Shin-ichi Tate

Alteration of the nucleosomal DNA path in the crystal structure of a human nucleosome core particle

Gene expression in eukaryotes depends upon positioning, mobility and packaging of nucleosomes; thus, we need the detailed information of the human nucleosome core particle (NCP) structure, which could clarify chromatin properties. Here, we report the 2.5 Å crystal structure of a human NCP. The overall structure is similar to those of other NCPs reported previously. However, the DNA path of human NCP is remarkably different from that taken within other NCPs with an identical DNA sequence. A comparison of the structural parameters between human and Xenopus laevis DNA reveals that the DNA path of human NCP consecutively shifts by 1 bp in the regions of superhelix axis location −5.0 to −2.0 and 5.0 to 7.0. This alteration of the human DNA path is caused predominantly by tight DNA–DNA contacts within the crystal. It is also likely that the conformational change in the human H2B tail induces the local alteration of the DNA path. In human NCP, the region with the altered DNA path lacks Mn2+ ions and the B-factors of the DNA phosphate groups are substantially high. Therefore, in contrast to the histone octamer, the nucleosomal DNA is sufficiently flexible and mobile and can undergo drastic conformational changes, depending upon the environment.

A comparative study of the solution structures of tachyplesin I and a novel anti-HIV synthetic peptide, T22 ([Tyr5,12, Lys7]-polyphemusin II), determined by nuclear magnetic resonance

The solution structure of tachyplesin I, which was isolated from membrane acid extracts of the hemocytes from the Japanese horseshoe crab (Tachypleus tridentatus), was determined by nuclear magnetic resonance (NMR) and distance geometry calculation. Tachyplesin I takes an antiparallel beta-sheet structure with a type-II beta-turn. Recently, among more than 20 synthetic peptides associated with tachyplesin and its isopeptide (polyphemusin), we found that a novel compound, which we designated as T22 ([Tyr5,12, Lys7]-polyphemusin II), strongly inhibited the human immunodeficiency virus (HIV)-1-induced cytopathic effect and viral antigen expression. The solution structure of T22 was investigated using NMR, and its secondary structure was confirmed to be similar to that of tachyplesin I. The anti-parallel beta-sheet structure and the several amino-acid side chains on the plane of the beta-sheet of T22 are thought to be associated with the expression of anti-HIV activity.

Assembly and Disassembly of Nucleosome Core Particles Containing Histone Variants by Human Nucleosome Assembly Protein I

ABSTRACT Histone variants play important roles in the maintenance and regulation of the chromatin structure. In order to characterize the biochemical properties of the chromatin structure containing histone variants, we investigated the dynamic status of nucleosome core particles (NCPs) that were assembled with recombinant histones. We found that in the presence of nucleosome assembly protein I (NAP-I), a histone chaperone, H2A-Barr body deficient (H2A.Bbd) confers the most flexible nucleosome structure among the mammalian histone H2A variants known thus far. NAP-I mediated the efficient assembly and disassembly of the H2A.Bbd-H2B dimers from NCPs. This reaction was accomplished more efficiently when the NCPs contained H3.3, a histone H3 variant known to be localized in the active chromatin, than when the NCPs contained the canonical H3. These observations indicate that the histone variants H2A.Bbd and H3.3 are involved in the formation and maintenance of the active chromatin structure. We also observed that acidic histone binding proteins, TAF-I/SET and B23.1, demonstrated dimer assembly and disassembly activity, but the efficiency of their activity was considerably lower than that of NAP-I. Thus, both the acidic nature of NAP-I and its other functional structure(s) may be essential to mediate the assembly and disassembly of the dimers in NCPs.

Tautomerism of histidine 64 associated with proton transfer in catalysis of carbonic anhydrase.

The imidazole 15N signals of histidine 64 (His64), involved in the catalytic function of human carbonic anhydrase II (hCAII), were assigned unambiguously. This was accomplished by incorporating the labeled histidine as probes for solution NMR analysis, with 15N at ring-Nδ1 and Nϵ2, 13Cat ring-Cϵ1, 13C and 15N at all carbon and nitrogen, or 15N at the amide nitrogen and the labeled glycine with 13C at the carbonyl carbon. Using the pH dependence of ring-15N signals and a comparison between experimental and simulated curves, we determined that the tautomeric equilibrium constant (KT) of His64 is 1.0, which differs from that of other histidine residues. This unique value characterizes the imidazole nitrogen atoms of His64 as both a general acid (a) and base (b): its ϵ2-nitrogen as (a) releases one proton into the bulk, whereas itsδ1-nitrogen as (b) extracts another proton from a water molecule within the water bridge coupling to the zinc-bound water inside the cave. This accelerates the generation of zinc-bound hydroxide to react with the carbon dioxide. Releasing the productive bicarbonate ion from the inside separates the water bridge pathway, in which the next water molecules move into beside zinc ion. A new water molecule is supplied from the bulk to near the δ1-nitrogen of His64. These reconstitute the water bridge. Based on these features, we suggest here a catalytic mechanism for hCAII: the tautomerization of His64 can mediate the transfers of both protons and water molecules at a neutral pH with high efficiency, requiring no time- or energy-consuming processes.

Location of phosphorylation site and DNA-binding site of a positive regulator, OmpR, involved in activation of the osmoregulatory genes of Escherichia coli

The OmpR protein of Escherichia coli is a positive regulator involved in activation of the ompF and ompC genes which encode the major outer membrane proteins OmpF and OmpC, respectively. By employing recombinant DNA techniques, we isolated the N‐ and C‐terminal halves of the OmpR molecule. From the results of biochemical analyses of these fragments, it was concluded that the N‐terminal portion contains a site involved in phosphorylation by an OmpR‐specific protein kinase EnvZ, whereas the C‐terminal part possesses a DNA‐binding site for the ompC and ompF promoters.

Solution structure of human acidic fibroblast growth factor and interaction with heparin-derived hexasaccharide

Fibroblast growth factors (FGFs) bind to extracellular matrices, especially heparin-like carbohydrates of heparansulfate proteoglycans which stabilize FGFs to protect against inactivation by heat, acid, proteolysis and oxidation. Moreover, binding of FGFs to cell surface proteoglycans promotes to form oligomers, which is essential for receptor oligomerization and activation. In the present study, we determined the solution structure of acidic FGF using a series of triple resonance multi-dimensional NMR experiments and simulated annealing calculations. Furthermore, we prepared the sample complexed with a heparin-derived hexasaccharide which is a minimum unit for aFGF binding. From the chemical shift differences between free aFGF and aFGF-heparin complex, we concluded that the major heparin binding site was located on the regions 110–131 and 17–21. The binding sites are quite similar to those observed for bFGF-heparin hexasaccharide complex, showing that both FGFs recognize heparin- oligosaccharides in a similar manner.

Location of DNA-binding segment of a positive regulator, OmpR, involved in activation of the ompF and ompC genes of Escherichia coli

OmpR protein is a positive regulator involved in activation of the ompF and ompC genes which encode the major outer membrane proteins OmpF and OmpC, respectively. OmpR protein is considered to have a two‐domain structure. In the present study we isolated a C‐terminal fragment of the OmpR molecule, which bound to a specific promoter sequence of the ompF gene. We conclude that the C‐terminal portion of the OmpR protein contains a DNA‐binding site.

Preparation and heteronuclear 2D NMR spectroscopy of a DNA dodecamer containing a thymidine residue with a uniformly 13C-labeled deoxyribose ring

Summary[13C5]-2-Deoxy-d-ribose, synthesized from [13C6]-d-glucose (98% 13C), was coupled with thymine to give [1′,2′,3′,4′,5′-13C5]-thymidine (T) in an 18% overall yield. The thymidine was converted to the 3′-phosphoramidite derivative and was then incorporated into a dodecamer 5′-d(CGCGAATTCGCG)-3′ by solid-phase DNA synthesis. Preparation of 0.24 μmole of the labeled dodecamer, which is sufficient for a single NMR sample, consumed only 25 mg of glucose. By virtue of the 13C labels, all of the 1H-1H vicinal coupling constants in the sugar moieties were accurately determined by HCCH-E.COSY.

Phosphorylated Intrinsically Disordered Region of FACT Masks Its Nucleosomal DNA Binding Elements

FACT is a heterodimer of SPT16 and SSRP1, which each contain several conserved regions in the primary structure. The interaction of FACT with nucleosomes induces chromatin remodeling through the combinatorial action of its distinct functional protein regions. However, there is little mechanistic insight into how these regions cooperatively contribute to FACT functions, particularly regarding the recognition of nucleosomal DNA. Here, we report the identification of novel phosphorylation sites of Drosophila melanogaster FACT (dFACT) expressed in Sf9 cells. These sites are densely concentrated in the acidic intrinsically disordered (ID) region of the SSRP1 subunit and control nucleosomal DNA binding by dFACT. This region and the adjacent segment of the HMG domain form weak electrostatic intramolecular interactions, which is reinforced by the phosphorylation, thereby blocking DNA binding competitively. Importantly, this control mechanism appears to support rapid chromatin transactions during early embryogenesis through the dephosphorylation of some sites in the maternally transmitted dSSRP1.

Proline cis/trans-Isomerase Pin1 Regulates Peroxisome Proliferator-activated Receptor γ Activity through the Direct Binding to the Activation Function-1 Domain

The important roles of a nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) are widely accepted in various biological processes as well as metabolic diseases. Despite the worldwide quest for pharmaceutical manipulation of PPARγ activity through the ligand-binding domain, very little information about the activation mechanism of the N-terminal activation function-1 (AF-1) domain. Here, we demonstrate the molecular and structural basis of the phosphorylation-dependent regulation of PPARγ activity by a peptidyl-prolyl isomerase, Pin1. Pin1 interacts with the phosphorylated AF-1 domain, thereby inhibiting the polyubiquitination of PPARγ. The interaction and inhibition are dependent upon the WW domain of Pin1 but are independent of peptidyl-prolyl cis/trans-isomerase activity. Gene knockdown experiments revealed that Pin1 inhibits the PPARγ-dependent gene expression in THP-1 macrophage-like cells. Thus, our results suggest that Pin1 regulates macrophage function through the direct binding to the phosphorylated AF-1 domain of PPARγ.