Reconstitution into liposomes of the B°-like glutamine-neutral amino acid transporter from renal cell plasma membrane

Na⁺ dependent [³H]glutamine uptake was found in liposomes reconstituted with solubilized rat kidney brush border in the presence of intraliposomal K⁺. The reconstituted system was optimised with respect to the critical parameters of the cyclic detergent removal procedure, i.e., the detergent used for the solubilization, the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. Time dependent [³H]glutamine accumulation in proteoliposomes occurred only in the presence of external Na⁺and internal K⁺. The transporter showed low if there is any tolerance towards the substitution of Na⁺ or K⁺ for other cations. Valinomycin strongly stimulated the transport indicating that it is electrogenic. Intraliposomal glutamine had no effect. From the dependence of the transport rate on the Na⁺ concentration cooperativity index close to 1 was derived, indicating that 1 Na⁺ should be involved in the cotransport with glutamine. The electrogenicity of the transport originated from the Na⁺ transport. Optimal rate of 0.1 mM [³H]glutamine uptake was found in the presence of 50 mM intraliposomal K-gluconate. At higher K-gluconate concentrations the transport rate decreased. The activity of the reconstituted transporter was pH dependent with optimal function in the range pH 6.5-7.0. [³H]glutamine (and [³H]leucine) uptake was inhibited by all the neutral but not by the positively or negatively charged amino acids. The sulfhydryl reagents HgCl₂, mersalyl, p-hydroxymercuribenzoate and the substrate analogue 2-aminobicyclo[2,2,1]heptane-2-carboxylate strongly inhibited the transporter, whereas the amino acid analogue α-(methylamino)isobutyrate had no effect. The inhibition by mersalyl was protected by the presence of the substrate. On the basis of the Na⁺ dependence, the electrogenic transport mode and the specificity towards the amino acids, the reconstituted transporter was classified as B°-like.     

Reconstitution into liposomes of the glutamine/amino acid transporter from renal cell plasma membrane: functional characterization, kinetics and activation by nucleotides

The glutamine/amino acid transporter was solubilized from rat renal apical plasma membrane (brush-border membrane) with C12E8 and reconstituted into liposomes by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite XAD-4. The reconstitution was optimised with respect to the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. The reconstituted glutamine/amino acid transporter catalysed a first-order antiport reaction stimulated by external, not internal, Na+. Optimal activity was found at pH 7.0. The sulfhydryl reagents HgCl2, mersalyl and p-hydroxymercuribenzoate and the amino acids alanine, serine, threonine, cysteine, asparagine, methionine and valine strongly inhibited the transport, whereas the amino acid analogue methylaminoisobutyrate had no effect. Glutamine, alanine, serine, asparagine, threonine were efficiently translocated from outside to inside and from inside to outside the proteoliposomes as well. Cysteine and valine were translocated preferentially from outside to inside. The Km for glutamine on the external and internal side of the transporter was 0.47 and 11 mM, respectively; the values were not influenced by the type of the counter substrate. The transporter is functionally asymmetrical and it is unidirectionally inserted into the proteoliposomal membrane with an orientation corresponding to that of the native membrane. By a bisubstrate kinetic analysis of the glutamine antiport, a random simultaneous mechanism was found. The glutamine antiport was strongly stimulated by internal nucleoside triphosphates and, to a lower extent, by pyrophoshate. The reconstituted glutamine/amino acid transporter functionally corresponds to the ASCT2 protein.     

Reconstitution into liposomes and functional characterization of the carnitine transporter from renal cell plasma membrane

The carnitine transporter was solubilized from rat renal apical plasma membrane (brush-border membrane) with C₁₂E₈ and reconstituted into liposomes by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite XAD-4. The reconstitution was optimised with respect to the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. The reconstituted carnitine transporter catalysed a first-order antiport reaction (carnitine/carnitine or carnitine/substrate) stimulated by external, not internal, Na⁺, with a positive cooperativity. Na⁺ was co-transported with carnitine. Optimal activity was found between pH 5.5 and pH 6.0. The sulfhydryl reagents MTSES, MTSET and mercurials strongly inhibited the transport. Substrate analogues inhibited the transport; the most effective were acylcarnitines and betaine, followed by dimethylglicine, tetraethylammonium and arginine. Besides carnitine, only acylcarnitines and betaine were efficiently translocated. The Km for carnitine on the external and internal side of the transporter was 0.08 and 1.2 mM, respectively. The transporter is asymmetrical and it is unidirectionally inserted into the proteoliposomal membrane with an orientation corresponding to that of the native membrane. The reconstituted carnitine transporter corresponds, very probably, to the OCTN2 protein.     

Reconstitution into liposomes and functional characterization of the carnitine transporter from renal cell plasma membrane

The carnitine transporter was solubilized from rat renal apical plasma membrane (brush-border membrane) with C12E8 and reconstituted into liposomes by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite XAD-4. The reconstitution was optimised with respect to the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. The reconstituted carnitine transporter catalysed a first-order antiport reaction (carnitine/carnitine or carnitine/substrate) stimulated by external, not internal, Na+, with a positive cooperativity. Na+ was co-transported with carnitine. Optimal activity was found between pH 5.5 and pH 6.0. The sulfhydryl reagents MTSES, MTSET and mercurials strongly inhibited the transport. Substrate analogues inhibited the transport; the most effective were acylcarnitines and betaine, followed by dimethylglicine, tetraethylammonium and arginine. Besides carnitine, only acylcarnitines and betaine were efficiently translocated. The Km for carnitine on the external and internal side of the transporter was 0.08 and 1.2 mM, respectively. The transporter is asymmetrical and it is unidirectionally inserted into the proteoliposomal membrane with an orientation corresponding to that of the native membrane. The reconstituted carnitine transporter corresponds, very probably, to the OCTN2 protein.     

The glutamine/amino acid transporter (ASCT2) reconstituted in liposomes: Transport mechanism, regulation by ATP and characterization of the glutamine/glutamate antiport

The glutamine/amino acid transporter solubilized from rat renal apical plasma membrane (brush-border membrane) with C₁₂E₈ and reconstituted into liposomes has been previously identified as the ASCT2 transporter. The reconstituted transporter catalyses an antiport reaction in which external glutamine and Na⁺ are cotransported in exchange with internal glutamine (or other amino acids). The glutamine-Na⁺ cotransport occurred with a 1:1 stoichiometry. The concentration of Na⁺ did not influence the Km for glutamine and vice versa. Experimental data obtained by a bi-substrate analysis of the glutamine-Na⁺ cotransport, together with previous report on the glutamine_ex/glutamine_in pseudo bi-reactant analysis, indicated that the transporter catalyses a three-substrate transport reaction with a random simultaneous mechanism. The presence of ATP in the internal compartment of the proteoliposomes led to an increase of the Vmax of the transport and to a decrease of the Km of the transporter for external Na⁺. The reconstituted glutamine/amino acid transporter was inhibited by glutamate; the inhibition was more pronounced at acidic pH. A kinetic analysis revealed that the inhibition was competitive with respect to glutamine. Glutamate was also transported in exchange with glutamine. The external Km of the transporter for glutamate (13.3 mM) was slightly higher than the internal one (8.3 mM). At acidic pH the external but not the internal Km decreased. According with the Km values, glutamate should be transported preferentially from inside to outside in exchange for external glutamine and Na⁺.     

Functional reconstitution into liposomes and characterization of the carnitine transporter from rat liver microsomes

The carnitine transporter was solubilized from rat liver microsomes with Triton X-100 and reconstituted into liposomes, after addition of Triton X-114, by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite (Bio-Beads SM 2). The reconstitution was optimized with respect to the detergent/phospholipid ratio, the protein concentration, and the number of passages through a single Amberlite column. The reconstituted carnitine transporter catalyzed a first-order uniport reaction inhibited by HgCl₂ and DIDS. The IC₅₀ for HgCl₂ was 0.16 ± 0.03 mM. The reconstituted transporter also catalyzed carnitine efflux from the proteoliposomes; the efflux was stimulated by externally added long-chain acylcarnitines. Besides carnitine, ornithine, arginine, glutamine and lysine were taken up by the reconstituted liposomes with lower efficiency respect to carnitine. Optimal activity was found at pH 8.0. The Km for carnitine on the external side of the transporter was 10.9 ± 0.16 mM. The activation energy of the carnitine transport derived by Arrhenius plot was 16.1 kJ/mol.     

Probing electron transfer reactions between two azurins from Alcaligenes xylosoxidans GIFU 1051 with optically active Ru complexes as molecular recognition probes: Importance of the 43rd residue

Electron transfer reactions between optically-active Ru^II/III complexes incorporating (S)-/(R)-amino acids, and the two azurins, azurin-1 (az-1Cu) and azurin-2 (az-2Cu) isolated from Alcaligenes xylosoxidans GIFU 1051, have been studied to probe molecular recognition sites on the two azurins. The Ru^II/III complexes are K[Ru^II(L)(bpy)] and [Ru^III(L)(bpy)], and have a tripodal ligand (L) derived from the (S)-/(R)-amino acids, which are in turn exchanged for other functional substituent groups, such as (S)-/(R)-phenylalanine, -leucine, -valine, -alanine, and -glutamic acid (L = (S)-/(R)-BCMPA, -BCMLE, -BCMVA, -BCMAL, and -BCMGA). In the oxidation reaction of az-1Cu^I promoted by the Ru^III complexes, the kinetic parameters exhibited enantio- and stereo-selectivities, while the same reaction of az-2Cu^I was less enantio- and stereo-selective. These differences suggest that the processes of formation of the activated states are different for the two azurins. On the other hand, such a difference has not been observed for az-1 and az-2 with respect to the reduction reactions promoted by both azurins Cu^II by the Ru^II complexes within the experimental error. This suggests that the neutrality of the Ru complexes is important for precise molecular recognition of azurins. His117 has been proposed as the electron transfer site. The local structures in the vicinity of the His117 side chain in the two azurins, are essentially identical with the exception of the 43rd residue, Val43 and Ala43 for az-1 and az-2, respectively. Electron transfer reactions between Ru^III complexes and a mutant azurin, V43A-az-1, were also carried out. Interestingly, the activation parameters estimated were very similar to those of az-2, indicating that the 43rd residue acts as the electron transfer site in azurins and provides rationalization for the different mechanisms of az-1 and az-2 in redox reactions.     

Homology model of the Na+/proline transporter PutP of Escherichia coli and its functional implications

Na⁺/solute symporters are essential membrane integrated proteins that couple the flow of Na⁺ ions driven by electrochemical Na⁺ gradients to the transport of solutes across biological membranes. Here, we used a combination of molecular modeling techniques and evolutionary conservation analysis to construct and validate a first model of the Na⁺/proline symporter PutP of Escherichia coli based on the crystal structure of the bacterial Na⁺/galactose symporter vSGLT. Ligand docking experiments were employed to gain information about residues involved in proline binding. The proposed model is consistent with the available experimental data and was further validated by amino acid substitutions and kinetic and protein chemical analyses. Combination of the results of molecular modeling and functional studies predicts the location and organization of the Na⁺ and proline binding sites. Remarkably, as proposed computationally and discovered here experimentally, residues Y140, W244, and Y248 of transmembrane segments 4 and 7 are found to be particularly important for PutP function and suggested to participate in proline binding and/or gating.     

Mutational analysis of histidine residues in the human proton-coupled amino acid transporter PAT1

The proton-coupled amino acid transporter 1 (PAT1) represents a major route by which small neutral amino acids are absorbed after intestinal protein digestion. The system also serves as a novel route for oral drug delivery. Having shown that H⁺ affects affinity constants but not maximal velocity of transport, we investigated which histidine residues are obligatory for PAT1 function. Three histidine residues are conserved among the H⁺-coupled amino acid transporters PAT1 to 4 from different animal species. We individually mutated each of these histidine residues and compared the catalytic function of the mutants with that of the wild type transporter after expression in HRPE cells. His-55 was found to be essential for the catalytic activity of hPAT1 because the corresponding mutants H55A, H55N and H55E had no detectable l-proline transport activity. His-93 and His-135 are less important for transport function since H93N and H135N mutations did not impair transport function. The loss of transport function of His-55 mutants was not due to alterations in protein expression as shown both by cell surface biotinylation immunoblot analyses and by confocal microscopy. We conclude that His-55 might be responsible for binding and translocation of H⁺ in the course of cellular amino acid uptake by PAT1.     

Isolation and characterization of the plasma membrane from the yeast Pichia pastoris

Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production.     

Substrate specificity and products of side-reactions catalyzed by jasmonate:amino acid synthetase (JAR1)

Jasmonate:amino acid synthetase (JAR1) is involved in the function of jasmonic acid (JA) as a plant hormone. It catalyzes the synthesis of several JA-amido conjugates, the most important of which appears to be JA-Ile. Structurally, JAR1 is a member of the firefly luciferase superfamily that comprises enzymes that adenylate various organic acids. This study analyzed the substrate specificity of recombinant JAR1 and determined whether it catalyzes the synthesis of mono- and dinucleoside polyphosphates, which are side-reaction products of many enzymes forming acyl approximately adenylates. Among different oxylipins tested as mixed stereoisomers for substrate activity with JAR1, the highest rate of conversion to Ile-conjugates was observed for (+/-)-JA and 9,10-dihydro-JA, while the rate of conjugation with 12-hydroxy-JA and OPC-4 (3-oxo-2-(2Z-pentenyl)cyclopentane-1-butyric acid) was only about 1-2% that for (+/-)-JA. Of the two stereoisomers of JA, (-)-JA and (+)-JA, rate of synthesis of the former was about 100-fold faster than for (+)-JA. Finally, we have demonstrated that (1) in the presence of ATP, Mg(2+), (-)-JA and tripolyphosphate the ligase produces adenosine 5'-tetraphosphate (p(4)A); (2) addition of isoleucine to that mixture halts the p(4)A synthesis; (3) the enzyme produces neither diadenosine triphosphate (Ap(3)A) nor diadenosine tetraphosphate (Ap(4)A) and (4) Ap(4)A cannot substitute ATP as a source of adenylate in the complete reaction that yields JA-Ile.     

Purification,crystal structure determination and functional characterization of type III antifreeze proteins from the European eelpout Zoarces viviparus

Antifreeze proteins (AFPs) are essential components of many organisms adaptation to cold temperatures. Fish type III AFPs are divided into two groups, SP isoforms being much less active than QAE1 isoforms. Two type III AFPs from Zoarces viviparus, a QAE1 (ZvAFP13) and an SP (ZvAFP6) isoform, are here characterized and their crystal structures determined. We conclude that the higher activity of the QAE1 isoforms cannot be attributed to single residues, but rather a combination of structural effects. Furthermore both ZvAFP6 and ZvAFP13 crystal structures have water molecules around T18 equivalent to the tetrahedral-like waters previously identified in a neutron crystal structure. Interestingly, ZvAFP6 forms dimers in the crystal, with a significant dimer interface. The presence of ZvAFP6 dimers was confirmed in solution by native electrophoresis and gel filtration. To our knowledge this is the first report of dimerization of AFP type III proteins.     

Different modes of membrane permeabilization by two RTX toxins: HlyA from Escherichia coli and CyaA from Bordetella pertussis

This study clarifies the membrane disruption mechanisms of two bacterial RTX toxins: αhemolysin (HlyA) from Escherichia coli and a highly homologous adenylate cyclase toxin (CyaA) from Bordetella pertussis. For this purpose, we employed a fluorescence requenching method using liposomes (extruded through filters of different pore size — 1000 nm, 400 nm or 100 nm) with encapsulated fluorescent dye/quencher pair ANTS/DPX. We showed that both toxins induced a graded leakage of liposome content with different selectivities α for DPX and ANTS. In contrast to HlyA, CyaA exhibited a higher selectivity for cationic quencher DPX, which increased with vesicle diameter. Large unilamellar vesicles (LUV₁₀₀₀) were found to be more suitable for distinguishing between high α values whereas smaller ones (LUV₁₀₀) were more appropriate for discriminating an all-or-none leakage (α = 0) from the graded leakage with low values of α. While disrupting LUV₁₀₀₀, CyaA caused a highly cation-selective leakage (α ~ 15) whereas its mutated form with decreased channel K⁺/Cl⁻ selectivity due to two substitutions in a predicted transmembrane segment (CyaA-E509K + E516K) exhibited much lower selectivity (α ∼ 6). We concluded that the fluorescence requenching method in combination with different size of liposomes is a valuable tool for characterization of pore-forming toxins and their variants.     

Ferredoxin-NADP+ reductase from Plasmodium falciparum undergoes NADP+-dependent dimerization and inactivation: functional and crystallographic analysis

The completion of the Plasmodium falciparum genome sequence has recently promoted the search for new antimalarial drugs. More specifically, metabolic pathways of the apicoplast, a key organelle for survival of the parasite, have been recognized as potential targets for the development of specific new antimalarial agents. As most apicomplexan parasites, P. falciparum displays a plant-type ferredoxin-NADP(+) reductase, yielding reduced ferredoxin for essential biosynthetic pathways in the apicoplast. Here we report a molecular, kinetic and ligand binding characterization of the recombinant ferredoxin-NADP(+) reductase from P. falciparum, in the light of current data available for plant ferredoxin-NADP(+) reductases. In parallel with the functional characterization, we describe the crystal structures of P. falciparum ferredoxin-NADP(+) reductase in free form and in complex with 2'-phospho-AMP (at 2.4 and 2.7 A resolution, respectively). The enzyme displays structural properties likely to be unique to plasmodial reductases. In particular, the two crystal structures highlight a covalent dimer, which relies on the oxidation of residue Cys99 in two opposing subunits, and a helix-coil transition that occurs in the NADP-binding domain, triggered by 2'-phospho-AMP binding. Studies in solution show that NADP(+), as well as 2'-phospho-AMP, promotes the formation of the disulfide-stabilized dimer. The isolated dimer is essentially inactive, but full activity is recovered upon disulfide reduction. The occurrence of residues unique to the plasmodial enzyme, and the discovery of specific conformational properties, highlight the NADP-binding domain of P. falciparum ferredoxin-NADP(+) reductase as particularly suited for the rational development of antimalarial compounds.     

H2O2 induces rapid biophysical and permeability changes in the plasma membrane of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the diffusion rate of hydrogen peroxide (H₂O₂) through the plasma membrane decreases during adaptation to H₂O₂ by means of a mechanism that is still unknown. Here, evidence is presented that during adaptation to H₂O₂ the anisotropy of the plasma membrane increases. Adaptation to H₂O₂ was studied at several times (15min up to 90min) by applying the steady-state H₂O₂ delivery model. For wild-type cells, the steady-state fluorescence anisotropy increased after 30min, or 60min, when using 2-(9-anthroyloxy) stearic acid (2-AS), or diphenylhexatriene (DPH) membrane probe, respectively. Moreover, a 40% decrease in plasma membrane permeability to H₂O₂ was observed at 15min with a concomitant two-fold increase in catalase activity. Disruption of the ergosterol pathway, by knocking out either ERG3 or ERG6, prevents the changes in anisotropy during H₂O₂ adaptation. H₂O₂ diffusion through the plasma membrane in S. cerevisiae cells is not mediated by aquaporins since the H₂O₂ permeability constant is not altered in the presence of the aquaporin inhibitor mercuric chloride. Altogether, these results indicate that the regulation of the plasma membrane permeability towards H₂O₂ is mediated by modulation of the biophysical properties of the plasma membrane.     

The many faces of calmodulin in cell proliferation,programmed cell death,autophagy, and cancer

Calmodulin (CaM) is a ubiquitous Ca^2 + receptor protein mediating a large number of signaling processes in all eukaryotic cells. CaM plays a central role in regulating a myriad of cellular functions via interaction with multiple target proteins. This review focuses on the action of CaM and CaM-dependent signaling systems in the control of vertebrate cell proliferation, programmed cell death and autophagy. The significance of CaM and interconnected CaM-regulated systems for the physiology of cancer cells including tumor stem cells, and processes required for tumor progression such as growth, tumor-associated angiogenesis and metastasis are highlighted. Furthermore, the potential targeting of CaM-dependent signaling processes for therapeutic use is discussed.     

Grifolin, a potential antitumor natural product from the mushroom Albatrellus confluens, inhibits tumor cell growth by inducing apoptosis in vitro

Grifolin is a natural biologically active substance isolated from the fresh fruiting bodies of the mushroom Albatrellus confluens. Here, for the first time, we describe a novel activity of grifolin, namely its ability to inhibit the growth of tumor cells by the induction of apoptosis. Grifolin strongly inhibited the growth of tumor cell lines: CNE1, HeLa, MCF7, SW480, K562, Raji and B95-8. Analysis of acridine orange (AO)/ethidium bromide (EB) staining and flow cytometry showed that grifolin possessed apoptosis induction activity to CNE1, HeLa, MCF7 and SW480. Furthermore, the cytochrome c release from mitochondria was detected by confocal microscopy in CNE1 cells after a 12h treatment with grifolin. The increase of caspase-8, 9, 3 activities revealed that caspase was a key mediator of the apoptotic pathway induced by grifolin, and the underexpression of Bcl-2 and up-regulation of Bax resulted in the increase of Bax: Bcl-2 ratio, suggesting that Bcl-2 family involved in the control of apoptosis. Owing to the combination of the significant antitumor activity by inducing apoptosis and natural abundance of the compound, grifolin holds the promise of being an interesting antitumor agent that deserves further laboratory and in vivo exploration.     

Structural Insights into Substrate Specificity in Variants of N-Acetylneuraminic Acid Lyase Produced by Directed Evolution

The substrate specificity of Escherichia coli N-acetylneuraminic acid lyase was previously switched from the natural condensation of pyruvate with N-acetylmannosamine, yielding N-acetylneuraminic acid, to the aldol condensation generating N-alkylcarboxamide analogues of N-acetylneuraminic acid. This was achieved by a single mutation of Glu192 to Asn. In order to analyze the structural changes involved and to more fully understand the basis of this switch in specificity, we have isolated all 20 variants of the enzyme at position 192 and determined the activities with a range of substrates. We have also determined five high-resolution crystal structures: the structures of wild-type E. coli N-acetylneuraminic acid lyase in the presence and in the absence of pyruvate, the structures of the E192N variant in the presence and in the absence of pyruvate, and the structure of the E192N variant in the presence of pyruvate and a competitive inhibitor (2R,3R)-2,3,4-trihydroxy-N,N-dipropylbutanamide. All structures were solved in space group P2₁ at resolutions ranging from 1.65 Å to 2.2 Å. A comparison of these structures, in combination with the specificity profiles of the variants, reveals subtle differences that explain the details of the specificity changes. This work demonstrates the subtleties of enzyme-substrate interactions and the importance of determining the structures of enzymes produced by directed evolution, where the specificity determinants may change from one substrate to another.     

Structural and functional characterization of the Geobacillus copper nitrite reductase: Involvement of the unique N-terminal region in the interprotein electron transfer with its redox partner

The crystal structures of copper-containing nitrite reductase (CuNiR) from the thermophilic Gram-positive bacterium Geobacillus kaustophilus HTA426 and the amino (N)-terminal 68 residue-deleted mutant were determined at resolutions of 1.3 Å and 1.8 Å, respectively. Both structures show a striking resemblance with the overall structure of the well-known CuNiRs composed of two Greek key β-barrel domains; however, a remarkable structural difference was found in the N-terminal region. The unique region has one β-strand and one α-helix extended to the northern surface of the type-1 copper site. The superposition of the Geobacillus CuNiR model on the electron-transfer complex structure of CuNiR with the redox partner cytochrome c₅₅₁ in other denitrifier system led us to infer that this region contributes to the transient binding with the partner protein during the interprotein electron transfer reaction in the Geobacillus system. Furthermore, electron-transfer kinetics experiments using N-terminal residue-deleted mutant and the redox partner, Geobacillus cytochrome c₅₅₁, were carried out. These structural and kinetics studies demonstrate that the region is directly involved in the specific partner recognition.     

Inhibition of plasma membrane Ca-ATPase by CrATP. LaATP but not CrATP stabilizes the Ca-occluded state

The bidentate complex of ATP with Cr³⁺, CrATP, is a nucleotide analog that is known to inhibit the sarcoplasmic reticulum Ca²⁺-ATPase and the Na⁺,K⁺-ATPase, so that these enzymes accumulate in a conformation with the transported ion (Ca²⁺ and Na⁺, respectively) occluded from the medium. Here, it is shown that CrATP is also an effective and irreversible inhibitor of the plasma membrane Ca²⁺-ATPase. The complex inhibited with similar efficiency the Ca²⁺-dependent ATPase and the phosphatase activities as well as the enzyme phosphorylation by ATP. The inhibition proceeded slowly (T_1/2 = 30 min at 37 °C) with a K_i = 28 ± 9 μM. The inclusion of ATP, ADP or AMPPNP in the inhibition medium effectively protected the enzyme against the inhibition, whereas ITP, which is not a PMCA substrate, did not. The rate of inhibition was strongly dependent on the presence of Mg²⁺ but unaltered when Ca²⁺ was replaced by EGTA. In spite of the similarities with the inhibition of other P-ATPases, no apparent Ca²⁺ occlusion was detected concurrent with the inhibition by CrATP. In contrast, inhibition by the complex of La³⁺ with ATP, LaATP, induced the accumulation of phosphoenzyme with a simultaneous occlusion of Ca²⁺ at a ratio close to 1.5 mol/mol of phosphoenzyme. The results suggest that the transport of Ca²⁺ promoted by the plasma membrane Ca²⁺-ATPase goes through an enzymatic phospho-intermediate that maintains Ca²⁺ ions occluded from the media. This intermediate is stabilized by LaATP but not by CrATP.