Design and expression of cysteine-bearing hydrophobic polypeptides and their self-assembling properties with bacteriochlorophyll a derivatives as a mimic of bacterial photosynthetic antenna complexes. Effect of steric confinement and orientation of the polypeptides on the pigment/polypeptide assembly process

A series of cysteine-bearing hydrophobic polypeptides analogous to a light-harvesting one betapolypeptide (LH1beta) from the LH1 complex from the purple photosynthetic bacterium, Rhodobacter sphaeroides, was synthesized using an Escherichia coli expression system. The cysteine was placed in the C- or N-terminal regions of the polypeptide to investigate the influence of steric confinement and orientation of the polypeptides via disulfide linkages as they were self-assembled with zinc-substituted bacteriochlorophyll a ([Zn]-BChl a). The polypeptides were expressed as water-soluble fusion proteins with maltose-binding protein (MBP). The fusion proteins formed a subunit-type complex with the [Zn]-BChl a in an n-octyl-beta-d-glucopyranoside (OG) micellar solution regardless of the cross-links or the cleavage of the cysteines, judging from absorption, CD, and fluorescence spectra. Following treatment with trypsin, the polypeptides were detached from the MBP portion. Such trypsin-digested polypeptides formed a subunit-type LH complex at 25 degrees C, which also showed that the disulfide linkage was not crucial for the subunit formation. When a polypeptide having cysteine on the C-terminus was assembled at 4 degrees C, the Qy absorption band was remarkably red-shifted to approximately 836 nm, suggesting that the cleavage of the large MBP portion liberates the polypeptides to form the progressive type of complex similar to LH1-type complex. The trypsin-treated polypeptides bearing cysteines in both terminal regions, which are randomly cross-linked, did not form the LH1-type complex under oxidative conditions but did form the complex under reductive conditions. This observation suggests that the polypeptide orientation strongly influences the LH1-type complex formation. The progressive assembly from the subunit to the holo-LH1-type complex following cleavage of MBP portion in a lipid bilayer is also briefly discussed.     

Mapping peptides correlated with transmission of intrasteric inhibition and allosteric activation in human cystathionine beta-synthase

Cystathionine beta-synthase plays a key role in the intracellular disposal of homocysteine and is the single most common locus of mutations associated with homocystinuria. Elevated levels of homocysteine are correlated with heart disease, Alzheimer's and Parkinson's diseases, and neural tube defects. Cystathionine beta-synthase is modular and subjected to complex regulation, but insights into the structural basis of this regulation are lacking. We have employed hydrogen exchange mass spectrometry to map peptides whose motions are correlated with transmission of intrasteric inhibition and allosteric activation. The mass spectrometric data provide an excellent correlation between kinetically and conformationally distinguishable states of the enzyme. We also demonstrate that a pathogenic regulatory domain mutant, D444N, is conformationally locked in one of two states sampled by the wild type enzyme. Our hydrogen exchange data identify surfaces that are potentially involved in the juxtaposition of the regulatory and catalytic domains and form the basis of a docked structural model for the full-length enzyme.     

Transient formation of a neutral ubisemiquinone radical and subsequent intramolecular electron transfer to pyrroloquinoline quinone in the Escherichia coli membrane-integrated glucose dehydrogenase

The membrane-bound quinoprotein glucose dehydrogenase (mGDH) in Escherichia coli contains pyrroloquinoline quinone (PQQ) and participates in the direct oxidation of D-glucose to D-gluconate by transferring electrons to ubiquinone (UQ). To elucidate the mechanism of ubiquinone reduction by mGDH, we applied a pulse radiolysis technique to mGDH with or without bound UQ8. With the UQ8-bound enzyme, a hydrated electron reacted with mGDH to form a transient species with an absorption maximum at 420 nm, characteristic of formation of a neutral ubisemiquinone radical. Subsequently, the decay of the absorbance at 420 nm was accompanied by an increase in the absorbance at 370 nm. Experiments with the PQQ-free apoenzyme showed no such subsequent absorption changes, although ubisemiquinone was formed. These results indicate that a pathway for an intramolecular electron transfer from ubisemiquinone radical at the UQ8 binding site to PQQ exists in mGDH. The first-order rate constant of this process was calculated to be equal to 1.2 x 10(3) s(-1). These findings are consistent with our proposal that during the catalytic cycle of mGDH the bound UQ8 mediates electron transfer from the reduced PQQ to UQ8 pools.     

Specific inhibition of hepatitis C virus replication by cyclosporin A

The difficulty in eradicating hepatitis C virus (HCV) infection is attributable to the limited treatment options against the virus. Recently, cyclosporin A (CsA), a widely used immunosuppressive drug, has been reported to be effective against HCV infection [J. Gastroenterol. 38 (2003) 567], although little is understood about the mechanism of its action against HCV. In this study, we investigated the anti-viral effects of CsA using an HCV replicon system. Human hepatoma Huh7 cells were transfected with an HCV replicon expressing a chimeric gene encoding a luciferase reporter and neomycin phosphotransferase (Huh7/Rep-Feo). Treatment of the Huh7/Rep-Feo cells with CsA resulted in suppression of the replication of the HCV replicon in a dose-dependent manner, with an IC50 of approximately 0.5 microg/ml. There were no changes in the rate of cell growth or viability, suggesting that the effect of CsA against HCV is specific and not due to cytotoxicity. In contrast, FK506, another immunosuppressive drug, did not suppress HCV replication. CsA did not activate interferon-stimulated gene responses, suggesting that its action is independent of that of interferon. In conclusion, CsA inhibits HCV replication in vitro specifically at clinical concentrations. Further defining its mode of action against HCV replication potentially may be important for identifying novel molecular targets to treat HCV infection.     

In vivo evidence that peptide vaccination can induce HLA-DR-restricted CD4+ T cells reactive to a class I tumor peptide

Vaccination with class I tumor peptides has been performed to induce tumor-reactive CD8(+) T cells in vivo. However, the kinds of immune responses that vaccination might elicit in patients are not fully understood. In this study we tried to elucidate the mechanisms by which vaccination of class I binding tumor peptides into an HLA-A2(+) lung cancer patient elicited dramatic amounts of IgG1 and IgG2 specific to a nonamer peptide, ubiquitin-conjugated enzyme variant Kua (UBE2V)(43-51). The UBE2V(43-51) peptide contains cysteine at the sixth position. HLA-DR-restricted and UBE2V(43-51) peptide-recognizing CD4(+) T cells were induced from postvaccination, but not from prevaccination, PBMCs of the cancer patient. In addition, a CD4(+) T cell line (UB-2) and its clone (UB-2.3), both of which recognize the UBE2V(43-51) peptide in the context of HLA-DRB1*0403 molecules, were successfully established from postvaccination PBMCs. The peptide vaccination increased the frequency of peptide-specific T cells, especially CD4(+) T cells. In contrast, mass spectrometric analysis revealed that the vaccinated UBE2V(43-51) peptide contained both monomeric and dimeric forms. Both forms, fractionated by reverse phase HPLC, were recognized by UB-2 and UB-2.3 cells. Recognition by these CD4(+) T cells was observed despite the addition of a reduction reagent or the fixation of APC. Overall, these results indicate that vaccination with class I tumor peptides can induce HLA-DR-restricted CD4(+) T cells in vivo and elicit humoral immune responses, and that a cysteine-containing peptide can be recognized by CD4(+) T cells not only as a monomer, but also as a dimer.     

Crystal structures of creatininase reveal the substrate binding site and provide an insight into the catalytic mechanism

Creatininase from Pseudomonas putida is a member of the urease-related amidohydrolase superfamily. The crystal structure of the Mn-activated enzyme has been solved by the single isomorphous replacement method at 1.8A resolution. The structures of the native creatininase and the Mn-activated creatininase-creatine complex have been determined by a difference Fourier method at 1.85 A and 1.6 A resolution, respectively. We found the disc-shaped hexamer to be roughly 100 A in diameter and 50 A in thickness and arranged as a trimer of dimers with 32 (D3) point group symmetry. The enzyme is a typical Zn2+ enzyme with a binuclear metal center (metal1 and metal2). Atomic absorption spectrometry and X-ray crystallography revealed that Zn2+ at metal1 (Zn1) was easily replaced with Mn2+ (Mn1). In the case of the Mn-activated enzyme, metal1 (Mn1) has a square-pyramidal geometry bound to three protein ligands of Glu34, Asp45, and His120 and two water molecules. Metal2 (Zn2) has a well-ordered tetrahedral geometry bound to the three protein ligands of His36, Asp45, and Glu183 and a water molecule. The crystal structure of the Mn-activated creatininase-creatine complex, which is the first structure as the enzyme-substrate/inhibitor complex of creatininase, reveals that significant conformation changes occur at the flap (between the alpha5 helix and the alpha6 helix) of the active site and the creatine is accommodated in a hydrophobic pocket consisting of Trp174, Trp154, Tyr121, Phe182, Tyr153, and Gly119. The high-resolution crystal structure of the creatininase-creatine complex enables us to identify two water molecules (Wat1 and Wat2) that are possibly essential for the catalytic mechanism of the enzyme. The structure and proposed catalytic mechanism of the creatininase are different from those of urease-related amidohydrolase superfamily enzymes. We propose a new two-step catalytic mechanism possibly common to creatininases in which the Wat1 acts as the attacking nucleophile in the water-adding step and the Wat2 acts as the catalytic acid in the ring-opening step.     

High-resolution crystal structure of Arthrobacter aurescens chondroitin AC lyase: an enzyme-substrate complex defines the catalytic mechanism

Chondroitin lyases (EC 4.2.2.4 and EC 4.2.2.5) are glycosaminoglycan-degrading enzymes that act as eliminases. Chondroitin lyase AC from Arthrobacter aurescens (ArthroAC) is known to act on chondroitin 4-sulfate and chondroitin 6-sulfate but not on dermatan sulfate. Like other chondroitin AC lyases, it is capable of cleaving hyaluronan. We have determined the three-dimensional crystal structure of ArthroAC in its native form as well as in complex with its substrates (chondroitin 4-sulfate tetrasaccharide, CS(tetra) and hyaluronan tetrasaccharide) at resolution varying from 1.25 A to 1.9A. The primary sequence of ArthroAC has not been previously determined but it was possible to determine the amino acid sequence of this enzyme from the high-resolution electron density maps and to confirm it by mass spectrometry. The enzyme-substrate complexes were obtained by soaking the substrate into the crystals for varying lengths of time (30 seconds to ten hours) and flash-cooling the crystals. The electron density map for crystals soaked in the substrate for as short as 30 seconds showed the substrate clearly and indicated that the ring of central glucuronic acid assumes a distorted boat conformation. This structure strongly supports the lytic mechanism where Tyr242 acts as a general base that abstracts the proton from the C5 position of glucuronic acid while Asn183 and His233 neutralize the charge on the glucuronate acidic group. Comparison of this structure with that of chondroitinase AC from Flavobacterium heparinum (FlavoAC) provides an explanation for the exolytic and endolytic mode of action of ArthroAC and FlavoAC, respectively.     

Structural and enzymatic properties of 1-aminocyclopropane-1-carboxylate deaminase homologue from Pyrococcus horikoshii

1-Aminocyclopropane-l-carboxylate deaminase (ACCD) is a pyridoxal 5/-phosphate dependent enzyme that shows deaminase activity toward ACC, a precursor of plant hormone ethylene. ACCD from some soil bacteria has been reported to be able to break the cyclopropane ring of ACC to yield a-ketobutyrate and ammonia. We reported the crystal structure of ACCD from the yeast Hansenula saturnus in the absence/presence of substrate ACC, and proposed its ingenious reaction mechanisms. In order to study the enzyme further, we overexpressed the ACCD homologue protein (phAHP) from the fully decoded hyperthermophilic archearon, Pyrococcus horikoshii OT3. However, phAHP does not show ACCD activity at high temperature as well as at room temperature, though it has significant sequence similarity. Instead of ACCD activity, the GC-MS analysis and enzymatic method show that phAHP has deaminase activity toward L and D-serine. Here, we present the crystal structures of the native and ACC-complexed phAHP. The overall topology of the phAHP structure is very similar to that of ACCD; however, critical differences were observed around the active site. Here, the differences of enzymatic activity between phAHP and ACCD are discussed based on the structural differences of these two proteins. We suggest that the catalytic disagreement between these two enzymes comes from the difference of the residues near the pyridine ring of pyridoxal 5'-phosphate (PLP), not the difference of the catalytic residues themselves. We also propose a condition necessary in the primary sequence to have ACCD activity.     

Dose-different effects of orexin-A on the renal sympathetic nerve and blood pressure in urethane-anesthetized rats

Previous studies have demonstrated that central injection of orexin-A affects renal sympathetic nerve activity (RSNA) and blood pressure (BP) in both anesthetized and unanesthetized rats. In the present study, we examined, using urethane-anesthetized rats, the dose-dependent effects of intravenous (iv) or intralateral cerebral ventricular (LCV) injection of various doses of orexin-A on RSNA and BP. We found that injection of a low dose of orexin-A (10 ng iv or 0.01 ng LCV) suppressed RSNA and BP significantly. Conversely, a high dose (1000 ng iv or 10 ng LCV) of orexin-A elevated both RSNA and BP significantly. Pretreatment with either iv or LCV injection of thioperamide, a histaminergic H(3)-receptor antagonist, eliminated the effects of a low dose of orexin-A on both RSNA and BP. Both iv and LCV injection of diphenhydramine, a histaminergic H(1)-receptor antagonist, abolished the effects of a high dose of orexin-A on RSNA and BP. Furthermore, bilateral lesions of the hypothalamic suprachiasmatic nucleus (SCN) abolished the effects of both low and high doses of orexin-A on RSNA and BP. These findings suggest that orexin-A affects RSNA and BP in a dose-dependent manner and that the SCN and histaminergic nerve may be involved in the dose-different effects of orexin-A in rats.