April Curtiss

Role of two arginine residues in domain II, loop 2 of Cry1Ab and Cry1Ac Bacillus thuringiensis delta-endotoxin in toxicity and binding to Manduca sexta and Lymantria dispar aminopeptidase N.

Two arginine residues (368–369) of Cry1Ab and Cry1Ac were mutated to alanine, glutamic acid and lysine by site‐directed mutagenesis. Insecticidal activities of the mutant toxins on Manduca sexta and Lymantria dispar larvae were examined. Cry1Ac mutant toxins (c)RR‐AA and (c)RR‐EE and Cry1Ab mutant toxins (b)RR‐AA and (b)RR‐EE showed great reductions in toxicity against both insects. In contrast, conservatively changed (c)RR‐KK and (b)RR‐KK mutants did not alter toxicity to either insect. Binding assays with brush border membrane vesicles (BBMVs) prepared from L. dispar midguts demonstrated that (c)RR‐AA, (c)RR‐EE, (b)RR‐AA and (b)RR‐EE bound with lower affinities compared with their respective wild‐type toxins. To M. sexta BBMVs, (c)RR‐AA and (c)RR‐EE showed great reductions in BBMV binding. However, (b)RR‐AA and (b)RR‐EE did not alter BBMV competition patterns, despite their reduced toxicity. Further binding assays were performed with aminopeptidase N (APN) purified from L. dispar and M. sexta BBMVs using surface plasmon resonance (BIAcore). Direct correlation between toxicity and APN binding was observed for the mutant toxins using this technique. The inconsistency between BBMV and APN binding data with Cry1Ab to M. sexta suggests the possibility of a different Cry1Ab toxin‐binding mechanism or the importance of another receptor in M. sexta.

The interaction of nitrotyrosine-83 plastocyanin with cytochromes f and c: pH dependence and the effect of an additional negative charge on plastocyanin

Spinach plastocyanin was selectively modified using tetranitromethane which incorporates a nitro group ortho to the hydroxyl group of tyrosine 83 (Anderson, G.P., Draheim, J.E. and Gross, E.L. (1985) Biochim. Biophys. Acta 810, 123-131). This tyrosine residue has been postulated to be part of the cytochrome f binding site on plastocyanin. Since the hydroxyl moiety of nitrotyrosine 83 is deprotonated above its pK of 8.3, it provides a useful modification for studying the effect of an extra negative charge on the interaction of plastocyanin with cytochrome f. No effect on cytochrome f oxidation was observed at pH 7 under conditions in which the hydroxyl moiety is protonated. However, the rate of cytochrome f oxidation increased at pH values greater than 8, reaching a maximum at pH 8.6 and decreasing at still higher pH values. The increase was half-maximal at pH 8.3 which is the pK for the hydroxyl moiety on nitrotyrosine 83. In contrast, the rate of cytochrome f oxidation for control plastocyanin was independent of pH from pH 7 to 8.6. These results show that increasing the negative charge on plastocyanin at Tyr-83 increases the ability to react with cytochrome f, supporting the hypothesis that cytochrome f interacts with plastocyanin at this location. In contrast, the reaction of Ntyr-83 plastocyanin with mammalian cytochrome c was independent of pH, suggesting that its mode of interaction with plastocyanin is different from that of cytochrome f. A comparison of the effects of Ntyr-83 modification of plastocyanin with the carboxyl- and amino-group modifications reported previously suggests that plastocyanin binds to cytochrome f in such a way that electrons could be donated to plastocyanin at either of its two binding sites.

Mutations at the arginine residues in α8 loop of Bacillus thuringiensis δ-endotoxin Cry1Ac affect toxicity and binding to Manduca sexta and Lymantria dispar aminopeptidase N

The functional role of the α8 loop residues in domain II of Bacillus thuringiensis Cry1Ac toxin was examined. Alanine substitution mutations were introduced in the residues from 275 to 293. Among the mutant toxins, substitutions at R281 and R289 affected toxicity to Manduca sexta and Lymantria dispar. Loss of toxicity by these mutant toxins was well correlated with reductions in binding affinity for brush border membrane vesicles and the purified receptor, aminopeptidase N (APN), from both insects. These data suggest that the two arginine residues in the α8 loop region are important in toxicity and APN binding in L. dispar and M. sexta.

Chemical modification of spinach plastocyanin using 4-chloro-3,5-dinitrobenzoic acid: characterization of four singly-modified forms.

Chemical modification of plastocyanin was carried out using 4-chloro-3,5-dinitrobenzoic acid, which has the effect of replacing positive charges on amino groups with negatively charged carboxyl groups. Four singly-modified forms were obtained which were separated using anion exchange FPLC. The four forms were modified at the N-terminal valine and at lysines 54, 71 and 77. The rates of reaction with mammalian cytochrome c were increased for all four modified plastocyanins. In contrast, the rates of reaction with cytochrome f were inhibited for the forms modified at residues 1, 54 and 77, whereas no effect was observed for the form modified at residue 71. Modification had no effect on either the midpoint redox potential or the reaction with K3Fe(CN)6. These results are consistent with a model in which charged residues on plastocyanin located at or near the binding site for cytochrome f recognize the positively-charged binding site on cytochrome f. In contrast, charged residues located at points on plastocyanin distant from the cytochrome f binding site recognize the net negative charge on the cytochrome f molecule. Based on these considerations, Glu-68 may be within the interaction sphere of cytochrome f, suggesting that cytochrome f may donate electrons to plastocyanin at either Tyr-83 or His-87.

The role of a proline‐induced broken‐helix motif in α‐helix 2 of Bacillus thuringiensis δ‐endotoxins

Bacillus thuringiensis δ‐endotoxins (Cry proteins), are widely used for insect control and plant protection. They are water‐soluble proteins that insert into membranes forming ion channels. In most Cry toxins α‐helix 2 is broken by a highly conserved proline residue (Pro70 in Cry1Ab), generating a broken‐helix motif. The flexibility of the motif was altered through site‐directed mutagenesis. It was found that increasing the flexibility of the motif decreased the stability, the ion transport ability and the toxicity of the protein. By removing the broken‐helix motif, the biological properties were restored to a wild type level.

Chemical Modification of the Lysine Residues on Plastocyanin and the Effect on Cytochrome f.

Plastocyanin (1) (PC) is a 10.5 kD copper protein which is located in the lumen of the thylakoid where it shuttles electrons between cytochrome f (cyt f) and P700. PC contains two potential binding sites for reaction partners (2). Negatively-charged molecules bind at Site 1 is His 87 (Site 1) at the top of the PC molecule as depicted in Fig. 1. Site 2 is located in the vicinity of Tyr 83 (Site 2) which is surrounded by a patch of highly-conserved negatively-charged residues (3). Positively-charged molecules such as cobalt phenanthroline (3) and cytochrome c (4) bind to this site.