Ribonucleotide reductase as one important target of [Tris(1,10-phenanthroline)lanthanum(III)] trithiocyanate (KP772).

KP772 is a new lanthanum complex containing three 1,10-phenathroline molecules. Recently, we have demonstrated that the promising in vitro and in vivo anticancer properties of KP772 are based on p53-independent G(0)G(1) arrest and apoptosis induction. A National Cancer Institute (NCI) screen revealed significant correlation of KP772 activity with that of the ribonucleotide reductase (RR) inhibitor hydroxyurea (HU). Consequently, this study aimed to investigate whether KP772 targets DNA synthesis in tumor cells by RR inhibition. Indeed, KP772 treatment led to significant reduction of cytidine incorporation paralleled by a decrease of deoxynucleoside triphosphate (dNTP) pools. This strongly indicates disruption of RR activity. Moreover, KP772 protected against oxidative stress, suggesting that this drug might interfere with RR by interaction with the tyrosyl radical in subunit R2. Additionally, several observations (e.g. increase of transferrin receptor expression and protective effect of iron preloading) indicate that KP772 interferes with cellular iron homeostasis. Accordingly, co-incubation of Fe(II) with KP772 led to generation of a coloured iron complex (Fe-KP772) in cell free systems. In electron paramagnetic resonance (EPR) measurements of mouse R2 subunits, KP772 disrupted the tyrosyl radical while Fe-KP772 had no significant effects. Moreover, coincubation of KP772 with iron-loaded R2 led to formation of Fe-KP772 suggesting chelation of RR-bound Fe(II). Summarizing, our data prove that KP772 inhibits RR by targeting the iron centre of the R2 subunit. As also Fe-KP772 as well as free lanthanum exert significant -though less pronounced- cytotoxic/static activities, additional mechanisms are likely to synergise with RR inhibition in the promising anticancer activity of KP772.

Chlamydial ribonucleotide reductase: tyrosyl radical function in catalysis replaced by the FeIII-FeIV cluster.

Ribonucleotide reductase (RNR) from Chlamydia trachomatis is a class I RNR composed of proteins R1 and R2. In protein R2, the tyrosine residue harboring the radical that is necessary for catalysis in other class I RNRs is replaced by a phenylalanine. Active C. trachomatis RNR instead uses the Fe(III)-Fe(IV) state of the iron cluster in R2 as an initiator of catalysis. The paramagnetic Fe(III)-Fe(IV) state, identified by (57)Fe substitution, becomes electron spin resonance detectable in samples that are frozen during conditions of ongoing catalysis. Its amount depends on the conditions for catalysis, such as incubation temperature and the R1/R2 ratio. The results link induction of the Fe(III)-Fe(IV) state with enzyme activity of chlamydial RNR. Based on these observations, a reaction scheme is proposed for the iron site. This scheme includes (i) an activation cycle involving reduction and an oxygen reaction in R2 and (ii) a catalysis cycle involving substrate binding and turnover in R1.

A stable FeIII-FeIV replacement of tyrosyl radical in a class I ribonucleotide reductase.

Ribonucleotide reductase (RNR) of Chlamydia trachomatis is a class I RNR enzyme composed of two homodimeric components, proteins R1 and R2. In class I RNR, R1 has the substrate binding site, whereas R2 has a diferric site and normally in its active form a stable tyrosyl free radical. C. trachomatis RNR is unusual, because its R2 component has a phenylalanine in the place of the radical carrier tyrosine. Replacing the tyrosyl radical, a paramagnetic Fe(III)-Fe(IV) species (species X, normally a transient intermediate in the process leading to radical formation) may provide the oxidation equivalent needed to start the catalytic process via long range electron transfer from the active site in R1. Here EPR spectroscopy shows that in C. trachomatis RNR, species X can become essentially stable when formed in a complete RNR (R1/R2/substrate) complex, adding further weight to the possible role of this species X in the catalytic reaction.

Unique physicochemical profile of beta-amyloid peptide variant Abeta1-40E22G protofibrils: conceivable neuropathogen in arctic mutant carriers.

A new early-onset form of Alzheimer's disease (AD) was described recently where a point mutation was discovered in codon 693 of the beta-amyloid (Abeta) precursor protein gene, the Arctic mutation. The mutation translates into a single amino acid substitution, glutamic acid-->glycine, in position 22 of the Abeta peptide. The mutation carriers have lower plasma levels of Abeta than normal, while in vitro studies show that Abeta1-40E22G protofibril formation is significantly enhanced. We have explored the nature of the Abeta1-40E22G peptide in more detail, in particular the protofibrils. Using size-exclusion chromatography (SEC) and circular dichroism spectroscopy (CD) kinetic and secondary structural characteristics were compared with other Abeta1-40 peptides and the Abeta12-28 fragment, all having single amino acid substitutions in position 22. We have found that Abeta1-40E22G protofibrils are a group of comparatively stabile beta-sheet-containing oligomers with a heterogeneous size distribution, ranging from >100 kDa to >3000 kDa. Small Abeta1-40E22G protofibrils are generated about 400 times faster than large ones. Salt promotes their formation, which significantly exceeds all the other peptides studied here, including the Dutch mutation Abeta1-40E22Q. Position 22 substitutions had significant effects on aggregation kinetics of Abeta1-40 and in Abeta12-28, although the qualitative aspects of the effects differed between the native peptide and the fragment, as no protofibrils were formed by the fragments. The rank order of protofibril formation of Abeta1-40 and its variants was the same as the rank order of the length of the nucleation/lag phase of the Abeta12-28 fragments, E22V>E22A?E22G>E22Q?E22, and correlated with the degree of hydrophobicity of the position 22 substituent. The molecular mass of peptide monomers and protofibrils were estimated better in SEC studies using linear rather than globular calibration standards. The characteristics of the Abeta1-40E22G suggest an important role for the peptide in the neuropathogenesis in the Arctic form of AD.

A left-handed 3(1) helical conformation in the Alzheimer Abeta(12-28) peptide.

We show for the first time that the secondary structure of the Alzheimer beta-peptide is in a temperature-dependent equilibrium between an extended left-handed 3(1) helix and a flexible random coil conformation. Circular dichroism spectra, recorded at 0.03 mM peptide concentration, show that the equilibrium is shifted towards increasing left-handed 3(1) helix structure towards lower temperatures. High resolution nuclear magnetic resonance (NMR) spectroscopy has been used to study the Alzheimer peptide fragment Abeta(12-28) in aqueous solution at 0 degrees C and higher temperatures. NMR translation diffusion measurements show that the observed peptide is in monomeric form. The chemical shift dispersion of the amide protons increases towards lower temperatures, in agreement with the increased population of a well-ordered secondary structure. The solvent exchange rates of the amide protons at 0 degrees C and pH 4.5 vary within at least two orders of magnitude. The lowest exchange rates (0.03-0.04 min(-1)) imply that the corresponding amide protons may be involved in hydrogen bonding with neighboring side chains.

Cell membrane translocation of the N-terminal (1-28) part of the prion protein.

The N-terminal (1-28) part of the mouse prion protein (PrP) is a cell penetrating peptide, capable of transporting large hydrophilic cargoes through a cell membrane. Confocal fluorescence microscopy shows that it transports the protein avidin (67kDa) into several cell lines. The (1-28) peptide has a strong tendency for aggregation and beta-structure formation, particularly in interaction with negatively charged phospholipid membranes. The findings have implications for how prion proteins with uncleaved signal peptides in the N-termini may enter into cells, which is important for infection. The secondary structure conversion into beta-structure may be relevant as a seed for the conversion into the scrapie (PrP(Sc)) form of the protein and its amyloidic transformation.

Tryptophan and tyrosine radicals in ribonucleotide reductase: a comparative high-field EPR study at 94 GHz.

Tryptophan radicals, which are generated in the reconstitution reaction of mutants Y122F and Y177W of subunit R2 apoprotein of E. coli and mouse ribonucleotide reductase (RNR), respectively, with Fe(2+) and oxygen, are investigated by high-field EPR at 94 GHz and compared with the tyrosine radicals occurring in the respective wild-type proteins. For the first time, accurate g-values are obtained for protein-associated neutral tryptophan free radicals, which show only a small anisotropy. The apparent hyperfine patterns observed in frozen solutions are very similar for tryptophan and tyrosine radicals in mouse subunit R2 at conventional X-band EPR. The radicals can, however, be discriminated by their different g-tensors using high-field EPR. Tryptophan radicals were postulated as reaction intermediates in the proposed radical transfer pathway of RNR. Furthermore, the data obtained here for the electronic structure of protein-associated tryptophan neutral free radicals are important for identification and understanding of the functional important tryptophan radicals which occur in other enzymes, e.g., DNA photolyase and cytochrome c peroxidase, where they are magnetically coupled to other radicals or to a metal center.

Cellular internalization of a cargo complex with a novel peptide derived from the third helix of the islet-1 homeodomain. Comparison with the penetratin peptide.

Cellular translocation into a human Bowes melanoma cell line was investigated and compared for penetratin and pIsl, two peptides that correspond to the third helices of the related homeodomains, from the Antennapedia transcription factor of Drosophila and the rat insulin-1 gene enhancer protein, respectively. Both biotinylated peptides internalized into the cells with similar efficacy, yielding an analogous intracellular distribution. When a large cargo protein, 63 kDa avidin, was coupled to either peptide, efficient cellular uptake for both the peptide-protein complexes was observed. The interactions between each peptide and SDS micelles were studied by fluorescence spectroscopy and acrylamide quenching of the intrinsic tryptophan (Trp) fluorescence. Both peptides interacted strongly and almost identically with the membrane mimicking environment. Compared to penetratin, the new transport peptide pIsl has only one Trp residue, which simplifies the interpretation of the fluorescence spectra and in addition has a native Cys residue, which may be used for alternative coupling reactions of cargoes of different character.

Mutagenesis and computer modelling approach to study determinants for recognition of signal peptides by the mitochondrial processing peptidase.

Determinants for the recognition of a mitochondrial presequence by the mitochondrial processing peptidase (MPP) have been investigated using mutagenesis and bioinformatics approaches. All plant mitochondrial presequences with a cleavage site that was confirmed by experimental studies can be grouped into three classes. Two major classes contain an arginine residue at position -2 or -3, and the third class does not have any conserved arginines. Sequence logos revealed loosely conserved cleavage motifs for the first two classes but no significant amino acid conservation for the third class. Investigation of processing determinants for a class III precursor, Nicotiana plumbaginifolia F1beta precursor of ATP synthase (pF1beta), was performed using a series of pF1beta presequence mutants and mutant presequence peptides derived from the C-terminal portion of the presequence. Replacement of -2 Gln by Arg inhibited processing, whereas replacement of either the most proximally located -5 Arg or -15 Arg by Leu had only a low inhibitory effect. The C-terminal portion of the pF1beta presequence forms a helix-turn-helix structure. Mutations disturbing or prolonging the helical element upstream of the cleavage site inhibited processing significantly. Structural models of potato MPP and the C-terminal pF1beta presequence peptide were built by homology modelling and empirical conformational energy search methods, respectively. Molecular docking of the pF1beta presequence peptide to the MPP model suggested binding of the peptide to the negatively charged binding cleft formed by the alpha-MPP and beta-MPP subunits in close proximity to the H111XXE114H115X(116-190)E191 proteolytic active site on beta-MPP. Our results show for the first time that the amino acid at the -2 position, even if not an arginine, as well as structural properties of the C-terminal portion of the presequence are important determinants for the processing of a class III precursor by MPP.

Specific metal-ion binding sites in a model of the P4-P6 triple-helical domain of a group I intron.

Divalent metal ions play a crucial role in RNA structure and catalysis. Phosphorothioate substitution and manganese rescue experiments can reveal phosphate oxygens interacting specifically with magnesium ions essential for structure and/or activity. In this study, phosphorothioate interference experiments in combination with structural sensitive circular dichroism spectroscopy have been used to probe molecular interactions underlying an important RNA structural motif. We have studied a synthetic model of the P4-P6 triple-helical domain in the bacteriophage T4 nrdB group I intron, which has a core sequence analogous to the Tetrahymena ribozyme. Rp and Sp sulfur substitutions were introduced into two adjacent nucleotides positioned at the 3' end of helix P6 (U452) and in the joining region J6/7 (U453). The effects of sulfur substitution on triple helix formation in the presence of different ratios of magnesium and manganese were studied by the use of difference circular dichroism spectroscopy. The results show that the pro-Sp oxygen of U452 acts as a ligand for a structurally important magnesium ion, whereas no such effect is seen for the pro-Rp oxygen of U452. The importance of the pro-Rp and pro-Sp oxygens of U453 is less clear, because addition of manganese could not significantly restore the triple-helical interactions within the isolated substituted model systems. The interpretation is that U453 is so sensitive to structural disturbance that any change at this position hinders the proper formation of the triple helix.

Mammalian p53R2 protein forms an active ribonucleotide reductase in vitro with the R1 protein, which is expressed both in resting cells in response to DNA damage and in proliferating cells.

Recently, a homologue of the small subunit of mammalian ribonucleotide reductase (RNR) was discovered, called p53R2. Unlike the well characterized S phase-specific RNR R2 protein, the new form was induced in response to DNA damage by the p53 protein. Because the R2 protein is specifically degraded in late mitosis and absent in G0/G1 cells, the induction of the p53R2 protein may explain how resting cells can obtain deoxyribonucleotides for DNA repair. However, no direct demonstration of RNR activity of the p53R2 protein was presented and furthermore, no corresponding RNR large subunit was identified. In this study we show that recombinant, highly purified human and mouse p53R2 proteins contain an iron-tyrosyl free radical center, and both proteins form an active RNR complex with the human and mouse R1 proteins. UV irradiation of serum-starved, G0/G1-enriched mouse fibroblasts, stably transformed with an R1 promoter-luciferase reporter gene construct, caused a 3-fold increase in luciferase activity 24 h after irradiation, paralleled by an increase in the levels of R1 protein. Taken together, our data indicate that the R1 protein can function as the normal partner of the p53R2 protein and that an R1-p53R2 complex can supply resting cells with deoxyribonucleotides for DNA repair.

The anaerobic ribonucleotide reductase from Lactococcus lactis. Interactions between the two proteins NrdD and NrdG.

Deoxyribonucleotide synthesis by anaerobic class III ribonucleotide reductases requires two proteins, NrdD and NrdG. NrdD contains catalytic and allosteric sites and, in its active form, a stable glycyl radical. This radical is generated by NrdG with its [4Fe-4S](+) cluster and S-adenosylmethionine. We now find that NrdD and NrdG from Lactobacillus lactis anaerobically form a tight alpha(2)beta(2) complex, suggesting that radical generation by NrdG and radical transfer to the specific glycine residue of NrdD occurs within the complex. Activated NrdD was separated from NrdG by anaerobic affinity chromatography on dATP-Sepharose without loss of its glycyl radical. NrdD alone then catalyzed the reduction of CTP with formate as the electron donor and ATP as the allosteric effector. The reaction required Mg(2+) and was stimulated by K(+) but not by dithiothreitol. Thus NrdD is the actual reductase, and NrdG is an activase, making class III reductases highly similar to pyruvate formate lyase and its activase and suggesting a common root for the two anaerobic enzymes during early evolution. Our results further support the contention that ribonucleotide reduction during transition from an RNA world to a DNA world started with a class III-like enzyme from which other reductases evolved when oxygen appeared on earth.

Interaction and structure induction of cell-penetrating peptides in the presence of phospholipid vesicles.

Certain short peptides, which are able to translocate across cell membranes with a low lytic activity, can be useful as carriers (vectors) for hydrophilic molecules. We have studied three such cell penetrating peptides: pAntp ('penetratin'), pIsl and transportan. pAntp and pIsl originate from the third helix of homeodomain proteins (Antennapedia and Isl-1, respectively). Transportan is a synthetic chimera (galanin and mastoparan). The peptides in the presence of various phospholipid vesicles (neutral and charged) and SDS micelles have been characterized by spectroscopic methods (fluorescence, EPR and CD). The dynamics of pAntp were monitored using an N-terminal spin label. In aqueous solution, the CD spectra of the three peptides show secondary structures dominated by random coil. With phospholipid vesicles, neutral as well as negatively charged, transportan gives up to 60% alpha-helix. pAntp and pIsl bind significantly only to negatively charged vesicles with an induction of around 60% beta-sheet-like secondary structure. With all three peptides, SDS micelles stabilize a high degree of alpha-helical structure. We conclude that the exact nature of any secondary structure induced by the membrane model systems is not directly correlated with the common transport property of these translocating peptides.

The position of the cell penetrating peptide penetratin in SDS micelles determined by NMR.

Penetratin is a 16 residue peptide, RQI KIWFQ NRRMK WKK-amide, with the ability to penetrate cell membranes and a sequence taken from the homeodomain of the Drosophila Antennapedia transcription factor. 600 MHz 1H-nuclear magnetic resonance has been used to study the structure and location of penetratin interacting with a sodium dodecyl sulphate micelle. The positioning of penetratin in the micelle was studied by adding paramagnetic probes (Mn2+ ions, 5-doxyl and 12-doxyl stearic acid) to the solvent. The results show that the peptide is a straight helix positioned with its C-terminus deep inside the micelle and its N-terminus near the surface of the micelle.

Restoring proper radical generation by azide binding to the iron site of the E238A mutant R2 protein of ribonucleotide reductase from Escherichia coli.

The enzyme activity of Escherichia coli ribonucleotide reductase requires the presence of a stable tyrosyl free radical and diiron center in its smaller R2 component. The iron/radical site is formed in a reconstitution reaction between ferrous iron and molecular oxygen in the protein. The reaction is known to proceed via a paramagnetic intermediate X, formally a Fe(III)-Fe(IV) state. We have used 9.6 GHz and 285 GHz EPR to investigate intermediates in the reconstitution reaction in the iron ligand mutant R2 E238A with or without azide, formate, or acetate present. Paramagnetic intermediates, i.e. a long-living X-like intermediate and a transient tyrosyl radical, were observed only with azide and under none of the other conditions. A crystal structure of the mutant protein R2 E238A/Y122F with a diferrous iron site complexed with azide was determined. Azide was found to be a bridging ligand and the absent Glu-238 ligand was compensated for by azide and an extra coordination from Glu-204. A general scheme for the reconstitution reaction is presented based on EPR and structure results. This indicates that tyrosyl radical generation requires a specific ligand coordination with 4-coordinate Fe1 and 6-coordinate Fe2 after oxygen binding to the diferrous site.

Secondary structure and position of the cell-penetrating peptide transportan in SDS micelles as determined by NMR.

Transportan is a 27-residue peptide (GWTLN SAGYL LGKIN LKALA ALAKK IL-amide) which has the ability to penetrate into living cells carrying a hydrophilic load. Transportan is a chimeric peptide constructed from the 12 N-terminal residues of galanin in the N-terminus with the 14-residue sequence of mastoparan in the C-terminus and a connecting lysine. Circular dichroism studies of transportan and mastoparan show that both peptides have close to random coil secondary structure in water. Sodium dodecyl sulfate (SDS) micelles induce 60% helix in transportan and 75% helix in mastoparan. The 600 MHz (1)H NMR studies of secondary structure in SDS micelles confirm the helix in mastoparan and show that in transportan the helix is localized to the mastoparan part. The less structured N-terminus of transportan has a secondary structure similar to that of the same sequence in galanin [Ohman, A., et al. (1998) Biochemistry 37, 9169-9178]. The position of mastoparan and transportan relative to the SDS micelle surface was studied by adding spin-labeled 5-doxyl- or 12-doxyl-stearic acid or Mn2+ to the peptide/micelle system. The combined results show that the peptides are for the most part buried in the SDS micelles. Only the C-terminal parts of both peptides and the central segment connecting the two parts of transportan are clearly surface exposed. For mastoparan, the secondary chemical shifts of the amide protons were found to vary periodically and display a pattern almost identical to those reported for mastoparan in phospholipid bicelles [Vold, R., et al. (1997) J. Biomol. NMR 9, 329-335], indicating similar structures and interactions in the two membrane-mimicking environments.

Accurate measurement of translational diffusion coefficients: a practical method to account for nonlinear gradients.

For NMR probes equipped with pulsed field gradient coils, which are not optimized for gradient linearity, the precision and accuracy of experimentally measured translational diffusion coefficients are limited by the linearity of the gradient pulses over the sample volume. This study shows that the accuracy and precision of measured diffusion coefficients by the Stejskal--Tanner spin-echo pulsed field gradient experiment can be significantly improved by mapping the gradient z-profile and by using the mapped calibration parameters in the data analysis. For practical applications the gradient distribution may be approximated by a truncated linear distribution defined by minimum and maximum values of the gradient. By including the truncated linear gradient distribution function in the Stejskal--Tanner equation, the systematic deviation between the fitted curve and the experimental attenuation curve decreases by an order of magnitude. The gradient distribution may be calibrated using an intense NMR signal from a sample with a known diffusion coefficient. The diffusion coefficient of an unknown sample may then be determined from a two-parameter fit, using the known gradient distribution function.

An FTIR and CD study of the structural effects of G-tract length and sequence context on DNA conformation in solution.

Fourier transform infrared (FTIR) and CD spectroscopy have been used to investigate the structural effects of G-tract length and flanking sequence on the conformation of DNA G-tracts in aqueous solution. Particularly, a possible predisposition for A-form features has been probed, since this may be important for protein-DNA interactions. Five different G-tract-containing DNA duplexes have been studied: d[CATGGCCATG](2), d[CATGGGCCCATG](2), d[CATGGGGCCCCATG](2,) d[AGGGGCCCCT](2), and d[TGGGGCCCCA](2). In addition, a DNA duplex lacking a G-tract center was probed (d[CATATGCATATG](2)). The CD and FTIR results show that the G-tract-containing sequences are all in a dominating B-DNA conformation in solution. However, certain spectral variations reflect structural effects of sequence context and G-tract length. CD spectra and FTIR results in the 1800-1500 cm(-1) region show that the base-stacking pattern is greatly affected by the sequence context. The FTIR backbone 1250-1000 cm(-1) region shows the antisymmetric non-bridging phosphate vibration around 1225 cm(-1) in all sequences, demonstrating the overall B-conformation of the backbone. The FTIR sugar 900-800 cm(-1) region shows variable contributions of two bands around 865 cm(-1) and 840 cm(-1), reflecting the N and S-type of sugar pucker. The relative intensities of the 865 cm(-1) and 840 cm(-1) bands have been proposed in the literature to quantitatively yield the contribution of N and S-type of sugar pucker, respectively. This correlation is supported by the present study. Furthermore, the contributions of N-type sugar in the DNA sequences studied indicate structural propensities that agree with trends in reported crystal structures of the same sequences: (1) d[CATGGCCATG](2), for which FTIR shows the lowest contribution of N-type sugar puckering in solution, crystallizes in a B-like conformation; (2) d[AGGGGCCCCT](2), with the highest degree of N-type sugar puckering of all the sequences studied, crystallizes in an A-like conformation; (3) d[CATGGGCCCATG](2), with an N-type contribution intermediate between that of d[CATGGCCATG](2) and d[AGGGGCCCCT](2), crystallizes in an A/B intermediate conformation.