-NH2 group of Lys45 is a short distance from the substrate Cα atom. The α-proton abstraction from d-serine by Lys45 and the elimination of the hydroxyl group seem to occur with the assistance of the zinc ion, resulting in the strict reaction specificity.

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 Å 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).