Spectroscopic and ITC study of the conformational change upon Ca2+-binding in TnC C-lobe and TnI peptide complex from Akazara scallop striated muscle

Akazara scallop (Chlamys nipponensis akazara) troponin C (TnC) of striated adductor muscle binds only one Ca²⁺ ion at the C-terminal EF-hand motif (Site IV), but it works as the Ca²⁺-dependent regulator in adductor muscle contraction. In addition, the scallop troponin (Tn) has been thought to regulate muscle contraction via activating mechanisms that involve the region spanning from the TnC C-lobe (C-lobe) binding site to the inhibitory region of the TnI, and no alternative binding of the TnI C-terminal region to TnC because of no similarity between second TnC-binding regions of vertebrate and the scallop TnIs. To clarify the Ca²⁺-regulatory mechanism of muscle contraction by scallop Tn, we have analyzed the Ca²⁺-binding properties of the complex of TnC C-lobe and TnI peptide, and their interaction using isothermal titration microcalorimetry, nuclear magnetic resonance, circular dichroism, and gel filtration chromatography. The results showed that single Ca²⁺-binding to the Site IV leads to a structural transition not only in Site IV but also Site III through the structural network in the C-lobe of scallop TnC. We therefore assumed that the effect of Ca²⁺-binding must lead to a change in the interaction mode between the C-lobe of TnC and the TnI peptide. The change should be the first event of the transmission of Ca²⁺ signal to TnI in Tn ternary complex.     

Different sensitivity of Phragmites australis and Glyceria maxima to high availability of ammonium-N

The ability to cope with NH₄⁺-N was studied in the littoral helophytes Phragmites australis and Glyceria maxima, species commonly occupying fertile habitats rich in NH₄⁺ and often used in artificial wetlands. In the present study, Glyceria growth rate was reduced by 16% at 179 μM NH₄⁺-N, and the biomass production was reduced by 47% at 3700 μM NH₄⁺-N compared to NO₃⁻-N. Similar responses were not found in Phragmites. The amounts (mg g⁻¹ dry wt) of starch and total non-structural carbohydrates (TNC) in rhizomes were significantly lower in NH₄⁺ (8.9; 12.2 starch; 20.1; 41.9 TNC) compared to NO₃⁻ treated plants (28.0; 15.6 starch; 58.5; 56.3 TNC) in Phragmites and Glyceria, respectively. In addition, Glyceria showed lower amounts (mg g⁻¹ dry wt) of soluble sugars, TNC, K⁺, and Mg²⁺ in roots under NH₄⁺ (5.6; 14.3; 20.6; 1.9) compared to NO₃⁻ nutrition (11.6; 19.9; 37.9; 2.9, for soluble sugars, TNC, K⁺, and Mg²⁺, respectively), while root internal levels of NH₄⁺ and Ca²⁺ (0.29; 4.6 mg g⁻¹ dry wt, mean of both treatments) were only slightly affected. In Phragmites, no changes in soluble sugars, TNC, Ca²⁺, K⁺, and Mg²⁺ contents of roots (7.3; 14.9; 5.1; 17.3; 2.6 mg g⁻¹ dry wt, means of both treatments) were found in response to treatments. The results, therefore, indicate a more pronounced tolerance towards high NH₄⁺ supply in Phragmites compared to Glyceria, although the former may be susceptible to starch exhaustion in NH₄⁺-N nutrition. In contrast, Glyceria's ability to colonize fertile habitats rich in NH₄⁺ is probably related to the avoidance strategy due to shallow rooting or to the previously described ability to cope with high NH₄⁺ levels when P availability is high and NO₃⁻ is also provided.     

Ca2+-dependent Muscle Dysfunction Caused by Mutation of the Caenorhabditis elegans Troponin T-1 Gene

We have investigated the functions of troponin T (CeTnT-1) in Caenorhabditis elegans embryonic body wall muscle. TnT tethers troponin I (TnI) and troponin C (TnC) to the thin filament via tropomyosin (Tm), and TnT/Tm regulates the activation and inhibition of myosin-actin interaction in response to changes in intracellular [Ca²⁺]. Loss of CeTnT-1 function causes aberrant muscle trembling and tearing of muscle cells from their exoskeletal attachment sites (Myers, C.D., P.-Y. Goh, T. StC. Allen, E.A. Bucher, and T. Bogaert. 1996. J. Cell Biol. 132:1061–1077). We hypothesized that muscle tearing is a consequence of excessive force generation resulting from defective tethering of Tn complex proteins. Biochemical studies suggest that such defective tethering would result in either (a) Ca²⁺-independent activation, due to lack of Tn complex binding and consequent lack of inhibition, or (b) delayed reestablishment of TnI/TnC binding to the thin filament after Ca²⁺ activation and consequent abnormal duration of force. Analyses of animals doubly mutant for CeTnT-1 and for genes required for Ca²⁺ signaling support that CeTnT-1 phenotypes are dependent on Ca²⁺ signaling, thus supporting the second model and providing new in vivo evidence that full inhibition of thin filaments in low [Ca²⁺] does not require TnT.     

Spectroscopic studies of the chlorophyll d containing photosystem I from the cyanobacterium, Acaryochloris marina

Absorbance difference spectroscopy and redox titrations have been applied to investigate the properties of photosystem I from the chlorophyll d containing cyanobacterium Acaryochloris marina. At room temperature, the (P740⁺ − P740) and (F_A/B⁻ − F_A/B) absorbance difference spectra were recorded in the range between 300 and 1000 nm while at cryogenic temperatures, (P740⁺A₁⁻ − P740A₁) and (³P740 − P740) absorbance difference spectra have been measured. Spectroscopic and kinetic evidence is presented that the cofactors involved in the electron transfer from the reduced secondary electron acceptor, phylloquinone (A₁⁻), to the terminal electron acceptor and their structural arrangement are virtually identical to those of chlorophyll a containing photosystem I. The oxidation potential of the primary electron donor P740 of photosystem I has been reinvestigated. We find a midpoint potential of 450 ± 10 mV in photosystem I-enriched membrane fractions as well as in thylakoids which is very similar to that found for P700 in chlorophyll a dominated organisms. In addition, the extinction difference coefficient for the oxidation of the primary donor has been determined and a value of 45,000 ± 4000 M^− 1 cm^− 1 at 740 nm was obtained. Based on this value the ratio of P740 to chlorophyll is calculated to be 1:~ 200 chlorophyll d in thylakoid membranes. The consequences of our findings for the energetics in photosystem I of A. marina are discussed as well as the pigment stoichiometry and spectral characteristics of P740.     

Femtosecond primary charge separation in Synechocystis sp. PCC 6803 photosystem I

The ultrafast (< 100 fs) conversion of delocalized exciton into charge-separated state between the primary donor P700 (bleaching at 705 nm) and the primary acceptor A₀ (bleaching at 690 nm) in photosystem I (PS I) complexes from Synechocystis sp. PCC 6803 was observed. The data were obtained by application of pump-probe technique with 20-fs low-energy pump pulses centered at 720 nm. The earliest absorbance changes (close to zero delay) with a bleaching at 690 nm are similar to the product of the absorption spectrum of PS I complex and the laser pulse spectrum, which represents the efficiency spectrum of the light absorption by PS I upon femtosecond excitation centered at 720 nm. During the first ∼ 60 fs the energy transfer from the chlorophyll (Chl) species bleaching at 690 nm to the Chl bleaching at 705 nm occurs, resulting in almost equal bleaching of the two forms with the formation of delocalized exciton between 690-nm and 705-nm Chls. Within the next ∼ 40 fs the formation of a new broad band centered at ∼ 660 nm (attributed to the appearance of Chl anion radical) is observed. This band decays with time constant simultaneously with an electron transfer to A₁ (phylloquinone). The subtraction of kinetic difference absorption spectra of the closed (state P700⁺A₀A₁) PS I reaction center (RC) from that of the open (state P700A₀A₁) RC reveals the pure spectrum of the P700⁺A₀⁻ ion-radical pair. The experimental data were analyzed using a simple kinetic scheme: An* [(PA₀)*A₁ P⁺A₀⁻A₁] P⁺A₀A₁⁻, and a global fitting procedure based on the singular value decomposition analysis. The calculated kinetics of transitions between intermediate states and their spectra were similar to the kinetics recorded at 694 and 705 nm and the experimental spectra obtained by subtraction of the spectra of closed RCs from the spectra of open RCs. As a result, we found that the main events in RCs of PS I under our experimental conditions include very fast (< 100 fs) charge separation with the formation of the P700⁺A₀⁻A₁ state in approximately one half of the RCs, the ∼ 5-ps energy transfer from antenna Chl* to P700A₀A₁ in the remaining RCs, and ∼ 25-ps formation of the secondary radical pair P700⁺A₀A₁⁻.     

Inhibition of calcium-calmodulin complex formation by vasorelaxant basic dipeptides demonstrated by in vitro and in silico analyses

Background

Tryptophan-histidine (Trp-His) was found to suppress the activity of the Ca^2 +/calmodulin (CaM)-dependent protein kinases II (CaMKII), which requires the Ca^2 +-CaM complex for an initial activation. In this study, we attempted to clarify whether Trp-His inhibits Ca^2 +-CaM complex formation, a CaMKII activator.

Methods

The ability of Trp-His and other peptides to inhibit Ca^2 +-CaM complex formation was investigated by a Ca^2 +-encapsulation fluorescence assay. The peptide-CaM interactions were illustrated by molecular dynamic simulation.

Results

We showed that Trp-His inhibited Ca^2 +-CaM complex formation with a 1:1 binding stoichiometry of the peptide to CaM, considering that Trp-His reduced Hill coefficient of Ca^2 +-CaM binding from 2.81 to 1.92. His-Trp also showed inhibitory activity, whereas Trp + His, 3-methyl His-Trp, and Phe-His did not show significant inhibitory activity, suggesting that the inhibitory activity was due to a peptide skeleton (irrespective of the sequence), a basic amino acid, a His residue, the N hydrogen atom of its imidazole ring, and Trp residue. In silico studies suggested the possibility that Trp-His and His-Trp interacted with the Ca^2 +-binding site of CaM by forming hydrogen bonds with key Ca^2 +-binding residues of CaM, with a binding free energy of − 49.1 and − 68.0 kJ/mol, respectively.

Conclusions

This is the first study demonstrating that the vasoactive dipeptide Trp-His possesses inhibitory activity against Ca^2 +-CaM complex formation, which may elucidate how Trp-His inhibited CaMKII in a previous study.

General significance

The results provide a basic idea that could lead to the development of small peptides binding with high affinity to CaM and inhibiting Ca^2 +-CaM complex formation in the future.     

Regulation of Oscillatory Contraction in Insect Flight Muscle by Troponin

Insect indirect flight muscle is activated by sinusoidal length change, which enables the muscle to work at high frequencies, and contracts isometrically in response to Ca²⁺. Indirect flight muscle has two TnC isoforms: F1 binding a single Ca²⁺ in the C-domain, and F2 binding Ca²⁺ in the N- and C-domains. Fibres substituted with F1 produce delayed force in response to a single rapid stretch, and those with F2 produce isometric force in response to Ca²⁺. We have studied the effect of TnC isoforms on oscillatory work. In native Lethocerus indicus fibres, oscillatory work was superimposed on a level of isometric force that depended on Ca²⁺ concentration. Maximum work was produced at pCa 6.1; at higher concentrations, work decreased as isometric force increased. In fibres substituted with F1 alone, work continued to rise as Ca²⁺ was increased up to pCa 4.7. Fibres substituted with various F1:F2 ratios produced maximal work at a ratio of 100:1 or 50:1; a higher proportion of F2 increased isometric force at the expense of oscillatory work. The F1:F2 ratio was 9.8:1 in native fibres, as measured by immunofluorescence, using isoform-specific antibodies. The small amount of F2 needed to restore work to levels obtained for the native fibre is likely to be due to the relative affinity of F1 and F2 for TnH, the Lethocerus homologue of TnI. Affinity of TnC isoforms for a TnI fragment of TnH was measured by isothermal titration calorimetry. The K_d was 1.01 μM for F1 binding and 22.7 nM for F2. The higher affinity of F2 can be attributed to two TnH binding sites on F2 and a single site on F1. Stretch may be sensed by an extended C-terminal domain of TnH, resulting in reversible dissociation of the inhibitory sequence from actin during the oscillatory cycle.     

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.     

B2 receptor-mediated dual effect of bradykinin on proximal tubule Na-ATPase: Sequential activation of the phosphoinositide-specific phospholipase Cβ/protein kinase C and Ca-independent phospholipase A2 pathways

In a previous paper we showed that bradykinin (BK), interacting with its B₂ receptor, inhibits proximal tubule Na⁺-ATPase activity but does not change (Na⁺ + K⁺)ATPase activity. The aim of this paper was to investigate the molecular mechanisms involved in B₂-mediated modulation of proximal tubule Na⁺-ATPase by BK. To abolish B₁ receptor-mediated effects, all experiments were carried out in the presence of (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu), des-Arg⁹-[Leu⁸]-BK (DALBK), a specific antagonist of B₁ receptor. A dual effect on the Na⁺-ATPase activity through the B₂ receptor was found: short incubation times (1-10 min) stimulate the enzyme activity; long incubation times (10-60 min) inhibit it. The stimulatory effect of BK is mediated by activation of phosphoinositide-specific phospholipase C β (PI-PLCβ)/protein kinase C (PKC); its inhibitory action is mediated by Ca²⁺-independent phospholipase A₂ (iPLA₂). Prior activation of the PI-PLCβ/PKC pathway is required to activate the iPLA₂-mediated inhibitory phase. These results reveal a new mechanism by which BK can modulate renal sodium excretion: coupling between B₂ receptor and activation of membrane-associated iPLA₂.     

A novel arsenate reductase from the bacterium Thermus thermophilus HB27: Its role in arsenic detoxification

Microorganisms living in arsenic-rich geothermal environments act on arsenic with different biochemical strategies, but the molecular mechanisms responsible for the resistance to the harmful effects of the metalloid have only partially been examined. In this study, we investigated the mechanisms of arsenic resistance in the thermophilic bacterium Thermus thermophilus HB27. This strain, originally isolated from a Japanese hot spring, exhibited tolerance to concentrations of arsenate and arsenite up to 20 mM and 15 mM, respectively; it owns in its genome a putative chromosomal arsenate reductase (TtarsC) gene encoding a protein homologous to the one well characterized from the plasmid pI258 of the Gram + bacterium Staphylococcus aureus. Differently from the majority of microorganisms, TtarsC is part of an operon including genes not related to arsenic resistance; qRT-PCR showed that its expression was four-fold increased when arsenate was added to the growth medium. The gene cloning and expression in Escherichia coli, followed by purification of the recombinant protein, proved that TtArsC was indeed a thioredoxin-coupled arsenate reductase with a k_cat/K_M value of 1.2 × 10⁴ M^− 1 s^− 1. It also exhibited weak phosphatase activity with a k_cat/K_M value of 2.7 × 10^− 4 M^− 1 s^− 1. The catalytic role of the first cysteine (Cys7) was ascertained by site-directed mutagenesis. These results identify TtArsC as an important component in the arsenic resistance in T. thermophilus giving the first structural–functional characterization of a thermophilic arsenate reductase.     

Experimental evaluation of the pathogenicity of Perkinsusolseni in juvenile Manila clams Ruditapes philippinarum

We evaluated the pathogenicity of Perkinsus olseni towards the Manila clam, Ruditapes philippinarum, by an experimental challenge. For production of prezoosporangia of P. olseni, we injected uninfected Manila clams with cells of a pure strain of P. olseni and reared them for 7 d. Prezoosporangia were isolated from the soft tissue of the injected clams after culturing in Ray’s fluid thioglycollate medium. Hatchery-reared, uninfected juvenile clams (3-10 mm shell length) were challenged by immersion in one of two concentrations of a prezoosporangial suspension of P. olseni for 6 d. The challenged clams had significantly higher mortality at both the concentrations than the unchallenged clams. The mortality due to infection dose-dependently began approximately 4 weeks and 7 weeks after challenge in the higher and lower concentrations, respectively. This is the first experimental evidence that P. olseni causes direct mortality in Manila clams. The lethal level of infection was estimated at approximately 10⁷ pathogen cells/g soft tissue weight.     

The complete mitogenome of Apocheima cinerarius (Lepidoptera: Geometridae: Ennominae) and comparison with that of other lepidopteran insects

The complete mitochondrial genome (mitogenome) of a female flightless geometrid moth Apocheima cinerarius was found to be 15,722 bp in length, containing 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a control region. The A + T content of the complete mitogenome is 80.83%. The AT skew value ([A − T] / [A + T]) is 0.027. The 13 PCGs of the mitogenome start with typical ATN codons, except for cox1 with the start codon CGA. All the tRNA genes have typical cloverleaf secondary structures, except for trnSer(AGN). The secondary structures of rrnL and rrnS were predicted. Six structural domains including conserved regions (IV, V) and variable regions (I, II, III, VI) were identified in the secondary structure of rrnL. The secondary structure of rrnS consists of 3 structural domains. The control region of A. cinerarius begins with conserved motifs of “ATAGA” + 19-bp poly T. It also contains a microsatellite-like (TA)₂₆, a stem-and-loop structure, and a poly-A stretch. Phylogenetic analysis showed that Geometroidea is more closely related to Bombycoidea than to Noctuoidea. A. cinerarius is more closely related to Biston panterinaria than to Phthonandria atrilineata, which is in accordance with the conventional morphology-based classification.     

The dynamics of mitochondrial Ca fluxes

We have investigated the kinetics of mitochondrial Ca²⁺ influx and efflux and their dependence on cytosolic [Ca²⁺] and [Na⁺] using low-Ca²⁺-affinity aequorin. The rate of Ca²⁺ release from mitochondria increased linearly with mitochondrial [Ca²⁺] ([Ca²⁺]_M). Na⁺-dependent Ca²⁺ release was predominant al low [Ca²⁺]_M but saturated at [Ca²⁺]_M around 400 μM, while Na⁺-independent Ca²⁺ release was very slow at [Ca²⁺]_M below 200 μM, and then increased at higher [Ca²⁺]_M, perhaps through the opening of a new pathway. Half-maximal activation of Na⁺-dependent Ca²⁺ release occurred at 5-10 mM [Na⁺], within the physiological range of cytosolic [Na⁺]. Ca²⁺ entry rates were comparable in size to Ca²⁺ exit rates at cytosolic [Ca²⁺] ([Ca²⁺]_c) below 7 μM, but the rate of uptake was dramatically accelerated at higher [Ca²⁺]_c. As a consequence, the presence of [Na⁺] considerably reduced the rate of [Ca²⁺]_M increase at [Ca²⁺]_c below 7 μM, but its effect was hardly appreciable at 10 μM [Ca²⁺]_c. Exit rates were more dependent on the temperature than uptake rates, thus making the [Ca²⁺]_M transients to be much more prolonged at lower temperature. Our kinetic data suggest that mitochondria have little high affinity Ca²⁺ buffering, and comparison of our results with data on total mitochondrial Ca²⁺ fluxes indicate that the mitochondrial Ca²⁺ bound/Ca²⁺ free ratio is around 10- to 100-fold for most of the observed [Ca²⁺]_M range and suggest that massive phosphate precipitation can only occur when [Ca²⁺]_M reaches the millimolar range.     

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.     

Structural and stoichiometric determinants of Ca release-activated Ca (CRAC) channel Ca-dependent inactivation

Depletion of intracellular Ca^2 + stores in mammalian cells results in Ca^2 + entry across the plasma membrane mediated primarily by Ca^2 + release-activated Ca^2 + (CRAC) channels. Ca^2 + influx through these channels is required for the maintenance of homeostasis and Ca^2 + signaling in most cell types. One of the main features of native CRAC channels is fast Ca^2 +-dependent inactivation (FCDI), where Ca^2 + entering through the channel binds to a site near its intracellular mouth and causes a conformational change, closing the channel and limiting further Ca^2 + entry. Early studies suggested that FCDI of CRAC channels was mediated by calmodulin. However, since the discovery of STIM1 and Orai1 proteins as the basic molecular components of the CRAC channel, it has become apparent that FCDI is a more complex phenomenon. Data obtained using heterologous overexpression of STIM1 and Orai1 suggest that, in addition to calmodulin, several cytoplasmic domains of STIM1 and Orai1 and the selectivity filter within the channel pore are required for FCDI. The stoichiometry of STIM1 binding to Orai1 also has emerged as an important determinant of FCDI. Consequently, STIM1 protein expression levels have the potential to be an endogenous regulator of CRAC channel Ca^2 + influx. This review discusses the current understanding of the molecular mechanisms governing the FCDI of CRAC channels, including an evaluation of further experiments that may delineate whether STIM1 and/or Orai1 protein expression is endogenously regulated to modulate CRAC channel function, or may be dysregulated in some pathophysiological states.