Conformational change of Arabidopsis thaliana thioredoxin reductase after binding of pyridine nucleotide and thioredoxin

We have found that the binding of NADP+ (Kd = 0.86+/-0.11 microM) enhanced the FAD fluorescence of Arabidopsis thaliana NADPH:thioredoxin reductase (TR, EC 1.6.4.5) by 2 times, whereas the binding of 3-aminopyridine adenine dinucleotide phosphate (AADP+) (Kd < 0.1 microM) quenched the fluorescence by 20%. Thioredoxin (TRX) also enhanced the FAD fluorescence by 35%. The Kd of TR-NADP+ and TR-AADP+ complexes did not change in the presence of 45 microM TRX. Our findings imply that the binding of NADP+ and AADP+ at the NADP(H)-binding site of A. thaliana TR, and/or the binding of TRX in the vicinity of the catalytic disulfide increase the content of fluorescent FR conformer (NADP(H)-binding site adjacent to flavin). The different effects of NADP+ and AADP+ on FAD fluorescence intensity may be explained by the superposition of two opposite factors: i) increased content of fluorescent FR conformer upon binding of NADP+ or AADP+; ii) quenching of FAD fluorescence by electron-donating 3-aminopyridinium ring of AADP+.     

High affinity Grb2-SH3 domain ligand incorporating Cbeta-substituted prolines in a Sos-derived decapeptide

Peptide ligands that disrupt MAPK pathways are of great interest for a better understanding of these signalling cascades and represent therefore an attractive target to control cell degenerative processes. In that context, selective disruption of the upstream Grb2/Sos complex in the Ras/MAPK cascade has focused extensive work. The Sos PPII decapeptide, which interacts with the Grb2-SH3 domains, has been modified in various positions and the best inhibitors designed so far are either dimeric ligands or peptoid analogues of the VPPPVPPRRR sequence. We report the synthesis of new Grb2 ligands in which the key Val5 residue has been replaced by a cis C(beta)-substituted proline. Both fluorescence and ITC assays have been employed to measure the affinity of these substituted peptides for a recombinant Grb2 protein. Whereas proline in position 5 completely abolished the binding potency, a cis C(beta)-methyl-L-proline restored the affinity. Other cis C(beta)-proline substituents led to a complete loss of binding potency. Combining the best modifications: a cis C(beta)-methylproline 5, N-acetylation, C-carboxamide and dimerization yielded a 560-fold affinity enhancement compared to the wild-type VPPPVPPRRR sequence. This study shows that C(beta)-substituted prolines may constitute a new alternative for PPII ligands, combining entropy and enthalpy beneficial effects.     

Comparative activity of two cholecystokinin analogues with partial agonist activity: effects on food intake and brain monoamines

Two analogues of the C-terminal heptapeptide of cholecystokinin have been synthesized, in which the C-terminal phenylalanine residue has been replaced by a phenylethylester (JMV 180) or a phenylethylamide (JMV 170) group. They have been shown to present partial agonist CCK activity on pancreatic amylase release. In this study, the effects of the two peptides were investigated on food intake and brain monoamine metabolism after intraperitoneal (IP) and intracerebroventricular (ICV) administration. Neither peptide was active on feeding after IP administration but both decreased food intake after ICV injection, with a slightly higher potency for JMV 170. JMV 180 induced no change in monoamine metabolism whatever the route of administration. JMV 170 IP decreased cortical levels of dopamine and its metabolites. This effect was stronger after ICV injection and was accompanied by changes in serotonergic metabolism in the hypothalamus and cortex. Contrary to CCK8 S, which is more active on feeding after peripheral injection, the feeding effects of the analogues obtained by modification of the C-terminal phenylalanine residue appear to involve a central site of action. Furthermore, phenylethylamide substitution (JMV 170) gives rise to greater potency on monoaminergic variations than replacement with a phenylethylester (JMV 180) and the effect is enhanced following central administration.     

Oxidation-reduction and activation properties of chloroplast fructose 1,6-bisphosphatase with mutated regulatory site.

The concentration of Mg(2+) required for optimal activity of chloroplast fructose 1,6-bisphosphatase (FBPase) decreases when a disulfide, located on a flexible loop containing three conserved cysteines, is reduced by the ferredoxin/thioredoxin system. Mutation of either one of two regulatory cysteines in this loop (Cys155 and Cys174 in spinach FBPase) produces an enzyme with a S(0.5) for Mg(2+) (0.6 mM) identical to that observed for the reduced WT enzyme and significantly lower than the S(0.5) of 12.2 mM of oxidized WT enzyme. E(m) for the regulatory disulfide in WT spinach FBPase is -305 mV at pH 7.0, with an E(m) vs pH dependence of -59 mV/pH unit, from pH 5.5 to 8.5. Aerobic storage of the C174S mutant produces a nonphysiological Cys155/Cys179 disulfide, rendering the enzyme partially dependent on activation by thioredoxin. Circular dichroism spectra and thiol titrations provide supporting evidence for the formation of nonphysiological disulfide bonds. Mutation of Cys179, the third conserved cysteine, produces FBPase that behaves very much like WT enzyme but which is more rapidly activated by thioredoxin f, perhaps because the E(m) of the regulatory disulfide in the mutant has been increased to -290 mV (isopotential with thioredoxin f). Structural changes in the regulatory loop lower S(0.5) for Mg(2+) to 3.2 mM for the oxidized C179S mutant. These results indicate that opening the regulatory disulfide bridge, either through reduction or mutation, produces structural changes that greatly decrease S(0.5) for Mg(2+) and that only two of the conserved cysteines play a physiological role in regulation of FBPase.     

Oxidation-reduction properties of two engineered redox-sensitive mutant Escherichia coli malate dehydrogenases

Redox potentials for two inactivating intrasubunit disulfides that link helix-5 and helix-9 in mutant Escherichia coli malate dehydrogenases have been determined. The Em is -285 mV when cysteines are at positions 121 and 305 and -295 mV when the cysteines are at positions 122 and 305. Oxidation to the disulfide affects kcat but not Km values. In the single V121C and N122C mutants, the Cys in helix-5 affects the Km for oxalacetate. The pH optimum in the direction of malate formation is affected by the redox state of the enzyme. Clearly, a disulfide bond can and does form between Cys residues substituted into positions 121 or 122 in the nucleotide binding domain and 305 in the carbon substrate binding domain of this NAD-dependent malate dehydrogenase. Apparently, crosslinking the domains interferes with catalysis.