Activation and inhibition of cyclin-dependent kinase-2 by phosphorylation; a molecular dynamics study reveals the functional importance of the glycine-rich loop

Nanoseconds long molecular dynamics (MD) trajectories of differently active complexes of human cyclin-dependent kinase 2 (inactive CDK2/ATP, semiactive CDK2/Cyclin A/ATP, fully active pT160-CDK2/Cyclin A/ATP, inhibited pT14-; pY15-; and pT14,pY15,pT160-CDK2/Cyclin A/ATP) were compared. The MD simulations results of CDK2 inhibition by phosphorylation at T14 and/or Y15 sites provide insight into the structural aspects of CDK2 deactivation. The inhibitory sites are localized in the glycine-rich loop (G-loop) positioned opposite the activation T-loop. Phosphorylation of T14 and both inhibitory sites T14 and Y15 together causes ATP misalignment for phosphorylation and G-loop conformational change. This conformational change leads to the opening of the CDK2 substrate binding box. The phosphorylated Y15 residue negatively affects substrate binding or its correct alignment for ATP terminal phospho-group transfer to the CDK2 substrate. The MD simulations of the CDK2 activation process provide results in agreement with previous X-ray data.     

Regulation of cleavage by protein kinase C in Chaetopterus

We report that protein kinase C (PKC) plays a regulatory role in early cleavage in Chaetopterus eggs. Using Western blotting, we assayed the expression patterns of conventional PKCs (cPKC), novel PKCs (nPKC), and atypical PKCs (aPKC). During early development after fertilization, PKC protein levels varied independently by isoform. PKC protein expression during differentiation, without cleavage and after parthenogenetic activation, was very similar to that during normal development indicating that PKC gene expression does not require cellularization. Since PKC has been shown to regulate meiosis in this organism, we also assayed the membrane association of these isoforms as an indicator of their activation during meiosis and early cleavage. PKC-gamma transiently associated with membranes and therefore became activated before meiotic division and cleavage, whereas PKC-alpha and -beta transiently dissociated from membranes and therefore became inactivated at these times. Inhibition of these PKC isoforms by bisindolylmaleimide I had no effect on cleavage or early development to the trochophore larva, indicating that PKC-gamma activation is not essential for cleavage or early development. However, their persistent activation by thymeleatoxin blocked cleavage. The results indicate that the dissociation of PKC-alpha and/or -beta from the membrane fraction, and therefore their inactivation, is essential for normal cleavage. Elevated PKC activity is essential for nuclear envelope breakdown and spindle formation at meiosis I. By contrast, down-regulation of this activity is essential for cleavage after fertilization.     

Phylogeny and host-plant association in the leaf beetle genus Trirhabda LeConte (Coleoptera: Chrysomelidae)

The leaf beetle genus Trirhabda contains 26 described species from the United States and Canada, feeding on host plants from the families Asteraceae and Hydrophyllaceae. In this study, we present a phylogeny for the genus that was reconstructed from mitochondrial COI and 12S rRNA fragments, nuclear ITS2 rRNA, and morphological characters. Both parsimony and mixed-model Bayesian likelihood analyses were performed. Under both methods, the mitochondrial and nuclear partitions support the same backbone phylogeny, as do the combined data. The utility of the molecular data is contrasted with the low phylogenetic signal among morphological characters. The phylogeny was used to trace the evolution of the host-plant association in Trirhabda. The recovered phylogeny shows that although the host-plant association is phylogenetically conservative, Trirhabda experienced one shift to a distantly related host-plant family, 6 shifts between host-plant tribes, and 6 between genera within tribes. The phylogeny reveals that Trirhabda were plesiomorphically adapted to tolerate complex secondary compounds of its host plants and this adaptation is retained in Trirhabda species, as evidenced by multiple shifts from chemically simpler host plants back to the more complex host plants.     

Dynamics of endogenous cytokinin pools in tobacco seedlings: a modelling approach

Recent advances in cytokinin analysis have made it possible to measure the content of 22 cytokinin metabolites in the tissue of developing tobacco seedlings. Individual types of cytokinins in plants are interconverted to their respective forms by several enzymatic activities (5'-AMP-isopentenyltransferase, adenosine nucleosidase, 5'-nucleotidase, adenosine phosphorylase, adenosine kinase, trans-hydroxylase, zeatin reductase, beta-glucosidase, O-glucosyl transferase, N-glucosyl transferase, cytokinin oxidase). This paper reports modelling and measuring of the dynamics of endogenous cytokinins in tobacco plants grown on media supplemented with isopentenyl adenine (IP), zeatin (Z) and dihydrozeatin riboside (DHZR). Differences in phenotypes generated by the three cytokinins are shown and discussed, and the assumption that substrate concentration drives enzyme kinetics underpinned the construction of a simple mathematical model of cytokinin metabolism in developing seedlings. The model was tested on data obtained from liquid chromatography/tandem mass spectrometry cytokinin measurements on tobacco seedlings grown on Murashige and Skoog agar nutrient medium, and on plants grown in the presence of IP, Z and DHZR. A close match was found between measured and simulated data, especially after a series of iterative parameter searches, in which the parameters were set to obtain the best fit with one of the data sets.     

Effects of oleic acid and its congeners,elaidic and stearic acids,on the structural properties of phosphatidylethanolamine membranes

Fatty acid derivatives are abundant in biological membranes, mainly as components of phospholipids and cholesterol esters. Their presence, free or bound to phospholipids, modulates the lipid membrane behavior. The present study shows the differential influence of the C-18 fatty acids (FAs), oleic, elaidic, and stearic acids on the structural properties of phosphatidylethanolamine (PE). X-ray diffraction of PE-FA systems demonstrated that oleic acid (OA) produced important concentration-dependent alterations of the lipid membrane structure: it induced reductions of up to 20-23 degrees C in the lamellar-to-hexagonal transition temperature of 1-palmitoyl-2-oleoyl PE and dielaidoyl PE and regulated the dimensions of the hexagonal lattice. In contrast, elaidic and stearic acids did not markedly alter the phospholipid mesomorphism. The above effects were attributed to the different "molecular shape" of OA (with a kink at the middle of the molecule) with respect to their congeners, elaidic and stearic acids. The effects of free fatty acids (FFAs) on membrane structure are relevant for several reasons: i) some biological membranes contain very high levels of FFAs. ii) Mediterranean diets with high OA intake have been shown to exert protective effects against tumoral and hypertensive pathologies. iii) FFA derivatives have been developed as antitumoral and antihypertensive drugs.     

dUTPase and nucleocapsid polypeptides of the Mason-Pfizer monkey virus form a fusion protein in the virion with homotrimeric organization and low catalytic efficiency

Betaretroviruses encode dUTPase, an essential factor in DNA metabolism and repair, in the pro open reading frame located between gag and pol. Ribosomal frame-shifts during expression of retroviral proteins provide a unique possibility for covalent joining of nucleocapsid (NC) and dUTPase within Gag-Pro polyproteins. By developing an antibody against the prototype betaretrovirus Mason-Pfizer monkey virus dUTPase, we demonstrate that i) the NC-dUTPase fusion protein exists both within the virions and infected cells providing the only form of dUTPase, and ii) the retroviral protease does not cleave NC-dUTPase either in the virion or in vitro. We show that recombinant betaretroviral NC-dUTPase and dUTPase are both inefficient catalysts compared with all other dUTPases. Dynamic light scattering and gel filtration confirm that the homotrimeric organization, common among dUTPases, is retained in the NC-dUTPase fusion protein. The betaretroviral dUTPase has been crystallized and single crystals contain homotrimers. Oligonucleotide and Zn2+ binding is well retained in the fusion protein, which is the first example of acquisition of a functional nucleic acid binding module by the DNA repair factor dUTPase. Binding of the hexanucleotide ACTGCC or the octanucleotide (TG)4 to NC-dUTPase modulates enzymatic function, indicating that the low catalytic activity may be compensated by adequate localization.     

How FMN binds to anabaena apoflavodoxin: a hydrophobic encounter at an open binding site

Molecular recognition begins when two molecules approach and establish interactions of certain strength. The mechanisms of molecular recognition reactions between biological molecules are not well known, and few systems have been analyzed in detail. We investigate here the reaction between an apoprotein and its physiological cofactor (apoflavodoxin and flavin mononucleotide) that binds reversibly to form a non-covalent complex (flavodoxin) involved in electron transfer reactions. We have analyzed the fast binding reactions between the FMN cofactor (and shorter analogs) and wild type (and nine mutant apoflavodoxins where residues interacting with FMN in the final complex have been replaced). The x-ray structures of two such mutants are reported that show the mutations are well tolerated by the protein. From the calculated microscopic binding rate constants we have performed a Phi analysis of the transition state of complex formation that indicates that the binding starts by interaction of the isoalloxazine-fused rings in FMN with residues of its hydrophobic binding site. In contrast, the phosphate in FMN, known to contribute most to the affinity of the final holoflavodoxin complex, is not bound in the transition state complex. Both the effects of ionic strength and of phosphate concentration on the wild type complex rate constant agree with this scenario. As suggested previously by nmr data, it seems that the isoalloxazine-binding site may be substantially open in solution. Interestingly, although FMN is a charged molecule, electrostatic interactions seem not to play a role in directing the binding, unlike what has been reported for other biological complexes. The binding can thus be best described as a hydrophobic encounter at an open binding site.