Shigeru Negi

Cytochrome c polymerization by successive domain swapping at the C-terminal helix.

Cytochrome c (cyt c) is a stable protein that functions in a monomeric state as an electron donor for cytochrome c oxidase. It is also released to the cytosol when permeabilization of the mitochondrial outer membrane occurs at the early stage of apoptosis. For nearly half a century, it has been known that cyt c forms polymers, but the polymerization mechanism remains unknown. We found that cyt c forms polymers by successive domain swapping, where the C-terminal helix is displaced from its original position in the monomer and Met-heme coordination is perturbed significantly. In the crystal structures of dimeric and trimeric cyt c, the C-terminal helices are replaced by the corresponding domain of other cyt c molecules and Met80 is dissociated from the heme. The solution structures of dimeric, trimeric, and tetrameric cyt c were linear based on small-angle X-ray scattering measurements, where the trimeric linear structure shifted toward the cyclic structure by addition of PEG and (NH4)2HPO4. The absorption and CD spectra of high-order oligomers (∼40 mer) were similar to those of dimeric and trimeric cyt c but different from those of monomeric cyt c. For dimeric, trimeric, and tetrameric cyt c, the ΔH of the oligomer dissociation to monomers was estimated to be about -20 kcal/mol per protomer unit, where Met-heme coordination appears to contribute largely to ΔH. The present results suggest that cyt c polymerization occurs by successive domain swapping, which may be a common mechanism of protein polymerization.

New Redesigned Zinc‐Finger Proteins: Design Strategy and Its Application

The design of DNA-binding proteins for the specific control of the gene expression is one of the big challenges for several research laboratories in the post-genomic era. An artificial transcription factor with the desired DNA binding specificity could work as a powerful tool and drug to regulate the target gene. The zinc-finger proteins, which typically contain many fingers linked in a tandem fashion, are some of the most intensively studied DNA-binding proteins. In particular, the Cys(2)His(2)-type zinc finger is one of the most common DNA-binding motifs in eukaryotes. A simple mode of DNA recognition by the Cys(2)His(2)-type zinc-finger domain provides an ideal framework for designing proteins with new functions. Our laboratory has utilized several design strategies to create new zinc-finger peptides/proteins by redesigning the Cys(2)His(2)-type zinc-finger motif. This review focuses on the aspects of design strategies, mainly from our recent results, for the creation of artificial zinc-finger proteins, and discusses the possible application of zinc-finger technology for gene regulation and gene therapy.

Zn(II) Binding and DNA Binding Properties of Ligand-Substituted CXHH-Type Zinc Finger Proteins

CCHH-type zinc fingers are among the most common DNA binding motifs found in eukaryotes. In a previous report, we substituted the second ligand cysteine residue with aspartic acid, producing a Zn(II)-responsive transcription factor; this indicates that a ligand substitution is a possible design target of an engineered zinc finger peptide. Despite the importance of Zn(II) binding with respect to the folding and DNA binding properties of a zinc finger peptide, no study about the effects of ligand substitution on both Zn(II) binding and DNA binding properties has been reported. Here, we substituted a conserved cysteine (C) with other zinc-coordinated amino acid residues, histidine (H), aspartic acid (D), and glutamic acid (E), to create CXHH-type zinc finger peptides (X = C, H, D, and E). The Zn(II)-dependent conformational change was observed in all peptides; however, the Zn(II) binding affinity and metal coordination geometry of the peptides were different. Gel mobility shift assays showed that the Zn(II)-bound forms of the ligand-substituted derivatives retain DNA binding ability, while the DNA binding affinity decreased in the following manner: CCHH > CDHH > CEHH ≫ CHHH. The DNA binding sequence preferences of the ligand-substituted derivatives were similar to that of the wild type in the context of the full three-finger DNA-binding domain of transcription factor Zif268. These results indicate that artificial zinc finger proteins with various DNA binding affinities that respond to a diverse range of Zn(II) concentrations can be designed by substituting the Zn(II) ligand.

Feedback Response to Selective Depletion of Endogenous Carbon Monoxide in the Blood

The physiological roles of endogenous carbon monoxide (CO) have not been fully understood because of the difficulty in preparing a loss-of-function phenotype of this molecule. Here, we have utilized in vivo CO receptors, hemoCDs, which are the supramolecular 1:1 inclusion complexes of meso-tetrakis(4-sulfonatophenyl)porphinatoiron(II) with per-O-methylated β-cyclodextrin dimers. Three types of hemoCDs (hemoCD1, hemoCD2, and hemoCD3) that exhibit different CO-affinities have been tested as CO-depleting agents in vivo. Intraperitoneally administered hemoCD bound endogenous CO within the murine circulation, and was excreted in the urine along with CO in an affinity-dependent manner. The sufficient administration of hemoCD that has higher CO-affinity than hemoglobin (Hb) produced a pseudoknockdown state of CO in the mouse in which heme oxygenase-1 (HO-1) was markedly induced in the liver, causing the acceleration of endogenous CO production to maintain constant CO-Hb levels in the blood. The contents of free hemin and bilirubin in the blood plasma of the treated mice significantly increased upon removal of endogenous CO by hemoCD. Thus, a homeostatic feedback model for the CO/HO-1 system was proposed as follows: HemoCD primarily removes CO from cell-free CO-Hb. The resulting oxy-Hb is quickly oxidized to met-Hb by oxidant(s) such as hydrogen peroxide in the blood plasma. The met-Hb readily releases free hemin that directly induces HO-1 in the liver, which metabolizes the hemin into iron, biliverdin, and CO. The newly produced CO binds to ferrous Hb to form CO-Hb as an oxidation-resistant state. Overall, the present system revealed the regulatory role of CO for maintaining the ferrous/ferric balance of Hb in the blood.

Binding of multivalent anionic porphyrins to V3 loop fragments of an HIV-1 envelope and their antiviral activity.

Interactions of multivalent anionic porphyrins and their iron(III) complexes with cationic peptides, V3(Ba-L) and V3(IIIB), which correspond to those of the V3 loop regions of the gp120 envelope proteins of the HIV-1(Ba-L) and HIV-1(IIIB) strains, respectively, are studied by UV/Vis, circular dichroism, (1)H NMR, and EPR spectroscopy, a microcalorimetric titration method, and anti-HIV assays. Tetrakis(3,5-dicarboxylatophenyl)porphyrin (P1), tetrakis[4-(3,5-dicarboxylatophenylmethoxy)phenyl]porphyrin (P2), and their ferric complexes (Fe(III)P1 and Fe(III)P2) were used as the multivalent anionic porphyrins. P1 and Fe(III)P1 formed stable complexes with both V3 peptides (binding constant K>10(6) M(-1)) through combined electrostatic and van der Waals interactions. Coordination of the His residues in V3(Ba-L) to the iron center of Fe(III)P1 also played an important role in the complex stabilization. As P2 and Fe(III)P2 form self-aggregates in aqueous solution even at low concentrations, detailed analysis of their interactions with the V3 peptides could not be performed. To ascertain whether the results obtained in the model system are applicable to a real biological system, anti-HIV-1(BA-L) and HIV-1(IIIB) activity of the porphyrins is examined by multiple nuclear activation of a galactosidase indicator (MAGI) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. There is little correlation between chemical analysis and actual anti-HIV activity, and the size rather than the number of the anionic groups of the porphyrin is important for anti-HIV activity. All the porphyrins show high selectivity, low cytotoxicity, and high viral activity. Fe(III)P1 and Fe(III)P2 are used for the pharmacokinetic study. Half-lives of these iron porphyrins in serum of male Wistar rats are around 4 to 6 h owing to strong interaction of these porphyrins with serum albumin.

Novel zinc finger nuclease created by combining the Cys2His2- and His4-type zinc finger domains

To improve the DNA hydrolytic activity of the zinc finger nuclease, we have created a new artificial zinc finger nuclease (ZWH4) by connecting two distinct zinc finger domains possessing different types of Zn(II) binding sites (Cys(2)His(2)- and His(4)-types). The overall fold of ZWH4 is similar to that of the wild-type Sp1 zinc finger (Sp1(zf123)) as revealed by circular dichroism spectroscopy. The gel mobility shift assay demonstrated that ZWH4 binds to the GC box DNA, although the DNA-binding affinity is lower than that of Sp1(zf123). Evidently, ZWH4 hydrolyzes the covalently closed circular plasmid DNA (form I) containing the GC box (pBSGC) to the linear duplex DNA (form III) in the presence of a higher concentration (50 times) of the protein than DNA for a 24-h reaction. Of special interest is the fact that the novel mixed zinc finger protein containing the Cys(2)His(2)- and His(4)-type domains was first created. The present results provide the useful information for the redesign strategy of an artificial nuclease based on the zinc finger motif.

Effects of bulkiness and hydrophobicity of an aliphatic amino acid in the recognition helix of the GAGA zinc finger on the stability of the hydrophobic core and DNA binding affinity.

The GAGA factor of Drosophila melanogaster uses a single Cys 2His 2-type zinc finger for specific DNA binding. The conformation and DNA binding mode of the GAGA zinc finger are similar to those of other structurally characterized zinc fingers. In almost all Cys 2His 2-type zinc fingers, the fourth position of the DNA-recognizing helix is occupied by the Leu residue involved in the formation of the minimal hydrophobic core. However, no systematic study on the precise role of the Leu residue in the hydrophobic core formation and DNA binding function has been reported. In this study, the Leu residue is substituted with other aliphatic amino acids having different side chain lengths and hydrophobicities, namely, Ile, Val, Aib, and Ala. The metal binding properties were studied by UV-vis spectroscopy. The peptide conformations were examined by CD and NMR spectroscopies. Furthermore, the DNA binding ability was examined with a gel mobility shift assay. Though the Ile, Val, and Aib mutants exhibited conformations similar to those of the wild type, the DNA binding affinity decreased as the side chain length of the amino acid decreased. Interestingly, the Val mutant can bind to the cognate DNA, while Aib cannot, in spite of the similarity in their secondary structures based on the CD measurements. Variable-temperature NMR experiments clearly indicated differences in the stability of the hydrophobic core between the Val and Aib mutants. This study demonstrates that the bulkiness of the conserved aliphatic residue is important in the formation of the well-packed minimal hydrophobic core and proper ternary structure and that the hydrophobic core stabilization is apparently related to the DNA binding function of the GAGA zinc finger.

Hydrogen-Bonded Complexes of Carboxylate Anions and Dextrins in an Aprotic Polar Solvent

Complexation of p-methylbenzoate (p-CH3C6H4CO2−) and alkanoate anions (CnCO2−) with cyclodextrins (CDs) and acyclic dextrins (Gns) through hydrogen bonding in an aprotic polar solvent, [D6]DMSO, has been studied by means of 1H NMR spectroscopy. Although undissociated p-methylbenzoic acid, p-CH3C6H4CO2H, does not interact with dextrins, p-CH3C6H4CO2− binds through hydrogen-bonding interactions with fairly large binding constants (K) both to native CDs such as α-, β-, and γ-CDs and to Gns. Similar complexation was observed with CnCO2− anions. The K values are related to the basicity of the carboxylate anions. 1H NMR spectroscopy shows that the CO2− group of the guest interacts with the secondary OH groups at the vicinal 2- and 3-positions of the dextrins, while the primary OH groups do not participate at all. Formation of the hydrogen-bonded complex of β-CD and p-CH3C6H4CO2− is an entropically favorable process. Addition of a small amount of D2O suppresses the formation of the hydrogen-bonded complexes, suggesting that hydrogen-bonding interactions between simple hosts possessing dense OH groups as hydrogen-bond donors and guests with CO2− groups as hydrogen-bond acceptors hardly occur in aqueous solution.

Mechanism for binding to the flexible cavity of permethylatedα-cyclodextrin

The cavity of α-cyclodextrin (α-CDx) is too small to includeo-toluic acid (o-TA) while it is filled byp-toluic acid (p-TA) to form a relatively stable inclusion complex. Such strict selectivity is ascribed to a rigid structure of the α-CDx cavity which is stabilized by intramolecular hydrogen bonds between the O(2) hydroxyl groups and the O(3) hydroxyl groups of adjacent glucopyranose units. Meanwhile, the substrate selectivity of hexakis (2,3,6-tri-O-methyl)-α-CDx (TMe-α-CDx) remains somewhat obscure because of the flexible nature of its cavity. The absence of the intramolecular hydrogen bonds seems to cause the flexible nature of the TMe-α-CDx cavity leading to an induced-fit type inclusion. The structures of the inclusion complexes have been presented on the basis of the1H NMR data. The thermodynamic parameters indicate that the complexation of TMe-α-CDx witho-TA orp-TA is the entropically favorable process. The entropically favorable complexation of TA with TMe-α-CDx seems to occur through dehydration from the CO2H group of TA which is situated at the hydrophobic CDx cavity. The dipole-dipole interaction has been regarded as the force which dominates the orientation of the polar guest molecule in the CDx cavity.

An arginine residue instead of a conserved leucine residue in the recognition helix of the finger 3 of Zif268 stabilizes the domain structure and mediates DNA binding.

The Cys(2)His(2)-type zinc finger is a common DNA binding motif that is widely used in the design of artificial zinc finger proteins. In almost all Cys(2)His(2)-type zinc fingers, position 4 of the α-helical DNA-recognition site is occupied by a Leu residue involved in formation of the minimal hydrophobic core. However, the third zinc finger domain of native Zif268 contains an Arg residue instead of the conserved Leu. Our aim in the present study was to clarify the role of this Arg in the formation of a stable domain structure and in DNA binding by substituting it with a Lys, Leu, or Hgn, which have different terminal side-chain structures. Assessed were the metal binding properties, peptide conformations, and DNA-binding abilities of the mutants. All three mutant finger 3 peptides exhibited conformations and thermal stabilities similar to the wild-type peptide. In DNA-binding assays, the Lys mutant bound to target DNA, though its affinity was lower than that of the wild-type peptide. On the other hand, the Leu and Hgn mutants had no ability to bind DNA, despite the similarity in their secondary structures to the wild-type. Our results demonstrate that, as with the Leu residue, the aliphatic carbon side chain of this Arg residue plays a key role in the formation of a stable zinc finger domain, and its terminal guanidinium group appears to be essential for DNA binding mediated through both electrostatic interaction and hydrogen bonding with DNA phosphate backbone.