Structural basis of translation termination,rescue, and recycling in mammalian mitochondria

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Cation-pi interaction in a folded polypeptide.

Cationic and aromatic side chains from protein residues interact to stabilize tertiary structure. The stabilization energy originates in part from electrostatic attraction between the cation, and regions of high electron density in pi-orbitals of the aromatic group, leading to the name cation-pi interaction. The lysine and tyrosine containing peptide, N-acetyl-Pro-Pro-Lys-Tyr-Asp-Lys-NH(2), has near uv CD characteristic of tyrosine in a structured environment. Nuclear Overhauser effect (NOE), coupling constant, and ring current chemical shift constraints obtained with (1)H NMR confirm that the peptide (t6p) folds. Simulated annealing consistent with all NMR constraints produces a 40-structure ensemble for t6p with potential energies within one standard deviation of the lowest value observed. Calculated binding energies indicate that cation-pi and cation-phenolic OH interactions exists between the Lys3 and Tyr4 side chains in most of the structures. The t6p peptide in solution is a model for these interactions in a protein. A perturbing electric field from the cationic ground state charge intermingles the excited states of the aromatic group. This intermingling effect may provide a cation-pi signature effect in the tyrosine spectroscopy. The absorption and CD for the lowest energy electronic transitions of the tyrosine phenol were computed for the ensemble. Red-shifted peak energy and hypochromicity in the absorbance band, and decreasing rotational strength, correlates with increasing binding energy of the complex indicating the cation-pi spectroscopic signature. The ensemble average spectroscopic signature effects in t6p are small and in agreement with observation.     

Prediction of volatile anesthetic binding sites in proteins

Computational methods designed to predict and visualize ligand protein binding interactions were used to characterize volatile anesthetic (VA) binding sites and unoccupied pockets within the known structures of VAs bound to serum albumin, luciferase, and apoferritin. We found that both the number of protein atoms and methyl hydrogen, which are within approximately 8 A of a potential ligand binding site, are significantly greater in protein pockets where VAs bind. This computational approach was applied to structures of calmodulin (CaM), which have not been determined in complex with a VA. It predicted that VAs bind to [Ca(2+)](4)-CaM, but not to apo-CaM, which we confirmed with isothermal titration calorimetry. The VA binding sites predicted for the structures of [Ca(2+)](4)-CaM are located in hydrophobic pockets that form when the Ca(2+) binding sites in CaM are saturated. The binding of VAs to these hydrophobic pockets is supported by evidence that halothane predominantly makes contact with aliphatic resonances in [Ca(2+)](4)-CaM (nuclear Overhauser effect) and increases the Ca(2+) affinity of CaM (fluorescence spectroscopy). Our computational analysis and experiments indicate that binding of VA to proteins is consistent with the hydrophobic effect and the Meyer-Overton rule.     

A new model of selection in women's handball

The aim of the study was to assess the basic motor abilities that determine top performance in women's handball, and to identify test panel for primary selection at handball school. The study included 155 female attendants of the Split Handball School, mean age 12.5 years. Differences in the basic motor abilities between the subjects that developed into elite handball players after 7-year training process and those that abandoned handball for being unable to meet the competition criteria were evaluated by use of discriminative analysis. The former were found to have also been superior initially in all variables analyzed, and in arm coordination, overall body coordination, throw and jump explosive strength, arm movement frequency and repetitive trunk strength in particular. Motor superiority based on the abilities of coordination, explosive strength and speed determines performance in women's handball, qualifying these abilities as reliable selection criteria. Based on this study results, a new model of selection in women's handball, with fine arm coordination as the major limiting factor of performance, has been proposed.     

Synthesis, characterization and biological activity evaluation of Pt(II), Pd(II), Co(III) and Ni(II) complexes with N-heteroaromatic selenosemicarbazones

Five new complexes of Pt(II), Pd(II), Co(III) and Ni(II) with 2-pyridine(quinoline)carboxaldehyde selenosemicarbazones were synthesized and characterized. Crystal structures of Pt(II) complex with the pyridine derivative and Co(III) complex with the quinoline derivative were determined. In all complexes the ligands were coordinated through N₂Se donor atom set forming either square-planar (Pt, Pd) or octahedral (Co, Ni) geometry. All complexes showed biological activity.     

The First Propeller Domain of LRP6 Regulates Sensitivity to DKK1

The Wnt coreceptor LRP6 is required for canonical Wnt signaling. To understand the molecular regulation of LRP6 function, we generated a series of monoclonal antibodies against the extra cellular domain (ECD) of LRP6 and selected a high-affinity mAb (mAb135) that recognizes cell surface expression of endogenous LRP6. mAb135 enhanced Wnt dependent TCF reporter activation and antagonized DKK1 dependent inhibition of Wnt3A signaling, suggesting a role in modulation of LRP6 function. Detailed analysis of LRP6 domain mutants identified Ser 243 in the first propeller domain of LRP6 as a critical residue for mAb135 binding, implicating this domain in regulating the sensitivity of LRP6 to DKK1. In agreement with this notion, mAb135 directly disrupted the interaction of DKK1 with recombinant ECD LRP6 and a truncated form of the LRP6 ECD containing only repeats 1 and 2. Finally, we found that mAb135 completely protected LRP6 from DKK1 dependent internalization. Together, these results identify the first propeller domain as a novel regulatory domain for DKK1 binding to LRP6 and show that mAb against the first propeller domain of LRP6 can be used to modulate this interaction.     

The influence of potassium ion (K+) on digoxin-induced inhibition of porcine cerebral cortex Na+ / K+-ATPase

The in vitro influence of potassium ion modulations, in the concentration range 2 mM-500 mM, on digoxin-induced inhibition of porcine cerebral cortex Na+ / K+-ATPase activity was studied. The response of enzymatic activity in the presence of various K+ concentrations to digoxin was biphasic, thereby, indicating the existence of two Na+ / K+-ATPase isoforms, differing in the affinity towards the tested drug. Both isoforms showed higher sensitivity to digoxin in the presence of K+ ions below 20 mM in the medium assay. The IC50 values for high/low isoforms 2.77 x 10(-6) M / 8.56 x 10(-5) M and 7.06 x 10(-7) M / 1.87 x 10(-5) M were obtained in the presence of optimal (20 mM) and 2 mM K+, respectively. However, preincubation in the presence of elevated K+ concentration (50-500 mM) in the medium assay prior to Na+ / K+-ATPase exposure to digoxin did not prevent the inhibition, i.e. IC50 values for both isoforms was the same as in the presence of the optimal K+ concentration. On the contrary, addition of 200 mM K+ into the medium assay after 10 minutes exposure of Na+ / K+-ATPase to digoxin, showed a time-dependent recovery effect on the inhibited enzymatic activity. Kinetic analysis showed that digoxin inhibited Na+ / K+-ATPase by reducing maximum enzymatic velocity (Vmax) and Km, implying an uncompetitive mode of interaction.     

Elongation arrest by SecM via a cascade of ribosomal RNA rearrangements

In E. coli, the SecM nascent polypeptide causes elongation arrest, while interacting with 23S RNA bases A2058 and A749-753 in the exit tunnel of the large ribosomal subunit. We compared atomic models fitted by real-space refinement into cryo-electron microscopy reconstructions of a pretranslocational and SecM-stalled E. coli ribosome complex. A cascade of RNA rearrangements propagates from the exit tunnel throughout the large subunit, affecting intersubunit bridges and tRNA positions, which in turn reorient small subunit RNA elements. Elongation arrest could result from the inhibition of mRNA.(tRNAs) translocation, E site tRNA egress, and perhaps translation factor activation at the GTPase-associated center. Our study suggests that the specific secondary and tertiary arrangement of ribosomal RNA provides the basis for internal signal transduction within the ribosome. Thus, the ribosome may itself have the ability to regulate its progression through translation by modulating its structure and consequently its receptivity to activation by cofactors.     

Nuclear localization of tetrahydrobiopterin biosynthetic enzymes

Biosynthesis of the tetrahydrobiopterin (BH(4)) cofactor, essential for catecholamines and serotonin production and nitric oxide synthase (NOS) activity, requires the enzymes GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS), and sepiapterin reductase (SR). Upon studying the distribution of GTPCH and PTPS with polyclonal immune sera in cross sections of rat brain, prominent nuclear staining in many neurons was observed besides strong staining in peri-ventricular structures. Furthermore, localization studies in transgenic mice expressing a Pts-LacZ gene fusion containing the N-terminal 35 amino acids of PTPS revealed beta-galactosidase in the nucleus of neurons. In contrast, PTPS-beta-galactosidase was exclusively cytoplasmic in the convoluted kidney tubules but nuclear in other parts of the nephron, indicating again that nuclear targeting may occur only in specific cell categories. Furthermore, the N terminus of PTPS acts as a domain able to target the PTPS-beta-galactosidase fusion protein to the nucleus. In transiently transfected COS-1 cells, which do not express GTPCH and PTPS endogenously, we found cytoplasmic and nuclear staining for GTPCH and PTPS. To further investigate nuclear localization of all three BH(4)-biosynthetic enzymes, we expressed Flag-fusion proteins in transiently transfected COS-1 cells and analyzed the distribution by immunolocalization and sub-cellular fractionation using anti-Flag antibodies and enzymatic assays. Whereas 5-10% of total GTPCH and PTPS and approximately 1% of total SR were present in the nucleus, only GTPCH was confirmed to be an active enzyme in nuclear fractions. The in vitro studies together with the tissue staining corroborate specific nuclear localization of BH(4)-biosynthetic proteins with yet unknown biological function.