Masato Shimizu

CRYSTAL STRUCTURE OF PHO4 BHLH DOMAIN-DNA COMPLEX : FLANKING BASE RECOGNITION

The crystal structure of a DNA‐binding domain of PHO4 complexed with DNA at 2.8 Å resolution revealed that the domain folds into a basic–helix–loop–helix (bHLH) motif with a long but compact loop that contains a short α‐helical segment. This helical structure positions a tryptophan residue into an aromatic cluster so as to make the loop compact. PHO4 binds to DNA as a homodimer with direct reading of both the core E‐box sequence CACGTG and its 3′‐flanking bases. The 3′‐flanking bases GG are recognized by Arg2 and His5. The residues involved in the E‐box recognition are His5, Glu9 and Arg13, as already reported for bHLH/Zip proteins MAX and USF, and are different from those recognized by bHLH proteins MyoD and E47, although PHO4 is a bHLH protein.

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.

The use of heteronuclear cross-polarization for backbone assignment of 2H-, 15N- and 13C-labeled proteins: A pulse scheme for triple-resonance 4D correlation of sequential amide protons and 15N

SummaryA new four-dimensional pulse scheme is described for the main-chain assignment of proteins by means of the J connectivity of the amide proton and nitrogen resonances of adjacent residues. Since the new experiment, 4D CP-HN(COCA)NH, involves heteronuclear cross-polarization for magnetization transfer from 13C=O to 15N via 13Cα, a relatively strong WALTZ-16 decoupling rf field is applied to 13Cα during magnetization transfer. Consequently, 13Cα is effectively decoupled from its attached 2H in the case of deuterated proteins, in the absence of a decoupling rf field for 2H. This efficiently improves the sensitivity of the experiment through 13C line narrowing. The experiment was performed on a randomly 60% deuterated protein, and the sensitivity of the final 4D spectrum was found to be excellent.

15N labeling method of peptides using a thioredoxin gene fusion expression system: an application to ACTH-(1–24)

For structure analysis of peptides by multinuclear NMR, stable isotope‐labeled samples are required. A direct over‐expression system by E. coli cells does not work for that purpose because of rapid degradation of the peptides and/or the mRNA in host cells. We here developed an over‐expression system by means of thioredoxin gene fusion system. The fused protein composed of thioredoxin and the objective peptide was expressed in E. coli and then the peptide part was released by enterokinase. This system was successfully applied for the production of 15N‐labeled human adrenocorticotropic hormone fragment (ACTH‐(1‐24)) as needed for multinuclear NMR analysis.

Distinctive Solution Conformation of Phosphatase Inhibitor CPI-17 Substituted with Aspartate at the Phosphorylation-site Threonine Residue

We present solution NMR structures for wild-type and mutated forms of CPI-17, a phosphoinhibitor for protein phosphatase 1. Phosphorylation of Thr38 of CPI-17 produces a >1000-fold increase in inhibitory potency for myosin phosphatase. We compared the 1H-15N heteronuclear single quantum coherence spectroscopy (HSQC) chemical shifts of wild-type CPI-17, partially phosphorylated CPI-17 and CPI-17 with Thr38 replaced with Asp to introduce a negative charge. There was a switch in the protein conformation due to either Asp substitution or phosphorylation, so we determined the solution NMR structure of the CPI-17 T38D mutant as a model for the active (phospho-) conformation. The structures reveal a molecular switch in conformation that involves the rotation of two of the four helices in the four helix bundle. Despite this conformational switch, there was little increase in the inhibitory potency with T38D. We propose that for this inhibitor, a negative charge at residue 38 is sufficient to trigger an active conformation, but a phosphoryl group is required for full inhibitory potency against protein phosphatase-1.

Decrease in fluorescence lifetime by glycation of collagen and its application in determining advanced glycation end-products in human dentin

Advanced Glycation End-products (AGEs) are produced by the Maillard reaction, which causes cross-linking of collagen and results in changes in the mechanical properties of collagen tissues. Several types of AGE fluoresce, and measurement of this fluorescence is effective for determining the presence of AGEs. Because fluorescence intensity by steady-state fluorometry is affected by sample surface condition and light source, we focused on fluorescence lifetime measurement (FLM). We found that fluorescence lifetime of collagen gel decreased with glycation progress. In vivo application of FLM for determination of AGEs was confirmed in human dentin.

Influence of Nonenzymatic Glycation in Dentinal Collagen on Dental Caries

Advanced glycation end-products (AGEs) are generated via nonenzymatic glycation of dentinal collagen, resulting in accumulation of AGEs in dentin tissue. Since accumulated AGEs cause crosslinking between amino acid polypeptides in the collagen molecule and modify mechanical properties of dentinal collagen, the authors assumed that there would be a significant interaction between the generation of AGEs and progression of caries in dentin. To confirm such an interaction, spectroscopic imaging analyses (i.e., nanosecond fluorescence lifetime imaging and second harmonic generation light imaging) were performed in addition to biochemical and electron microscopic analyses in the present study. Seven carious human teeth were fixed in paraformaldehyde and cut longitudinally into 1-mm sections using a low-speed diamond saw for the following analyses. In transmission electron microscopy (TEM) analysis, nondecalcified specimens were embedded in epoxy resin and sliced into thin sections for observation. For the immunohistochemical analysis, the specimens were paraffin embedded after decalcification for 2 wk and sectioned with a microtome. Resultant sections were stained with anti-AGE and anticollagen antibodies. The demineralized specimens were used for spectroscopic analyses without additional treatment. For Western blotting analysis, specimens were separated into carious and sound dentin. Each specimen was homogenized with a bead crusher and an ultrasonic homogenizer and then treated with hydrochloric acid. In carious dentin, the collagen fibers showed an amorphous structure in the TEM image, and the AGEs were localized in the areas of bacterial invasion in the immunostaining image. The total amount of AGEs in carious dentin was higher than in sound dentin in Western blotting. The ultrastructure of type I collagen and total amount of AGEs varied markedly in the dentinal caries region. The fluorescence lifetime was shorter in the carious area than that in the sound areas, indicating an increase of AGEs in the carious area. The increase of AGEs could influence the progression of dentinal caries.

Phosphorylation-induced conformational change responsible for the function of a myosin phosphatase inhibitor, CPI-17

Abstract The structures of CPI-17 (Protein kinase-C dependent protein phosphatase-1 (PP1) inhibitor of 17 kDa) in an inactive and an active form have been determined by multidimensional NMR spectroscopy. Comparison of the two structures revealed how the molecular switch turns on at atomic resolution. Using the NMR structure of CPI-17 in the active form, the binding with catalytic domain of PP1 (PP1c) was simulated and the binding model is proposed in this report. When the phospho-Thr38 docks to the catalytic site of PP1, possible interactions for the tight binding are found; one is electrostatic interaction between a negatively charged cluster on phospho-CPI-17 and an acidic groove of PP1c, and the other is hydrophobic interaction between a hydrophobic surface area of phospho-CPI-17 and a hydrophobic groove of PP1c.