Synchrotron SAXS studies on the structural stability of Carcinus aestuarii hemocyanin in solution

The effect of GuHCl and of NaCl on the structural properties of the hemocyanin (Hc) from Carcinus aestuarii has been studied by small angle x-ray scattering (SAXS) using synchrotron radiation. SAXS data collected as a function of perturbant concentration have been used to analyze conformational states of hexameric holo and apoHc as well as the holo and apoforms of the monomeric subunit CaeSS2. In the case of the holoprotein in GuHCl, two concentration domains were identified: at lower concentration, the perturbant induces aggregation of Hc molecules, whereas at higher concentration the aggregates dissociate with concomitant denaturation of the protein. In contrast, with apoHc the denaturation occurs at rather low GuHCl, pointing to an important effect of the active site bound copper for the stabilization of Hc tertiary structure. The effects of NaCl are similar to those of GuHCl as far as CaeSS2 is concerned, namely oligomerization precedes denaturation, whereas in the case of the hexameric form no aggregation occurs. To improve data analysis, on the basis of the current models for Hc monomers and oligomers, the fraction of each aggregation state and/or unfolded protein has been determined by fitting experimental SAXS curves with form factors calculated from Monte Carlo methods. In addition, a global analysis has been carried out on the basis of a thermodynamic model involving an equilibrium between a monomer in a nativelike and denatured form as well as a class of equilibria among the monomer and other aggregates.     

Location of intrinsic and inducible phenoloxidase activity in molluscan hemocyanin

The phenoloxidase (PO) activity of the hemocyanins (Hcs) from two molluscan species, the gastropod Helix pomatia (Hp) and the cephalopod Sepia officinalis (So), was studied. With catechol as substrate the Hcs showed a weak o-diPO activity, which was moderately enhanced on limited proteolysis with subtilisin. The sites in the Hc molecules mainly responsible for this activity were identified. The highest intrinsic o-diPO activity and also by far the highest level of induction were found in the functional units (FUs) Hp f and So g, isolated from Hp beta-Hc and So Hc (subunit 2), respectively. The results thus support the earlier conclusion, made on the basis of sequence homology between molluscan Hcs, that Hp f and So g are functional and structural analogues. The subtilisin treatment of Hp f also induced monoPO activity, considered to be at the origin of browning of the sample.     

Phosphate closes the solution structure of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Mycobacterium tuberculosis

The 5-enolpyruvylshikimate-3-phosphate synthase catalyses the sixth step of the shikimate pathway that is responsible for synthesizing aromatic compounds and is absent in mammals, which makes it a potential target for drugs development against microbial diseases. Here, we report the phosphate binding effects at the structure of the 5-enolpyruvylshikimate-3-phosphate synthase from Mycobacterium tuberculosis. This enzyme is formed by two similar domains that close on each other induced by ligand binding, showing the occurrence of a large conformation change. We have monitored the phosphate binding effects using analytical ultracentrifugation, small angle X-ray scattering and, circular dichroism techniques. The low resolution results showed that the enzyme in the presence of phosphate clearly presented a more compact structure. Thermal-induced unfolding experiments followed by circular dichroism suggested that phosphate rigidified the enzyme. Summarizing, these data suggested that the phosphate itself is able to induce conformational change resulting in the closure movement in the M. tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase.     

Insights into the oligomerization process of the C-terminal domain of human plasma membrane Ca-ATPase

Plasma membrane calcium pumps (PMCAs) sustain a primary transport system for the specific removal of cytosolic calcium ions from eukaryotic cells. PMCAs are characterized by the presence of a C-terminal domain referred to as a regulatory domain. This domain is target of several regulatory mechanisms: activation by Ca²⁺-calmodulin complex and acidic phospholipids, phosphorylation by kinase A and C, proteolysis by calpain and oligomerization. As far as oligomerization is concerned, the C-terminal domain seems to be crucial for this process. We have cloned the C-terminal domain of the human PMCA isoform 1b, and characterized its properties in solution. The expressed protein maintains its tendency to oligomerize in aqueous solutions, but it is dissociated by amphipathic molecules such as diacylglycerol and sodium dodecyl sulphate. The presence of sodium dodecyl sulphate stabilizes the domain as a compact structure in monomeric form retaining the secondary structure elements, as shown by small angle neutron scattering and circular dichroism measurements. The importance of oligomerization for the regulation of PMCA activity and intracellular calcium concentration is discussed.     

Role of the tertiary structure in the diphenol oxidase activity of Octopus vulgaris hemocyanin

The functional differences between the oxygen transport protein Hemocyanin and the enzymes Tyrosinase and Catechol oxidase are believed to be governed, at least in part, by the tertiary structure, which differs in these molecules and controls the accessibility of their copper containing active site for substrate(s). Accordingly, Octopus vulgaris Hemocyanin catalyses the o-diphenol oxidation to o-quinone at a very low rate. The crystallographic structure of one of the functional units (called Odg) of O. dofleini Hemocyanin shows two domains, a mainly α-helical domain that directly binds the copper ions of the reaction center and a β-strand domain that precludes access to the active site to ligands bigger than molecular oxygen. In this work, we have first cleaved the whole protein and then purified different oxygen binding functional units from O. vulgaris Hemocyanin. These functional units were used in activity assays with l-DOPA, the paradigmatic substrate for Catechol oxidase. All functional units show a negligible enzymatic activity. The procedure to generate the functional units induces in only one of them a proteolytic cleavage. Amino terminal sequencing and mass spectroscopy of the fragments allow to place the cleavage site between the alpha and beta domains of the functional unit homologous to Odd, in the O. dofleini sequence. An increase, up to three orders of magnitude, of Tyrosinase-like activity was observed when the cleaved Odd-like was incubated with the substrate in the presence of trifluoroethanol or hexafluoroisopropanol.     

Structural roles of the active site iron(III) ions in catechol 1,2-dioxygenases and differential secondary structure changes in isoenzymes A and B from Acinetobacter radioresistens S13

The reversible active site metal ion removal process for two catechol 1,2-dioxygenase isoenzymes (IsoA and IsoB) isolated from Acinetobacter radioresistens S13 has been monitored using circular dichroism and fluorescence spectroscopic techniques. IsoA and IsoB are homodimers, containing one iron(III) ion per subunit. Their amino acid sequence identity is 48.4%. Previous experiments suggested that structural diversities could be responsible for the differential thermal and pH stabilities of the two isoenzymes and of their distinct demetallation kinetics. The far-UV CD spectra of IsoA and IsoB catechol 1,2-dioxygenases from A. radioresistens S13 provide information on their secondary structures. IsoB appears to have a content of alpha-helices higher than IsoA. Upon metal ion removal, both proteins reversibly lose part of their secondary structure following distinct pathways. CD spectra simulations allowed us to estimate the content of alpha-helices, beta-sheets, and turns for each isoenzyme and to monitor the secondary structure rearrangements. The metal ion withdrawal has large influence on the secondary structure: in particular a significant reduction of alpha-helices content is observed for both isoenzymes. Intrinsic fluorescence emission spectra clearly support such results, adding information on the local environment changes of the tryptophan residues. The positioning of Trp250 in IsoB has been shown to be of particular interest for monitoring the local structure changes occurring upon metal ion removal. For the first time these studies allow to underline the role of active site iron ions on dioxygenases folding and stability, further evidencing the differences in structural assembling between the two isoenzymes from A. radioresistens S13.     

Nascent structure in the kinase anchoring domain of microtubule-associated protein 2

Biological processes are often viewed as highly ordered interactions between well-folded protein domains. The specific interactions exhibited by certain highly abundant neuronal proteins such as microtubule-associated protein 2 (MAP2) and tau stand in stark contrast because these proteins do not show evidence of structure by standard biophysical assays, yet they do bind to specific targets. It is conceivable that there are regions of MAP2 and tau with propensity to form structural domains upon binding a target. To search for evidence of such regions, limited proteolysis experiments were carried out on MAP2c, the smallest MAP2 isoform. Increased protease resistance was observed around the binding site for the RII subunit of cAMP-dependent protein kinase. Protein constructs spanning this region were produced based on the long-lived tryptic fragments Ser44-Arg93 and Ile94-Arg182, and were probed for structure using spectroscopic methods. The results support the existence of regions of nascent structure in the N-terminal region of MAP2c, which are believed to contribute to its regulatory function.     

Site-directed mutagenesis of the conserved Ala348 and Gly350 residues at the putative active site of Bacillus kaustophilus leucine aminopeptidase

Leucine aminopeptidase (LAP) is an exopeptidase that catalyzes the hydrolysis of amino acid residues from the amino terminus of proteins and peptides. Sequence alignment shows that the conserved Ala348 and Gly350 residues of Bacillus kaustophilus LAP (BkLAP) are located right next to a coordinated ligand. We further investigated the roles of these two residues by performing computer modeling and site-directed mutagenesis. Based on the modeling, the carbonyl group of Ala348 interacts with Asn345 and Asn435, and that of Gly350 with Ile353 and Leu354, where these interactions might maintain the zinc-coordinated residues at their correct positions. Replacement of Ala348 with Arg resulted in a dramatic reduction in LAP activity. A complete loss of the activity was also observed in A348E, A348V, and the Gly350 variants. Measurement of intrinsic tryptophan fluorescence revealed alteration of the microenvironment of aromatic amino acid residues, while circular dichroism spectra were nearly identical for wild-type and all mutant enzymes. Protein modeling and site-directed mutagenesis suggest that residues Ala348 and Gly350 are essential for BkLAP in maintaining a stable active-site environment for the catalytic reaction.     

Involvement of glycan chains in the antigenicity of Rapana thomasiana hemocyanin

Functional unit (FU) RtH2-e from Rapana thomasiana hemocyanin (Hc) was degraded into small fragments with chymotrypsin. The glycopeptides were separated from the non-glycosylated peptides by chromatography on Concanavalin-A-Sepharose and characterized by mass spectrometry. The glycan part of the glycopeptides (all with common peptide stretch of 14 amino acids) consists of the classical trimannosyl-N,N-diacetylchitobiose core for N-glycosylation, predominantly extended with a unique tetrasaccharide that is branched on fucose. In inhibition ELISA experiments, the glycopeptides interfered in the complex formation between FU RtH2-e and rabbit antibodies against Rapana Hc (about 30% of inhibition). The inhibition also was retained after treatment of the glycopeptides with pronase in order to completely destroy the peptide part. The inhibitory effect of the non-glycosylated peptides, on the other hand, was very low. This study thus demonstrates that the glycans attached to FU RtH2-e contribute to the antigenicity of Rapana Hc.     

Structural analysis of the stalk subunit Vma5p of the yeast V-ATPase in solution

Vma5p (subunit C) of the yeast V-ATPase was produced in Escherichia coli and purified to homogeneity. Analysis of secondary structure by circular dichroism spectroscopy showed that Vma5p comprises 64% -helix and 17% β-sheet content. The molecular mass of this subunit, determined by gel filtration analysis and small angle X-ray scattering (SAXS), was approximately 51 ± 4 kDa, indicating a high hydration level of the protein in solution. The radius of gyration and the maximum size of Vma5p were determined to be 3.74 ± 0.03 and 12.5 ± 0.1 nm, respectively. Using two independent ab initio approaches, the first low-resolution shape of the protein was determined. Vma5p is an elongated boot-shaped particle consisting of two distinct domains. Co-reconstitution of Vma5p to V₁ without C from Manduca sexta resulted in a V₁–Vma5p hybrid complex and a 20% increase in ATPase hydrolysis activity.     

Asn124 of Cel5A from Hypocrea jecorina not only provides the N-glycosylation site but is also essential in maintaining enzymatic activity

To investigate the function of N-glycosylation of Cel5A (endoglucanase II) from Hypocrea jecorina, two N-glycosylation site deletion Cel5A mutants (rN124D and rN124H) were expressed in Saccharomyces cerevisiae. The weights of these recombinant mutants were 54 kDa, which were lower than that of rCel5A. This result was expected to be attributed to deglycosylation. The enzyme activity of rN124H was greatly reduced to 60.6% compared with rCel5A, whereas rN124D showed slightly lower activity (10%) than that of rCel5A. rN124D and rN124H showed different thermal stabilities compared with the glycosylated rCel5A, especially at lower pH value. Thermal stabilities were reduced and improved for rN124D and rN124H, respectively. Circular dichroism spectroscopy showed that the modification of secondary structure by mutation may be the reason for the change in enzymatic activity and thermal stability. [BMB Reports 2014; 47(5): 256-261]     

Conformational stability of recombinant manganese superoxide dismutase from the filamentous fungus Trichoderma reesei

Superoxide dismutases (SODs; EC 1.15.1.1) are part of the antioxidant system of aerobic organisms and are used as a defense against oxidative injury caused by reactive oxygen species (ROS). The cloning and sequencing of the 788-bp genomic DNA from Trichoderma reesei strain QM9414 (anamorph of Hypocrea jecorina) revealed an open reading frame encoding a protein of 212 amino acid residues, with 65-90% similarity to manganese superoxide dismutase from other filamentous fungi. The TrMnSOD was purified and shown to be stable from 20 to 90 °C for 1 h at pH from 8 to 11.5, while maintaining its biological activity.     

Transient binding of plastocyanin to its physiological redox partners modifies the copper site geometry

The transient complexes of plastocyanin with cytochrome f and photosystem I are herein used as excellent model systems to investigate how the metal sites adapt to the changes in the protein matrix in transient complexes that are involved in redox reactions. Thus, both complexes from the cyanobacterium Nostoc sp. PCC 7119 (former Anabaena sp. PCC 7119) have been analysed by X-ray absorption spectroscopy. Our data are consistent with a significant distortion of the trigonal pyramidal geometry of the Cu coordination sphere when plastocyanin binds to cytochrome f, no matter their redox states are. The resulting tetrahedral geometry shows a shortening of the distance between Cu and the S(delta) atom of its ligand Met-97, with respect to the crystallographic structure of free plastocyanin. On the other hand, when plastocyanin binds to photosystem I instead of cytochrome f, the geometric changes are not significant but a displacement in charge distribution around the metal centre can be observed. Noteworthy, the electronic density around the Cu atom increases or decreases when oxidised plastocyanin binds to cytochrome f or photosystem I, respectively, thus indicating that the protein matrix affects the electron transfer between the two partners during their transient interaction.     

Ferric ions involved in the flower color development of the Himalayan blue poppy, Meconopsis grandis

The Himalayan blue poppy, Meconopsis grandis, has sky blue-colored petals, although the anthocyanidin nucleus of the petal pigment is cyanidin. The blue color development in this blue poppy involving ferric ions was therefore studied. We analyzed the vacuolar pH, and the organic and inorganic components of the colored cells. A direct measurement by a proton-selective microelectrode revealed that the vacuolar pH value was 4.8. The concentrations of the total anthocyanins in the colored cells were around 5mM, and ca. three times more concentrated flavonols were detected. Fe was detected by atomic analysis of the colored cells, and the ratio of Fe to anthocyanins was ca. 0.8 eq. By mixing the anthocyanin, flavonol and metal ion components in a buffered aq. solution at pH 5.0, we were able to reproduce the same blue color; the visible absorption spectrum and CD were identical to those in the petals, with Fe(3+), Mg(2+) and flavonol being essential for the blue color. The blue pigment in Meconopsis should be a new type of metal complex pigment that is different from a stoichiometric supramolecular pigment such as commelinin or protocyanin.     

The active site of the Escherichia coli glycogen synthase is similar to the active site of retaining GT-B glycosyltransferases

Bacterial glycogen synthases transfer a glucosyl unit, retaining the anomeric configuration, from ADP-glucose to the non-reducing end of glycogen. We modeled the Escherichia coli glycogen synthase based on three glycosyltransferases with a GT-B fold. Comparison between the model and the structure of the active site of crystallized retaining GT-B glycosyltransferases identified conserved residues with the same topology. To confirm the importance of these residues predicted by the model, we studied them in E. coli glycogen synthase by site-directed mutagenesis. Mutations D137A, R300A, K305A, and H161A decreased the specific activity 8100-, 2600-, 1200-, and 710-fold, respectively. None of these mutations increased the Km for glycogen and only H161A and R300A had a higher Km for ADP-Glc of 11- and 8-fold, respectively. These residues were essential, validating the model that shows a strong similarity between the active site of E. coli glycogen synthase and the other retaining GT-B glycosyltransferases known to date.     

Mobility of the conserved glycine 155 is required for formation of the active plasmodial Pdx1 dodecamer

Background

Vitamin B6 synthesis requires a functional Pdx1 assembly that is dodecameric in vivo. We have previously shown that mutation of a catalytic lysine in the plasmodial Pdx1 protein results in a protein that is both inactive and hexameric in vitro.

Methods

Static and dynamic light scattering, circular dichroism, co-purification and enzyme assays are used to investigate the role of a glycine conserved in all Pdx1 family members.

Results

Static light scattering indicates that a glycine to alanine mutant is present as a hexamer in vitro. Subsequent circular dichroism experiments demonstrate that a significant change in secondary structure content is induced by this mutation. However, this mutant is still competent to bind and support Pdx2 activity.

Conclusions

As the mutated glycine occupies an unrestricted region of the Ramachandran plot the additional stereo-chemical restrictions imposed on alanine residues strongly support our hypothesis that significant structural rearrangement of Pdx1 is required during the transition from hexamer to dodecamer.

General significance

The presented results demonstrate that reduction in the mobility of this region in Pdx1 proteins is required for formation of the in vivo dodecamer, negatively affecting the activity of Pdx1, opening the possibility of allosteric Pdx1 inhibitors.     

Atomic resolution structures of native copper nitrite reductase from Alcaligenes xylosoxidans and the active site mutant Asp92Glu

We provide the first atomic resolution (<1.20 A) structure of a copper protein, nitrite reductase, and of a mutant of the catalytically important Asp92 residue (D92E). The atomic resolution where carbon-carbon bonds of the peptide become clearly resolved, remains a key goal of structural analysis. Despite much effort and technological progress, still very few structures are known at such resolution. For example, in the Protein Data Bank (PDB) there are some 200 structures of copper proteins but the highest resolution structure is that of amicyanin, a small (12 kDa) protein, which has been resolved to 1.30 A. Here, we present the structures of wild-type copper nitrite reductase (wtNiR) from Alcaligenes xylosoxidans (36.5 kDa monomer), the "half-apo" recombinant native protein and the D92E mutant at 1.04, 1.15 and 1.12A resolutions, respectively. These structures provide the basis from which to build a detailed mechanism of this important enzyme.     

Dynamics of the active site architecture in plant-type ferredoxin-NADP reductases catalytic complexes

Kinetic isotope effects in reactions involving hydride transfer and their temperature dependence are powerful tools to explore dynamics of enzyme catalytic sites. In plant-type ferredoxin-NADP⁺ reductases the FAD cofactor exchanges a hydride with the NADP(H) coenzyme. Rates for these processes are considerably faster for the plastidic members (FNR) of the family than for those belonging to the bacterial class (FPR). Hydride transfer (HT) and deuteride transfer (DT) rates for the NADP⁺ coenzyme reduction of four plant-type FNRs (two representatives of the plastidic type FNRs and the other two from the bacterial class), and their temperature dependences are here examined applying a full tunnelling model with coupled environmental fluctuations. Parameters for the two plastidic FNRs confirm a tunnelling reaction with active dynamics contributions, but isotope effects on Arrhenius factors indicate a larger contribution for donor–acceptor distance (DAD) dynamics in the Pisum sativum FNR reaction than in the Anabaena FNR reaction. On the other hand, parameters for bacterial FPRs are consistent with passive environmental reorganisation movements dominating the HT coordinate and no contribution of DAD sampling or gating fluctuations. This indicates that active sites of FPRs are more organised and rigid than those of FNRs. These differences must be due to adaptation of the active sites and catalytic mechanisms to fulfil their particular metabolic roles, establishing a compromise between protein flexibility and functional optimisation. Analysis of site-directed mutants in plastidic enzymes additionally indicates the requirement of a minimal optimal architecture in the catalytic complex to provide a favourable gating contribution.     

Growth, reproductive and photosynthetic responses to copper in the yellow-horned poppy, Glaucium flavum Crantz.

Glaucium flavum Crantz. is found in an anthropized coastal grassland at the joint estuary of the Tinto and Odiel rivers (SW Spain), growing under the influence of high levels of copper contamination derived from nearby petrochemical industries, with no obvious adverse affects on the performance of the plant. In addition, this species exhibits a series of ecological characteristics which may render it appropriate for use in the phytoremediation of contaminated areas. Nonetheless, the response of G. flavum to elevated copper concentrations has not been studied. A greenhouse experiment was conducted to investigate the effects of a range of Cu concentrations (0 to 47 mmol l⁻¹) on the growth, reproduction and photosynthetic performance of G. flavum, by measuring relative growth rate, fruit and seed production, chlorophyll fluorescence parameters, gas exchange and photosynthetic pigment concentrations. We also determined total copper, nitrogen, phosphorous, sulphur, calcium and magnesium concentrations. G. flavum survived with concentrations of up to 730 mg Cu kg⁻¹ DW in the leaves, when treated with 30 mmol Cu l⁻¹ (2000 mg l⁻¹). Quantum efficiency of PSII, net photosynthesis rate, as well as leaf Ca and Mg concentrations were all negatively affected by Cu concentrations greater than 9 mmol l⁻¹ in the nutrient solution. Our results indicate that the reduction in photosynthetic performance may be attributed to the adverse effect of excess Cu on the photosynthetic apparatus of the plant, both directly, via a decrease in pigment concentrations, and indirectly, via interference of Cu with Ca ions of PSII. Growth and seed production were only slightly affected by leaf tissue concentrations as high as 230 mg Cu kg⁻¹ dry mass, which suggests that this species could play an important role in phytoremediation of Cu-contaminated soils.     

Structure analysis of the protein translocating channel TatA in membranes using a multi-construct approach

The twin-arginine-translocase (Tat) can transport proteins in their folded state across bacterial or thylakoid membranes. In Bacillus subtilis the Tat-machinery consists of only two integral (inner) membrane proteins, TatA and TatC. Multiple copies of TatA are supposed to form the transmembrane channel, but little structural data is available on this 70-residue component. We used a multi-construct approach for expressing several characteristic fragments of TatA_d, to determine their individual structures and to cross-validate them comprehensively within the architecture of the full-length protein. Here, we report the design, high-yield expression, detergent-aided purification and lipid-reconstitution of five constructs of TatA_d, overcoming difficulties associated with the very different hydrophobicities and sizes of these membrane protein fragments. Circular dichroism (CD) and oriented CD (OCD) were used to determine their respective conformations and alignments in suitable, negatively charged phospholipid bilayers. CD spectroscopy showed an N-terminal α-helix, a central helical stretch, and an unstructured C-terminus, thus proving the existence of these secondary structures in TatA_d for the first time. The OCD spectra demonstrated a transmembrane orientation of the N-terminal α-helix and a surface alignment of the central amphiphilic helix in lipid bilayers, thus supporting the postulated topology model and function of TatA as a transmembrane channel.