The structure of gelsolin bound to ATP.

Calcium activation of the actin-modifying properties of gelsolin is sensitive to ATP. Here, we show that soaking calcium-free gelsolin crystals in ATP-containing media results in ATP occupying a site that spans the two pseudosymmetrical halves of the protein. ATP binding involves numerous polar and hydrophobic contacts and is identical for the two copies of gelsolin related by non-crystallographic symmetry within the crystal. The gamma-phosphate of ATP participates in several charge-charge interactions consistent with the preference of gelsolin for ATP, as a binding partner, over ADP. In addition, disruption of the ATP-binding site through Ca2+ activation of gelsolin reveals why ATP binds more tightly to the inactive molecule, and suggests how the binding of ATP may modulate the sensitivity of gelsolin to calcium ions. Similarities between the ATP and PIP2 interactions with the C-terminal half of gelsolin are evident from their overlapping binding sites and in that both molecules bind more tightly in the absence of calcium ions. We propose a model for how PIP2 may bind to calcium-free gelsolin based on the ATP-binding site.

Antiplasmodial activity of ferrocenyl chalcones: investigations into the role of ferrocene.

A series of ferrocenyl chalcones were synthesized and evaluated in vitro against Plasmodium falciparum (K1) in a [3H] hypoxanthine uptake assay. Appropriate size, electronic, lipophilic and electrochemical parameters were determined for QSAR analysis. The results showed that the location of ferrocene influenced the ease of oxidation of Fe2+ in ferrocene and the polarity of the carbonyl linkage. These parameters were found to influence antiplasmodial activity. A general trend was noted in which compounds with ferrocene adjacent to the carbonyl linkage (series A) were associated with more selective and potent antiplasmodial activities. These compounds had polarized carbonyl linkages, lower lipophilicities and ferrocene rings that were less readily oxidized. The most active analogue was 1-ferrocenyl-3-(4-nitrophenyl)prop-2-en-1-one (28) (IC50 4.6 microM, selectivity index 37 against KB3-1 cells). To understand how the redox properties of ferrocene might influence antiplasmodial activity, the oxidant properties of selected compounds were investigated in antioxidant (ABTS+) and EPR experiments. The incorporation of ferrocene in the chalcone template was found to enhance its role in processes that involved the quenching and generation of free radicals. Thus, ferrocene may participate in redox cycling and this process may contribute to the antiplasmodial activity of ferrocenyl chalcones. However, the extent to which this property is manifested is also influenced by other physicochemical properties (lipophilicity, polarity, and planarity) of the compound.

Essential tension and constructive destruction: the spindle checkpoint and its regulatory links with mitotic exit.

Replicated genetic material must be partitioned equally between daughter cells during cell division. The precision with which this is accomplished depends critically on the proper functioning of the mitotic spindle. The assembly, orientation and attachment of the spindle to the kinetochores are therefore constantly monitored by a surveillance mechanism termed the SCP (spindle checkpoint). In the event of malfunction, the SCP not only prevents chromosome segregation, but also inhibits subsequent mitotic events, such as cyclin destruction (mitotic exit) and cytokinesis. This concerted action helps to maintain temporal co-ordination among mitotic events. It appears that the SCP is primarily activated by either a lack of occupancy or the absence of tension at kinetochores. Once triggered, the inhibitory circuit bifurcates, where one branch restrains the sister chromatid separation by inhibiting the E3 ligase APC(Cdc20) (anaphase-promoting complex activated by Cdc20) and the other impinges on the MEN (mitotic exit network). A large body of investigations has now led to the identification of the control elements, their targets and the functional coupling among them. Here we review the emerging regulatory network and discuss the remaining gaps in our understanding of this effective mechanochemical control system.

A novel sequence in the coiled-coil domain of Stat3 essential for its nuclear translocation.

Stat3 is activated by cytokines and growth factors via specific tyrosine phosphorylation, dimerization, and nuclear translocation. However, the mechanism involved in its nuclear translocation is unclear. In this study, by systematic deletion and site-directed mutagenesis we identified Arg-214/215 in the alpha-helix 2 region of the coiled-coil domain of Stat3 as a novel sequence element essential for its nuclear translocation, stimulated by epidermal growth factor as well as by interleukin-6. Furthermore, we identified Arg-414/417 in the DNA binding domain as also required for the nuclear localization of Stat3. This sequence element corresponds to Lys-410/413 of Stat1, a reported sequence for Stat1 nuclear translocation. On the other hand, Leu-411 of Stat3, corresponding to Leu-407 of Stat1, a necessary residue for Stat1 nuclear transport, is not essential for Stat3 nuclear import. The mutant of Arg-214/215 or Arg-414/417 was shown to be tyrosyl-phosphorylated normally but failed to enter the nucleus in response to epidermal growth factor or interleukin-6. The defect, however, can be rescued by the wild-type Stat3 but cannot be compensated by these two mutants. Mutations on Arg-414/417, but not Arg-214/215, destroy the DNA binding activity of Stat3. Our data for the first time identified a sequence element located in the coiled-coil domain that is involved in the ligand-induced nuclear translocation of Stat3. This novel sequence together with a conserved sequence element in the DNA binding domain coordinates to mediate the nuclear translocation of Stat3.

Biochemical implications of sequence comparisons of the cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is both of medical significance in humans and of interest with regard to osmoregulation in aquatic organisms. CFTR is composed of five domains: two membrane-spanning domains, two nucleotide-binding domains, and a regulatory domain. Notwithstanding the plethora of information concerning the structure and function of CFTR, the biochemistry of many facets of CFTR are not completely understood. In this regard, we have performed a sequence alignment of representative vertebrate CFTR with the aim of generating hypotheses on the functional significance of conserved and variable residues. Postulates on function common to all organisms are: (i) Thr338 in the sixth transmembrane segment could have a function related to that of the pore-lining residue Lys335, and it is possible that Thr338 hydrogen bonds to Lys335, thus indirectly affecting anion permeability; (ii) the fragment (111)PDNKE could be an ion sensor; (iii) motifs in the two nucleotide-binding domains reflect differential ATP binding and hydrolysis; and (iv) an interaction in the R domain involving (765)RRQSVL and the C terminal end of the domain results in an inhibitory conformation. Major adaptations in fishes include variations in the postulated ion sensor (111)PDNKE, and the absence of a proline residue in the R domain with consequent higher chloride efflux.