Functional roles of metal binding domains of the Archaeoglobus fulgidus Cu(+)-ATPase CopA

CopA, a thermophilic membrane ATPase from Archaeoglobus fulgidus, drives the outward movement of Cu(+) or Ag(+) [Mandal et al. (2002) J. Biol. Chem. 277, 7201-7208]. This, as other P(IB)-ATPases, is characterized by a putative metal binding sequence (C(380)PC(382)) in its sixth transmembrane fragment and cytoplasmic metal binding sequences in its NH(2)- and COOH-terminal ends (C(27)AMC(30) and C(751)HHC(754)). Using isolated CopA, we have studied the functional role of these three putative metal binding domains. Replacement of transmembrane Cys residues by Ala results in nonfunctional enzymes that are unable to hydrolyze ATP. However, the CPC --> APA substituted enzyme binds ATP, indicating its correct folding and suggesting that enzyme turnover is prevented by the lack of metal binding to the transmembrane site. Replacement of C-terminal Cys by Ala (C(751,754)A) has no significant effect on ATPase activity, enzyme phosphorylation, apparent binding affinities of ligands, or E1-E2 equilibrium. In contrast, replacement of Cys in the N-terminal metal binding domain (N-MBD) (C(27,30)A) leads to 40% reduction in enzyme turnover. The C(27,30)A enzyme binds Cu(+), Ag(+), and ATP with the same high apparent affinities as the wild-type CopA. Evidence that N-MBD disruption has no effect on the E1-E2 equilibrium is provided by the normal interaction of ATP acting with low affinity and the unaffected IC(50) for vanadate inhibition observed in the C(27,30)A-substituted enzyme. However, replacement C(27,30)A slowed the dephosphorylation of the E2P(metal) form of the enzyme, suggesting a reduction in the rate of metal release. Other investigators have shown the Cu-dependent interaction of isolated N-MBDs from the Wilson disease Cu-ATPase with the ATP binding cytoplasmic domain [Tsivkovskii et al. (2001) J. Biol. Chem. 276, 2234-2242]. Therefore, the data suggest a regulatory mechanism in which the Cu-dependent N-MBD/ATP binding domain interaction would accelerate cation release, the enzyme rate-limiting step, and consequently Cu(+) transport.     

Backbone dynamics of rusticyanin: the high hydrophobicity and rigidity of this blue copper protein is responsible for its thermodynamic properties

Local dynamics and solute-solvent exchange properties of rusticyanin (Rc) from Thiobacillus ferrooxidans have been studied by applying heteronuclear ((1)H, (15)N) NMR spectroscopy. (15)N relaxation parameters have been determined for the reduced protein, and a model-free analysis has been applied. The high average value of the generalized order parameter, S(2) (0.93), indicates that Rc is very rigid. The analysis of cross correlation rates recorded in both the reduced and the oxidized forms conclusively proves that Rc possesses the same dynamic features in both oxidation states. The accessibility of backbone amide protons to the solvent at different time scales has also been studied by applying specific heteronuclear pulse sequences and by H(2)O/D(2)O exchange experiments. These experiments reveal that rusticyanin is extremely hydrophobic. The first N-35 amino acids, not present in the other BCPs, protect the beta-barrel core from its interaction with the solvent, and thus, this is one of the main factors contributing to the hydrophobicity. Both characteristics (high rigidity and hydrophobicity) are maintained in the metal ion surroundings.     

Characterization of a lactose permease mutant that binds IIAGlc in the absence of ligand

Enzyme IIA(Glc) of the Escherichia coli phosphoenolpyruvate:glucose phosphotransferase system plays a direct role in regulating inducible transport systems. Dephosphorylated IIA(Glc) binds directly to lactose permease in a reaction that requires binding of a galactosidic substrate. A double-Cys mutation (Ile129 --> Cys/Lys131 --> Cys) was introduced into helix IV of the permease near the IIA(Glc) binding site in cytoplasmic loop IV/V and in the vicinity of the galactoside binding site at the interface of helices IV, V, and VIII. The mutant no longer requires galactoside for IIA(Glc) binding as demonstrated by both a [(125)I]IIA(Glc) binding assay and a newly developed fluorescence anisotropy assay. Further characterization of the mutant shows that it binds substrate with high affinity, but is almost completely defective in all modes of translocation across the cytoplasmic membrane. The data are consistent with the interpretation that the double mutant is locked in an inward-facing conformation.     

C-terminal half of human centrin 2 behaves like a regulatory EF-hand domain

Human centrin 2 (HsCen2) is an EF-hand protein that plays a critical role in the centrosome duplication and separation during cell division. We studied the structural and Ca(2+)-binding properties of two C-terminal fragments of this protein: SC-HsCen2 (T94-Y172), covering two EF-hands, and LC-HsCen2 (M84-Y172), having 10 additional residues. Both fragments are highly disordered in the apo state but become better structured (although not conformationally homogeneous) in the presence of Ca(2+) and depending on the nature of the cations (K(+) or Na(+)) in the buffer. Only the longer C-terminal domain, in the Ca(2+)-saturated state and in the presence of Na(+) ions, was amenable to structure determination by nuclear magnetic resonance. The solution structure of LC-HsCen2 reveals an open two EF-hand structure, similar to the conformation of related Ca(2+)-saturated regulatory domains. Unexpectedly, the N-terminal helix segment (F86-T94) lies over the exposed hydrophobic cavity. This unusual intramolecular interaction increases considerably the Ca(2+) affinity and constitutes a useful model for the target binding.     

Transgene expression of green fluorescent protein and germ line transmission in cloned calves derived from in vitro-transfected somatic cells

In vitro transfection of cultured cells combined with nuclear transfer currently is the most effective procedure to produce transgenic livestock. In the present study, bovine primary fetal fibroblasts were transfected with a green fluorescent protein (GFP)-reporter transgene and used as nuclear donor cells in oocyte reconstructions. Because cell synchronization protocols are less effective after transfection, activated oocytes may be more suitable as hosts for nuclear transfer. To examine the role of host cytoplasm on transgene expression and developmental outcome, GFP-expressing fibroblasts were fused to oocytes reconstructed either before (metaphase) or after (telophase) activation. Expression of GFP was examined during early embryogenesis, in tissues of cloned calves, and again during embryogenesis, after passage through germ line using semen from the transgenic cloned offspring. Regardless of the kind of host cytoplasm used, GFP became detectable at the 8- to 16-cell stage, approximately 80 h after reconstruction, and remained positive at all later stages. After birth, although cloned calves obtained through both procedures expressed GFP in all tissues examined, expression levels varied both between tissues and between cells within the same tissue, indicating a partial shutdown of GFP expression during cellular differentiation. Moreover, nonexpressing fibroblasts derived from transgenic offspring were unable to direct GFP expression after nuclear transfer and development to the blastocyst stage, suggesting an irreversible silencing of transgenes. Nonetheless, GFP was expressed in approximately half the blastocysts obtained with sperm from a transgenic clone, confirming transmission of the transgene through the germ line.     

Self-assembly of Pseudomonas fluorescens lipase into bimolecular aggregates dramatically affects functional properties

It has been found that lipase from Pseudomonas fluorescens (PFL) is able to aggregate into bimolecular structures (MW around 66 kD) even at moderate enzyme concentrations. At very low enzyme concentrations and in the presence of detergents, the same enzyme displayed a unimolecular structure with a molecular weight of 33 kD. Both enzyme structures displayed different functional properties. First, the bimolecular structure was much more stable than the unimolecular species (the bimolecular structure maintained over 80% of initial activity after 72 hours at 45 degrees C, while the unimolecular structure retained only around 30% of initial activity after 4 hours of incubation under the same experimental conditions); and the bimolecular form presented a higher optimal T. Second, the unimolecular form showed a much lower K(M) for ethyl butyrate than the bimolecular form. Third, the interfacial activation in biphasic substrate-aqueous milieu was higher for the bimolecular form. Fourth, the unimolecular structure was less active but much more enantioselective than the unimolecular species in the model reaction used. It is proposed that the bimolecular aggregates of PFL might be formed by two open lipase molecules (mutual interfacial activation), in intimate contact, and that the bimolecular form represents an example of "pseudo-quaternary" structure.     

Interaction of secretory immunoglobulin A antibodies with Naegleria fowleri trophozoites and collagen type I

In this work, we analyzed the in vitro interaction of human secretory immunoglobulin A (sIgA) antibodies with Naegleria fowleri trophozoites and the capacity of these antibodies to inhibit amoeba adherence to collagen type I. We also studied N. fowleri antigens that are recognized by sIgA, using immunoblot assays. Immunocytochemical analysis of the interaction showed a redistribution of antigens on the surface of trophozoites by sIgA antibodies. Ultrastructural analysis of antibody-amoeba interaction showed that besides the patching and cap formation, parasites were capable of eliminating the antigen-antibody complex produced on the surface. sIgA antibodies were capable of inhibiting the in vitro adhesion of trophozoites to collagen type I. We suggest that nonsymptomatic infections by N. fowleri may stimulate a local specific immunity that prevents trophozoite adhesion and invasion of nasal mucosa.     

Structural features of an arabinogalactan gum exudates from Spondias dulsis (Anacardiaceae)

The tree Spondias dulcis, located in Venezuela, exudes a light-brown gum. The polysaccharide, isolated from the original gum, contains galactose, arabinose, mannose, rhamnose, glucuronic acid, and its 4-O-methyl derivative. Application of chemical methods, in combination with 1D and 2D NMR spectroscopy afforded interesting structural features of the gum polysaccharide. The unequivocal presence of rhamnose in the polymer structure was confirmed by chemical and spectral data [1H (1.03 ppm); 13C (16.92 ppm)]. Also confirmed was the existence of 3-O- and 6-O-substitutes galactose residues by the spectral data correlations observed in Heteronuclear Multiple Quantum Coherence (HMQC) and Heteronuclear Multiple Bond Correlation (HMBC). Also observed were unequivocal resonances for beta-D-glucuronic acid and its 4-O-methyl derivative, and the presence of 3-O-alpha-L-arabinofuranose and 3-O-beta-L-arabinopyranose residues.