Symmetrical stabilization of bound Ca2+ ions in a cooperative pair of EF-hands through hydrogen bonding of coordinating water molecules in calbindin D(9k)

Water molecules are found to complete the Ca2+ coordination sphere when a protein fails to provide enough ligating oxygens. Hydrogen bonding of these water molecules to the protein backbone or side chains may contribute favorably to the Ca2+ affinity, as suggested in an earlier study of two calbindin D(9k) mutants [E60D and E60Q; Linse et al. (1994) Biochemistry 33, 12478-12486]. To investigate the generality of this conclusion, another side chain, Gln 22, which hydrogen bonds to a Ca2+-coordinating water molecule in calbindin D(9k), was mutated. Two calbindin D(9k) mutants, (Q22E+P43M) and (Q22N+P43M), were constructed to examine the interaction between Gln 22 and the water molecule in the C-terminal calcium binding site II. Shortening of the side chain, as in (Q22N+P43M), reduces the affinity of binding two calcium ions by a factor of 18 at low ionic strength, whereas introduction of a negative charge, as in (Q22E+P43M), leads to a 12-fold reduction. In 0.15 M KCl, a 7-fold reduction in affinity was observed for both mutants. The cooperativity of Ca2+ binding increases for (Q22E+P43M), while it decreases for (Q22N+P43M). The rates of Ca2+ dissociation are 5.5-fold higher for the double mutants than for P43M at low ionic strength. For both mutants, reduced strength of hydrogen bonding to calcium-coordinating water molecules is a likely explanation for the observed effects on Ca2+ affinity and dissociation. In the apo forms, the (Q22E+P43M) mutant has lower stability toward urea denaturation than (Q22N+P43M) and P43M. 2D (1)H NMR and crystallographic experiments suggest that the structure of (Q22E+P43M) and (Q22N+P43M) is unchanged relative to P43M, except for local perturbations in the loop regions.     

Peptide-derived antagonists of the urokinase receptor. affinity maturation by combinatorial chemistry, identification of functional epitopes, and inhibitory effect on cancer cell intravasation

The high-affinity interaction between urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) plays an important role in pericellular plasminogen activation. Since proteolytic degradation of the extracellular matrix has an established role in tumor invasion and metastasis, the uPA-uPAR interaction represents a potential target for therapeutic intervention. By affinity maturation using combinatorial chemistry we have now developed and characterized a 9-mer, linear peptide antagonist of the uPA-uPAR interaction demonstrating specific, high-affinity binding to human uPAR (K(d) approximately 0.4 nM). Studies by surface plasmon resonance reveal that the off-rate for this receptor-peptide complex is comparable to that measured for the natural protein ligand, uPA. The functional epitope on human uPAR for this antagonist has been delineated by site-directed mutagenesis, and its assignment to loop 3 of uPAR domain III (Met(246), His(249), His(251), and Phe(256)) corroborates data previously obtained by photoaffinity labeling and provides a molecular explanation for the extreme selectivity observed for the antagonist toward human compared to mouse, monkey, and hamster uPAR. When human HEp-3 cancer cells were inoculated in the presence of this peptide antagonist, a specific inhibition of cancer cell intravasation was observed in a chicken chorioallantoic membrane assay. These data imply that design of small organic molecules mimicking the binding determinants of this 9-mer peptide antagonist may have a potential application in combination therapy for certain types of cancer.     

Distribution, abundance, behavior and metabolism of Periphylla periphylla, a mesopelagic coronate medusa in a Norwegian fjord

The distribution, behavior and metabolism of the mesopelagic jellyfish, Periphylla periphylla (Péron & Lesueur), were investigated in Lurefjorden, Norway. Field studies, conducted in 1998–1999 with plankton nets and a remotely operated vehicle, indicated that 80-90% of the dense (up to 2.5 m^–3) population migrated 200–400 m vertically each day throughout the year. In situ observations with red light revealed that swimming rates and feeding activity varied with age and time of day. Detection of turbulence and contact with surfaces caused this medusa to conceal one or all of its tentacles in the stomach or to shed nematocyst-laden tissue from the tentacles. Stomachs of medusae collected with nets were often full of prey entangled with the sloughed tissue. Stomachs of medusae captured individually with ROV samplers were empty or contained only a few prey in their stomachs (typically, 1–4 copepods Calanus spp. or chaetognaths Eukrohnia hamata Möbius per medusa). Low rates (0.4–5.6 l O₂ mg C^–1 h^–1) of oxygen consumption of P. periphylla suggested that this species was sustained by relatively few (1–34) prey d^–1.     

The proteoglycans aggrecan and Versican form networks with fibulin-2 through their lectin domain binding

Aggrecan, versican, neurocan, and brevican are important components of the extracellular matrix in various tissues. Their amino-terminal globular domains bind to hyaluronan, but the function of their carboxyl-terminal globular domains has long remained elusive. A picture is now emerging where the C-type lectin motif of this domain mediates binding to other extracellular matrix proteins. We here demonstrate that aggrecan, versican, and brevican lectin domains bind fibulin-2, whereas neurocan does not. As expected for a C-type lectin, the interactions are calcium-dependent, with K(D) values in the nanomolar range as measured by surface plasmon resonance. Solid phase competition assays with previously identified ligands demonstrated that fibulin-2 and tenascin-R bind the same site on the proteoglycan lectin domains. Fibulin-1 has affinity for the common site on versican but may bind to a different site on the aggrecan lectin domain. By using deletion mutants, the interaction sites for aggrecan and versican lectin domains were mapped to epidermal growth factor-like repeats in domain II of fibulin-2. Affinity chromatography and solid phase assays confirmed that also native full-length aggrecan and versican bind the lectin domain ligands. Electron microscopy confirmed the mapping and demonstrated that hyaluronan-aggrecan complexes can be cross-linked by the fibulins.     

Multiple origin of the tropical forest tree family Icacinaceae

Analyses of DNA sequences from four genes (ndhF, rbcL, atpB, and 18S rDNA) and morphological data show that the members of the tropical forest tree family Icacinaceae do not have a common origin. All of the genera earlier placed in Icacinaceae are euasterids but placed in the following three different orders: Garryales, Aquifoliales, and Apiales. Icacina and related genera are members of Garryales and, pending more data, are still best treated as Icacinaceae (sensu stricto). The genera related to Aquifoliales are placed in Cardiopteridaceae and a new family, Stemonuraceae. The genus Pennantia is a member of Apiales and the family Pennantiaceae is recognized. Morphological characters delimiting these groups are discussed. Twenty-six new ndhF sequences were obtained for the study (25 from former Icacinaceae and 1 from Cardiopteris).     

EPR investigation of the active site of recombinant human 5-lipoxygenase: inhibition by selenide

Lipoxygenases are a group of non-heme iron dioxygenases which catalyze the formation of lipid hydroperoxides from unsaturated fatty acids. 5-Lipoxygenase (5LO) is of particular interest for formation of leukotrienes and lipoxins, implicated in inflammatory processes. In this study, electron paramagnetic resonance (EPR) spectroscopy was used to investigate the active site iron of purified recombinant human 5-lipoxygenase (5LO), and to explore the action of selenide on 5LO. After oxidation by lipid hydroperoxides, 5LO exhibited axial EPR spectra typified by a signal at g = 6.2. However, removal of the lipid hydroperoxides, their metabolites, and the solvent ethanol from the samples resulted in a shift to more rhombic EPR spectra (g = 5.17 and g = 9.0). Thus, many features of 5LO and soybean lipoxygenase-1 EPR spectra were similar, indicating similar flexible iron ligand arrangements in these lipoxygenases. Selenide (1.5 microM) showed a strong inhibitory effect on the enzyme activity of 5LO. In EPR, selenide abolished the signal at g = 6.2, typical for enzymatically active 5LO. Lipid hydroperoxide added to selenide-treated 5LO could not reinstate the signal at g = 6.2, indicating an irreversible change of the coordination of the active site iron.     

Role of phosphoinositide 3OH-kinase in autocrine transformation by PDGF-BB

Phosphoinositide 3OH-kinases (PI3K) are a family of lipid kinases that activates signalling pathways important for migration, cytoskeletal rearrangements, and cell survival. These processes are important hallmarks in transformation. We have evaluated the functional role of PI3K for development of a transformed morphology and migratory responses of murine fibroblasts (NIH/sis and COL1A1/NIH3T3 cell lines) stimulated in an autocrine fashion by constitutive expression of platelet-derived growth factor-BB (PDGF-BB). We show that prolonged treatment with the specific PI3K inhibitor LY294002, induced a reversion of the transformed morphology, and prevented density-independent growth and focus formation. Functional PI3K was also required for development of the transformed morphology of NIH/sis and COL1A1/NIH3T3. Furthermore, treatment with LY294002 completely perturbed random migration of the cells. In addition our data show that, in the signalling pathways downstream of PI3K, activation of the small GTPase Rac was a prerequisite for the transformation signal. Our data also indicate the presence of a suramin-insensitive PI3K activity. Most likely this was due to the presence of a suramin-insensitive intracellular PDGFR pool that allowed activation of PI3K located in intracellular compartments. In conclusion these data show that intact PI3K activity was required for the morphological alterations and the enhanced migratory response that are hallmarks for PDGF induced autocrine transformation.     

Structural basis for enantiomer binding and separation of a common beta-blocker: crystal structure of cellobiohydrolase Cel7A with bound (S)-propranolol at 1.9 A resolution

Cellobiohydrolase Cel7A (previously called CBH 1), the major cellulase produced by the mould fungus Trichoderma reesei, has been successfully exploited as a chiral selector for separation of stereo-isomers of some important pharmaceutical compounds, e.g. adrenergic beta-blockers. Previous investigations, including experiments with catalytically deficient mutants of Cel7A, point unanimously to the active site as being responsible for discrimination of enantiomers.In this work the structural basis for enantioselectivity of basic drugs by Cel7A has been studied by X-ray crystallography. The catalytic domain of Cel7A was co-crystallised with the (S)-enantiomer of a common beta-blocker, propranolol, at pH 7, and the structure of the complex was determined and refined at 1. 9 A resolution. Indeed, (S)-propranolol binds at the active site, in glucosyl-binding subsites -1/+1. The catalytic residues Glu212 and Glu217 make tight salt links with the secondary amino group of (S)-propranolol. The oxygen atom attached to the chiral centre of (S)-propranolol forms hydrogen bonds to the nucleophile Glu212 O(epsilon1) and to Gln175 N(epsilon2), whereas the aromatic naphthyl moiety stacks with the indole ring of Trp376 in site +1. The bidentate charge interaction with the catalytic glutamate residues is apparently crucial, since no enantioselectivity has been obtained with the catalytically deficient mutants E212Q and E217Q.Activity inhibition experiments with wild-type Cel7A were performed in conditions close to those used for crystallisation. Competitive inhibition constants for (R)- and (S)-propranolol were determined at 220 microM and 44 microM, respectively, corresponding to binding free energies of 20 kJ/mol and 24 kJ/mol, respectively. The K(i) value for (R)-propranolol was 57-fold lower than the highest concentration, 12.5 mM, used in co-crystallisation experiments. Still several attempts to obtain a complex with the (R)-enantiomer have failed.By using cellobiose as a selective competing ligand, the retention of the enantiomers of propranolol on the chiral stationary phase (CSP) based on Cel7A mutant D214N were resolved into enantioselective and non- selective binding. The enantioselective binding was weaker for both enantiomers on D214N-CSP than on wild-type-CSP.