Asn183 in alpha5 is essential for oligomerisation and toxicity of the Bacillus thuringiensis Cry4Ba toxin

The proposed toxicity mechanism of the Bacillus thuringiensis Cry insecticidal proteins involves membrane penetration and lytic pore formation of the alpha4-alpha5 hairpins in the target larval midgut cell membranes. In this study, alanine substitutions of selected polar residues (Tyr(178), Gln(180), Asn(183), Asn(185), and Asn(195)) in the hydrophobic helix-alpha5 of the Cry4Ba mosquito-larvicidal protein were initially conducted via PCR-based directed mutagenesis. Upon IPTG induction, all the 130-kDa mutant protoxins were highly expressed in Escherichia coli as cytoplasmic inclusions, with yields similar to the wild-type protoxin. When E. coli cells expressing each mutant toxin were tested against Stegomyia aegypti mosquito larvae, the larvicidal activity of the N183A mutant was almost completely abolished whereas the four other mutants showed only a small reduction in toxicity. Additionally, replacements of this critical residue with various amino acids revealed that the uncharged polar residue at position 183 in alpha5 is crucial for larvicidal activity. Further characterisation of the N183K bio-inactive mutant revealed that the 65-kDa activated toxin was unable to form oligomers in lipid vesicles and its ability to induce the release of entrapped calcein from liposomes was much weaker than that of the wild-type toxin. These results suggest that the highly conserved Asn(183) located in the middle of the transmembrane alpha5 of Cry4Ba plays a crucial role in toxicity and toxin oligomerisation in the lipid membranes.     

Significance of the Conserved Tyr352 and Asp380 Residues in the Catalytic Activity of Bacillus stearothermophilus Aminopeptidase II as Evaluated by Site-directed Mutagenesis

The importance of the conserved Tyr352 and Asp380 residues of Bacillus stearothermophilus aminopeptidase II (AP-II) was investigated by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in recombinant Escherichia coli M15 cells and the 45-kD proteins were purified from the cell-free extracts by Ni(2+)-NTA resin. The specific activity for Tyr352 and Asp380 replacements was decreased by more than 3.5-fold. Detailed analysis of the kinetic consequences in the mutant proteins revealed that the K (m) values were increased 1.9- to 2.6-fold with respect to wild-type enzyme. Catalytic efficiencies (k (cat)/K (m)) of mutant proteins were between 3.5- and 31-fold lower than the corresponding value of the wild-type enzyme. Tryptophan emission fluorescence and circular dichroism spectra were nearly identical for wild-type and mutant enzymes. These results indicate that residues Tyr352 and Asp380 are essential for the proper function of AP-II.     

Cys31, Cys47, and Cys195 in BinA Are Essential for Toxicity of a Binary Toxin from Bacillus sphaericus

The mosquito larvicidal binary toxin produced by Bacillus sphaericus is composed of 2 proteins called BinA and BinB. While BinB acts as specificity determinant, BinA is expected to bind to BinB, translocates into cytosol, and exerts its activity via an unknown mechanism. To study the role of cysteine in BinA, 3 cysteine residues were substituted by alanine and serine. Substitution at Cys195 significantly reduced the toxin activity, whereas substitution at Cys31 and Cys47 abolished its toxicity. Intrinsic fluorescent analysis suggested that all mutant proteins should have similar tertiary structure to that of the wild type. Analysis of the mutant protein on sodium dodecyl sulfate–polyacrylamide gel electrophoresis with and without a reducing agent indicated that all 3 cysteine residues were not involved in disulfide bond formation within the BinA molecule. This is the first report to demonstrate that cysteine residues at 3 positions in BinA are required for full toxicity of the binary toxin. They may play a critical role during oligomerization or interaction between BinA and BinB to form the active complex.     

Amino acid substitutions in alphaA and alphaC of Cyt2Aa2 alter hemolytic activity and mosquito-larvicidal specificity

Cyt2Aa2 produced by Bacillus thuringiensis subsp. darmstadiensis exhibits in vitro cytolytic activity against broad range of cells but shows specific in vivo toxicity against larvae of Dipteran insects. To investigate the role of amino acids in alphaA and alphaC of this toxin, 3 single-point mutants (A61C, S108C and V109A) were generated. All 3 mutant proteins were highly produced as inclusion bodies that could be solubilized and activated by proteinase K similar to that of the wild type. Hemolytic activity of A61C and S108C mutants was significantly reduced whereas the V109A mutant showed comparable hemolytic activity to the wild type. Interestingly, the A61C mutant exhibited high larvicidal activity to both Aedes aegypti and Culex quinquefasciatus. S108C and V109A mutants showed low activity against C. quinquefasciatus but relatively high toxicity to A. aegypti. These results demonstrated for the first time that amino acids in alphaA and alphaC are involved in the selectivity of the Cyt toxin to the targeted organism.     

Specific mutations within the alpha4-alpha5 loop of the Bacillus thuringiensis Cry4B toxin reveal a crucial role for Asn-166 and Tyr-170

The widely accepted model for toxicity mechanisms of the Bacillus thuringiensis Cry delta-endotoxins suggests that helices alpha4 and alpha5 form a helix-loop-helix hairpin structure to initiate membrane insertion and pore formation. In this report, alanine substitutions of two polar amino acids (Asn-166 and Tyr-170) and one charged residue (Glu-171) within the alpha4-alpha5 loop of the 130-kDa Cry4B mosquito-larvicidal protein were initially made via polymerase chain reaction-based directed mutagenesis. As with the wild-type toxin, all of the mutant proteins were highly expressed in Escherichia coli as inclusion bodies upon isopropyl-beta-Dthiogalactopyranoside induction. When E. coli cells expressing each mutant toxin were assayed against Aedes aegypti mosquito larvae, the activity was almost completely abolished for N166A and Y170A mutations, whereas E171A showed only a small reduction in toxicity. Further analysis of these two critical residues by induction of specific mutations revealed that polarity at position 166 and highly conserved aromaticity at position 170 within the alpha4-alpha5 loop play a crucial role in the larvicidal activity of the Cry4B toxin.     

Structural requirements of the unique disulphide bond and the proline-rich motif within the alpha4-alpha5 loop for larvicidal activity of the Bacillus thuringiensis Cry4Aa delta-endotoxin

Both the disulphide bond (Cys192-Cys199) and the proline-rich motif (Pro193ProAsnPro196) in the long loop connecting the alpha4-alpha5 transmembrane hairpin of the Cry4Aa mosquito-larvicidal protein have been found to be unique among the Bacillus thuringiensis Cry delta-endotoxins. In this study, their structural requirements for larvicidal activity of the Cry4Aa toxin were investigated. C192A and C199A mutant toxins were initially generated and over-expressed in Escherichia coli cells as 130-kDa protoxins at levels comparable to that of the wild-type toxin. When their activities against Aedes aegypti larvae were determined, Escherichia coli cells expressing each mutant toxin retained the high-level toxicity. Further mutagenic analysis of the PPNP motif revealed that an almost complete loss in larvicidal activity was observed for the C199A/P193A double mutant, whereas a small reduction in toxicity was shown for the C199A/P194A and C199A/P196A mutants. Increasing the flexibility of the alpha4-alpha5 loop through C199A/P193G, C199A/P194G/P196A, C199A/P194A/P196G, and C199A/P194G/P196G mutations significantly decreased the larvicidal activity. Similar to the wild-type protoxin, all mutant toxins were structurally stable upon solubilisation and trypsin activation in carbonate buffer, pH 9.0. These findings are the first biological evidence for a structural function in larvicidal activity of the unique disulphide bridge as well as the proline-rich motif within the alpha4-alpha5 loop of the Cry4Aa toxin.     

Spare interactions of highly potent [Arg14,Lys15]nociceptin for cooperative induction of ORL1 receptor activation

[Arg¹⁴,Lys¹⁵]Nociceptin is a very potent for ORL1 receptor, showing a few times stronger binding activity and much more enhanced biological activity than endogenous nociceptin. This synergistic outcome has been suggested to be due to the interaction with the receptor aromatic and/or acidic amino acid residues crucial to receptor activation. In order to identify such receptor residues in the second ORL1 extracellular loop, we prepared a series of recombinant mutant receptors. The mutant receptor Gln205Ala was found to be as active as wild-type ORL1 for both nociceptin and [Arg¹⁴,Lys¹⁵]nociceptin. In contrast, Asp206Ala and Tyr207Ala exhibited considerably reduced activity for [Arg¹⁴,Lys¹⁵]nociceptin, exhibiting no synergistic activity enhancement. These results suggest that Asp206 and Tyr207 are directly involved in the interaction with nociceptin-[Arg¹⁴,Lys¹⁵]. Trp208Ala was found to bind strongly both nociceptin and [Arg¹⁴,Lys¹⁵]nociceptin, although it elicited no biological activity. All these results indicate that the consecutive amino acid residues Asp206, Tyr207, and Trp208 are critical to the activation of the ORL1 receptor, but not to nociceptin-binding.     

Structurally conserved aromaticity of Tyr249 and Phe264 in helix 7 is important for toxicity of the Bacillus thuringiensis Cry4Ba toxin

Functional elements of the conserved helix 7 in the poreforming domain of the Bacillus thuringiensis Cry delta- endotoxins have not yet been clearly identified. Here, we initially performed alanine substitutions of four highly conserved aromatic residues, Trp(243), Phe(246), Tyr(249) and Phe(264), in helix 7 of the Cry4Ba mosquito-larvicidal protein. All mutant toxins were overexpressed in Escherichia coli as 130-kDa protoxins at levels comparable to the wild-type. Bioassays against Stegomyia aegypti mosquito larvae revealed that only W243A, Y249A or F264A mutant toxins displayed a dramatic decrease in toxicity. Further mutagenic analysis showed that replacements with an aromatic residue particularly at Tyr(249) and Phe(264) still retained the high-level toxin activity. In addition, a nearly complete loss in larvicidal activity was found for Y249L/F264L or F264A/ Y249A double mutants, confirming the involvement in toxicity of both aromatic residues which face towards the same direction. Furthermore, the Y249L/F264L mutant was found to be structurally stable upon toxin solubilisation and trypsin digestion, albeit a small change in the circular dichroism spectrum. Altogether, the present study provides for the first time an insight into the highly conserved aromaticity of Tyr(249) and Phe(264) within helix 7 playing an important role in larvicidal activity of the Cry4Ba toxin.     

Site-directed mutagenesis of the conserved Ala348 and Gly350 residues at the putative active site of Bacillus kaustophilus leucine aminopeptidase

Leucine aminopeptidase (LAP) is an exopeptidase that catalyzes the hydrolysis of amino acid residues from the amino terminus of proteins and peptides. Sequence alignment shows that the conserved Ala348 and Gly350 residues of Bacillus kaustophilus LAP (BkLAP) are located right next to a coordinated ligand. We further investigated the roles of these two residues by performing computer modeling and site-directed mutagenesis. Based on the modeling, the carbonyl group of Ala348 interacts with Asn345 and Asn435, and that of Gly350 with Ile353 and Leu354, where these interactions might maintain the zinc-coordinated residues at their correct positions. Replacement of Ala348 with Arg resulted in a dramatic reduction in LAP activity. A complete loss of the activity was also observed in A348E, A348V, and the Gly350 variants. Measurement of intrinsic tryptophan fluorescence revealed alteration of the microenvironment of aromatic amino acid residues, while circular dichroism spectra were nearly identical for wild-type and all mutant enzymes. Protein modeling and site-directed mutagenesis suggest that residues Ala348 and Gly350 are essential for BkLAP in maintaining a stable active-site environment for the catalytic reaction.     

Targeted mutagenesis of loop residues in the receptor-binding domain of the Bacillus thuringiensis Cry4Ba toxin affects larvicidal activity

Loop residues in domain II of Bacillus thuringiensis Cry delta-endotoxins have been demonstrated to be involved in insecticidal specificity. In this study, selected residues in loops beta6-beta7 (S(387)SPS(390)), beta8-beta9 (S(410), N(411), T(413), T(415), E(417) and G(418)) and beta10-beta11 (D(454)YNS(457)) in domain II of the Cry4Ba mosquito-larvicidal protein were changed individually to alanine by PCR-based directed mutagenesis. All mutant toxins were expressed in Escherichia coli JM109 cells as 130-kDa protoxins at levels comparable to the wild type. Only E. coli cells that express the P389A, S410A, E417A, Y455A or N456A mutants exhibited a loss in toxicity against Aedes aegypti mosquito larvae of approximately 30% when compared to the wild type. In addition, E. coli cells expressing double mutants, S410A/E417A or Y455A/N456A, at wild-type levels revealed a significantly higher loss in larvicidal activity of approximately 70%. Similar to the wild-type protoxin, both double mutant toxins were structurally stable upon solubilisation and trypsin activation in carbonate buffer, pH 9.0. These results indicate that S(410) and E(417) in the beta8-beta9 loop, and Y(455) and N(456) in the beta10-beta11 loop are involved in larvicidal activity of the Cry4Ba toxin.     

Cloning and over-expression of thermostable Bacillus sp. YM55-1 aspartase and site-directed mutagenesis for probing a catalytic residue.

A thermostable aspartase gene (aspB) from Bacillus sp. YM55-1 was cloned and the gene sequenced. The aspB gene (1407 bp ORF) encodes a protein with a molecular mass of 51 627 Da, consisting of 468 amino-acid residues. An amino-acid sequence comparison revealed that Bacillus YM55-1 aspartase shared 71% homology with Bacillus subtilis aspartase and 49% with Escherichia coli and Pseudomonas fluorescens aspartases. The E. coli TK237/pUCASPB strain, which was obtained by transforming E. coli TK237 (aspartase-null strain) with a vector plasmid (pUCASPB) containing the cloned aspB gene, produced a large amount of the enzyme corresponding to > 10% of the total soluble protein. The over-expressed recombinant enzyme (native molecular mass: 200 kDa) was purified effectively and rapidly using heat treatment and affinity chromatography. In order to probe the catalytic residues of this enzyme, two conserved amino-acid residues, Lys183 and His134, were individually mutated to alanine. Although the tertiary structure of each mutant was estimated to be the same as that of wild-type aspartase in CD and fluorescence measurements, the Lys183Ala mutant lost its activity completely, whereas His134Ala retained full activity. This finding suggests that Lys183 may be involved in the catalytic activity of this thermostable Bacillus YM55-1 aspartase.     

Significance of the four carboxyl terminal amino acid residues of bovine pancreatic ribonuclease A for structural folding

The C-terminal amino acid residues of bovine pancreatic ribonuclease A (RNase A) form a core structure in the initial stage of the folding process that leads to the formation of the tertiary structure. In this paper, roles of the C-terminal four amino acids in the structure, function, and refolding were studied by use of recombinant mutant enzymes in which these residues were deleted or replaced. Purified mutant enzymes were analyzed for their secondary structure, thermal stability, and ability to regenerate from the denatured and reduced state. The C-terminal deleted mutant enzymes showed lower hydrolytic activity for C>p and nearly identical CD spectra compared with the wild-type enzyme. The rate of recovery of activity was significantly different among the C-terminal deleted mutant enzymes when air oxidation was employed in the absence of GSH and GSSG: the rates decreased in the order of des-124-, des-(123-124)-, and des-(122-124)-RNase A. It is noteworthy that the regeneration rates of mutant RNase A in the presence of GSH and GSSG were nearly the same. Des-(121-124)-RNase A failed to recover activity both in the presence and absence of glutathione, due to the mismatched formation of disulfide bonds. The mutant enzyme in which all of the C-terminal four amino acid residues were replaced by alanine residues showed lower hydrolytic activity and an indistinguishable CD spectrum compared with the wild-type enzyme, and also recovered its activity from the denatured and reduced state by air oxidation. The D121 mutant enzymes showed decreased hydrolytic activity and identical CD spectra compared with the wild type. The recovery rates of activity of D121A and D121K were determined to be lower than that of the wild-type enzyme, while the rate of recovery of D121E was comparable to that of the wild type. The C-terminal amino acids play a significant role in the formation of the correct disulfide bonds during the refolding process, and the interaction of amino acid residues and the existence of the main chain around the C-terminal region are both important for achieving the efficient packing of the RNase A molecule.     

Characterization of mutations in the GTP-binding domain of IF2 resulting in cold-sensitive growth of Escherichia coli

The infB gene encodes translation initiation factor IF2. We have determined the entire sequence of infB from two cold-sensitive Escherichia coli strains IQ489 and IQ490. These two strains have been isolated as suppressor strains for the temperature-sensitive secretion mutation secY24. The mutations causing the suppression phenotype are located within infB. The only variations from the wild-type (wt) infB found in the two mutant strains are a replacement of Asp409 with Glu in strain IQ489 and an insertion of Gly between Ala421 and Gly422 in strain IQ490. Both positions are located in the GTP-binding G-domain of IF2. A model of the G-domain of E.coli IF2 is presented in. Physiological quantities of the recombinant mutant proteins were expressed in vivo in E.coli strains from which the chromosomal infB gene has been inactivated. At 42 degrees C, the mutants sustained normal cell growth, whereas a significant decrease in growth rate was found at 25 degrees C for both mutants as compared to wt IF2 expressed in the control strain. Circular dichroism spectra were recorded of the wt and the two mutant proteins to investigate the structural properties of the proteins. The spectra are characteristic of alpha-helix dominated structure, and reveal a significant different behavior between the wt and mutant IF2s with respect to temperature-induced conformational changes. The temperature-induced conformational change of the wt IF2 is a two-state process. In a ribosome-dependent GTPase assay in vitro the two mutants showed practically no activity at temperatures below 10 degrees C and a reduced activity at all temperatures up to 45 degrees C, as compared to wt IF2. The results indicate that the amino acid residues, Asp409 and Gly422, are located in important regions of the IF2 G-domain and demonstrate the importance of GTP hydrolysis in translation initiation for optimal cell growth.     

Photoaffinity labeling and site-directed mutagenesis of rat squalene epoxidase

Squalene epoxidase (SE) (EC 1.14.99.7) is a flavin-requiring, non-cytochrome P-450 oxidase that catalyzes the conversion of squalene to (3S)-2,3-oxidosqualene. Photolabeling and site-directed mutagenesis were performed on recombinant rat SE (rrSE) to elucidate the location and roles of active-site residues important for catalysis. Two new benzophenone-containing analogs of NB-598, a nanomolar inhibitor of vertebrate SE, were synthesized in tritium-labeled form. These photoaffinity analogs (PDA-I and PDA-II) became covalently attached to SE when irradiated at 360 nm. Lys-C digestion and HPLC purification of [3H]PDA-I-labeled rrSE resulted in isolation of a single major peptide. MALDI-TOF mass spectrometry of this peptide indicated a covalent adduct between PDA-I and a tripeptide, Asp-Ile-Lys, beginning at Asp-426 of rat SE. Based on the labeling results, three mutant constructs were made. First, the D426A and K428A constructs showed a 5- to 8-fold reduction in SE activity compared with wild-type enzyme, while little change was observed in the I427A mutant. Second, a set of five mutant constructs was prepared for the conserved region based on the structure of the flavoprotein p-hydroxybenzoate hydroxylase (PHBH). Compared with wild-type, D284A and D407A showed less than 25% SE activity. This reduction also appeared to correlate with reduced affinity of the mutant proteins for FAD. Finally, each of the seven Cys residues of rrSE were individually mutated to Ala. Three Cys substitutions had no effect on SE activity, and substitutions at Cys-500 and Cys-533 showed a 50% lower SE activity. Mutations at Cys-490 and Cys-557 produced proteins with negligible SE activity, implicating these residues as being either structurally or catalytically essential. Chemical modification of wildtype and Cys mutants with a thiol-modifying reagent support the existence of a disulfide bond between Cys-490 and Cys-557.     

Relaxing the nicotinamide cofactor specificity of phosphite dehydrogenase by rational design

Homology modeling was used to identify two particular residues, Glu175 and Ala176, in Pseudomonas stutzeri phosphite dehydrogenase (PTDH) as the principal determinants of nicotinamide cofactor (NAD(+) and NADP(+)) specificity. Replacement of these two residues by site-directed mutagenesis with Ala175 and Arg176 both separately and in combination resulted in PTDH mutants with relaxed cofactor specificity. All three mutants exhibited significantly better catalytic efficiency for both cofactors, with the best kinetic parameters displayed by the double mutant, which had a 3.6-fold higher catalytic efficiency for NAD(+) and a 1000-fold higher efficiency for NADP(+). The cofactor specificity was changed from 100-fold in favor of NAD(+) for the wild-type enzyme to 3-fold in favor of NADP(+) for the double mutant. Isoelectric focusing of the proteins in a nondenaturing gel showed that the replacement with more basic residues indeed changed the effective pI of the protein. HPLC analysis of the enzymatic products of the double mutant verified that the reaction proceeded to completion using either substrate and produced only the corresponding reduced cofactor and phosphate. Thermal inactivation studies showed that the double mutant was protected from thermal inactivation by both cofactors, while the wild-type enzyme was protected by only NAD(+). The combined results provide clear evidence that Glu175 and Ala176 are both critical for nicotinamide cofactor specificity. The rationally designed double mutant might be useful for the development of an efficient in vitro NAD(P)H regeneration system for reductive biocatalysis.     

Regulation of glycogen synthase. Identification of residues involved in regulation by the allosteric ligand glucose-6-P and by phosphorylation

The major yeast glycogen synthase, Gsy2p, is inactivated by phosphorylation and activated by the allosteric ligand glucose-6-P. From studies of recombinant proteins, the control can be accommodated by a three-state model, in which unphosphorylated enzyme has intermediate activity (state II). Glucose-6-P increased V(max)/K(m) by about 2-fold (state III), whereas phosphorylation by the cyclin-dependent protein kinase Pcl10p/Pho85p decreased V(max)/K(m) by approximately 30-fold (state I). In the presence of glucose-6-P, state III is achieved regardless of phosphorylation state. The enzyme forms complexes in solution with the yeast glycogenin Glg2p, but this interaction appears not to affect control either by glucose-6-P binding or by phosphorylation. Scanning mutagenesis was applied to identify residues potentially involved in ligand binding. Of 22 mutant enzymes analyzed, seven were essentially inactive. Five mutant proteins were altered in their activation by glucose-6-P, and two were completely unaffected by the hexose phosphate. One of these, R586A/R588A/R591A (all three of the indicated Arg residues mutated to Ala), had wild-type activity and was normally inactivated by phosphorylation. A second mutant, R579A/R580A/R582A, had somewhat reduced V(max), but its activity was not greatly reduced by phosphorylation. The Arg residues in these two mutants are restricted to a highly conserved, 13-residue segment of Gsy2p that we propose to be important for glucose-6-P binding and/or the ability of the enzyme to undergo transitions between activity states.     

Phosphorylation by CK2 enhances the rapid light-induced degradation of phytochrome interacting factor 1 in Arabidopsis

The phytochrome family of sensory photoreceptors interacts with phytochrome interacting factors (PIFs), repressors of photomorphogenesis, in response to environmental light signals and induces rapid phosphorylation and degradation of PIFs to promote photomorphogenesis. However, the kinase that phosphorylates PIFs is still unknown. Here we show that CK2 directly phosphorylates PIF1 at multiple sites. α1 and α2 subunits individually phosphorylated PIF1 weakly in vitro. However, each of four β subunits strongly stimulated phosphorylation of PIF1 by α1 or α2. Mapping of the phosphorylation sites identified seven Ser/Thr residues scattered throughout PIF1. Ser/Thr to Ala scanning mutations at all seven sites eliminated CK2-mediated phosphorylation of PIF1 in vitro. Moreover, the rate of degradation of the Ser/Thr to Ala mutant PIF1 was significantly reduced compared with wild-type PIF1 in transgenic plants. In addition, hypocotyl lengths of the mutant PIF1 transgenic plants were much longer than the wild-type PIF1 transgenic plants under light, suggesting that the mutant PIF1 is suppressing photomorphogenesis. Taken together, these data suggest that CK2-mediated phosphorylation enhances the light-induced degradation of PIF1 to promote photomorphogenesis.     

Aromaticity of Tyr-202 in the alpha4-alpha5 loop is essential for toxicity of the Bacillus thuringiensis Cry4A toxin

The current model for the mechanism of action of the Bacillus thuringiensis Cry delta-endotoxins involves the penetration of the alpha4-alpha5 hairpin into the target midgut epithelial cell membranes, followed by pore formation. In this study, PCR-based mutagenesis was employed to identify a critical residue within the alpha4-alpha5 loop of the 130kDa Cry4A mosquito-larvicidal protein. Alanine-substitutions of two charged (Asp-198 and Asp-200) and four polar (Asn-190, Asn-195, Tyr-201 and Tyr-202) residues in the alpha4-alpha5 loop were performed. Like the wild-type, all of the mutant toxins were over-expressed as inclusion bodies in Escherichia coli. When E. coli cells expressing each mutant toxin were bioassayed against Aedes aegypti larvae, larvicidal activity was completely abolished for the substitution of only Tyr-202, while replacements at the other positions still retained a high level of toxicity. Further replacement of Tyr-202 with an aromatic side chain, phenylalanine, did not affect the toxicity. These results revealed a crucial role in toxin activity for the conserved aromatic residue at the 202 position within the alpha4-alpha5 loop of the Cry4A toxin.     

Crystal structures of MS2 coat protein mutants in complex with wild-type RNA operator fragments.

In MS2 assembly of phage particles results from an interaction between a coat protein dimer and a stem-loop of the RNA genome (the operator hairpin). Amino acid residues Thr45, which is universally conserved among the small RNA phages, and Thr59 are part of the specific RNA binding pocket and interact directly with the RNA; the former through a hydrogen bond, the latter through hydrophobic contacts. The crystal structures of MS2 protein capsids formed by mutants Thr45Ala and Thr59Ser, both with and without the 19 nt wild-type operator hairpin bound, are reported here. The RNA hairpin binds to these mutants in a similar way to its binding to wild-type protein. In a companion paper both mutants are shown to be deficient in RNA binding in an in vivo assay, but in vitro the equilibrium dissociation constant is significantly higher than wild-type for the Thr45Ala mutant. The change in binding affinity of the Thr45Ala mutant is probably a direct consequence of removal of direct hydrogen bonds between the protein and the RNA. The properties of the Thr59Ser mutant are more difficult to explain, but are consistent with a loss of non-polar contact.