Helix 4 Mutants of the Bacillus thuringiensis Insecticidal Toxin Cry1Aa Display Altered Pore-Forming Abilities

ABSTRACT The role played by α-helix 4 of the Bacillus thuringiensis toxin Cry1Aa in pore formation was investigated by individually replacing each of its charged residues with either a neutral or an oppositely charged amino acid by using site-directed mutagenesis. The majority of the resulting mutant proteins were considerably less toxic to Manduca sexta larvae than Cry1Aa. Most mutants also had a considerably reduced ability to form pores in midgut brush border membrane vesicles isolated from this insect, with the notable exception of those with alterations at amino acid position 127 (R127N and R127E), located near the N-terminal end of the helix. Introducing a negatively charged amino acid near the C-terminal end of the helix (T142D and T143D), a region normally devoid of charged residues, completely abolished pore formation. For each mutant that retained detectable pore-forming activity, reduced membrane permeability to KCl was accompanied by an approximately equivalent reduction in permeability to N-methyl-d-glucamine hydrochloride, potassium gluconate, sucrose, and raffinose and by a reduced rate of pore formation. These results indicate that the main effect of the mutations was to decrease the toxin's ability to form pores. They provide further evidence that α-helix 4 plays a crucial role in the mechanism of pore formation.

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Cysteine Scanning Mutagenesis of α4, a Putative Pore-Lining Helix of the Bacillus thuringiensis Insecticidal Toxin Cry1Aa

Applied and Environmental Microbiology ◽

10.1128/aem.00094-08 ◽

2008 ◽

Vol 74 (9) ◽

pp. 2565-2572 ◽

Cited By ~ 26

Author(s):

Frédéric Girard ◽

Vincent Vachon ◽

Gabrielle Préfontaine ◽

Lucie Marceau ◽

Yanhui Su ◽

...

Keyword(s):

Bacillus Thuringiensis ◽

Pore Formation ◽

Membrane Vesicles ◽

Significant Activity ◽

Amino Acid Residues ◽

Insect Larvae ◽

Insecticidal Toxin ◽

Cysteine Scanning ◽

Cysteine Scanning Mutagenesis ◽

Pore Lining

ABSTRACT Helix α4 of Bacillus thuringiensis Cry toxins is thought to line the lumen of the pores they form in the midgut epithelial cells of susceptible insect larvae. To define its functional role in pore formation, most of the α4 amino acid residues were replaced individually by a cysteine in the Cry1Aa toxin. The toxicities and pore-forming abilities of the mutated toxins were examined, respectively, by bioassays using neonate Manduca sexta larvae and by a light-scattering assay using midgut brush border membrane vesicles isolated from M. sexta. A majority of these mutants had considerably reduced toxicities and pore-forming abilities. Most mutations causing substantial or complete loss of activity map on the hydrophilic face of the helix, while most of those having little or only relatively minor effects map on its hydrophobic face. The properties of the pores formed by mutants that retain significant activity appear similar to those of the pores formed by the wild-type toxin, suggesting that mutations resulting in a loss of activity interfere mainly with pore formation.

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Mutations in Domain I Interhelical Loops Affect the Rate of Pore Formation by the Bacillus thuringiensis Cry1Aa Toxin in Insect Midgut Brush Border Membrane Vesicles

Applied and Environmental Microbiology ◽

10.1128/aem.02924-08 ◽

2009 ◽

Vol 75 (12) ◽

pp. 3842-3850 ◽

Cited By ~ 11

Author(s):

Geneviève Lebel ◽

Vincent Vachon ◽

Gabrielle Préfontaine ◽

Frédéric Girard ◽

Luke Masson ◽

...

Keyword(s):

Bacillus Thuringiensis ◽

Conformational Changes ◽

Brush Border ◽

Pore Formation ◽

Brush Border Membrane ◽

Membrane Vesicles ◽

Thiol Group ◽

Brush Border Membrane Vesicles ◽

Site Directed Mutagenesis ◽

Domain I

ABSTRACT Pore formation in the apical membrane of the midgut epithelial cells of susceptible insects constitutes a key step in the mode of action of Bacillus thuringiensis insecticidal toxins. In order to study the mechanism of toxin insertion into the membrane, at least one residue in each of the pore-forming-domain (domain I) interhelical loops of Cry1Aa was replaced individually by cysteine, an amino acid which is normally absent from the activated Cry1Aa toxin, using site-directed mutagenesis. The toxicity of most mutants to Manduca sexta neonate larvae was comparable to that of Cry1Aa. The ability of each of the activated mutant toxins to permeabilize M. sexta midgut brush border membrane vesicles was examined with an osmotic swelling assay. Following a 1-h preincubation, all mutants except the V150C mutant were able to form pores at pH 7.5, although the W182C mutant had a weaker activity than the other toxins. Increasing the pH to 10.5, a procedure which introduces a negative charge on the thiol group of the cysteine residues, caused a significant reduction in the pore-forming abilities of most mutants without affecting those of Cry1Aa or the I88C, T122C, Y153C, or S252C mutant. The rate of pore formation was significantly lower for the F50C, Q151C, Y153C, W182C, and S252C mutants than for Cry1Aa at pH 7.5. At the higher pH, all mutants formed pores significantly more slowly than Cry1Aa, except the I88C mutant, which formed pores significantly faster, and the T122C mutant. These results indicate that domain I interhelical loop residues play an important role in the conformational changes leading to toxin insertion and pore formation.

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Impact of evolutionary selection on dynamic behavior of MCAK protein

10.1101/2020.12.04.412650 ◽

2020 ◽

Author(s):

Shiwani Limbu

Keyword(s):

Amino Acid ◽

Amino Acid Position ◽

Coding Region ◽

Microtubule Depolymerization ◽

Protein Coding ◽

Microtubule Binding ◽

Evolutionary Selection ◽

Family Protein ◽

Α Helix

AbstractKinesins of class 13 (kinesin-13s), also known as KinI family proteins, are non-motile microtubule binding kinesin proteins. Mitotic centromere-associated kinesin (MCAK), a member of KinI family protein, diffuses along the microtubule and plays a key role in microtubule depolymerization. Here we have demonstrated the role of evolutionary selection in MCAK protein coding region in regulating its dynamics associated with microtubule binding and stability. Our results indicate that evolutionary selection within MCAK motor domain at amino acid position 440 in carnivora and artiodactyla order results in significant change in the dynamics of α – helix and loop 11, indicating its likely impact on changing the microtubule binding and depolymerization process. Furthermore, evolutionary selections at amino acid position 600, 617 and 698 are likely to affect MCAK stability. A deeper understanding of evolutionary selections in MCAK can reveal the mechanism associated with change in microtubule dynamics within eutherian mammals.

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Monitoring of Bacillus Thuringiensis Cry3Aa Toxin Pore Formation using Artificial Bilayer Array with Fused Brush Border Membrane Vesicles from Colorado Potato Beetle Larvae

Biophysical Journal ◽

10.1016/j.bpj.2016.11.2829 ◽

2017 ◽

Vol 112 (3) ◽

pp. 523a

Author(s):

Ekaterina Zaitseva ◽

Gerhard Baaken ◽

Victor M. Ruiz-Arroyo ◽

Inmaculada García-Robles ◽

Camila Ochoa-Campuzano ◽

...

Keyword(s):

Bacillus Thuringiensis ◽

Colorado Potato Beetle ◽

Brush Border ◽

Pore Formation ◽

Brush Border Membrane ◽

Membrane Vesicles ◽

Brush Border Membrane Vesicles ◽

Potato Beetle

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A membrane associated metalloprotease cleaves Cry3Aa Bacillus thuringiensis toxin reducing pore formation in Colorado potato beetle brush border membrane vesicles

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/j.bbamem.2007.06.014 ◽

2007 ◽

Vol 1768 (9) ◽

pp. 2293-2299 ◽

Cited By ~ 24

Author(s):

C. Rausell ◽

C. Ochoa-Campuzano ◽

A.C. Martínez-Ramírez ◽

A. Bravo ◽

M.D. Real

Keyword(s):

Bacillus Thuringiensis ◽

Colorado Potato Beetle ◽

Brush Border ◽

Pore Formation ◽

Brush Border Membrane ◽

Membrane Vesicles ◽

Brush Border Membrane Vesicles ◽

Bacillus Thuringiensis Toxin ◽

Potato Beetle

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Lipid-induced Pore Formation of the Bacillus thuringiensis Cry1Aa Insecticidal Toxin

The Journal of Membrane Biology ◽

10.1007/s002320010070 ◽

2001 ◽

Vol 180 (3) ◽

pp. 195-203 ◽

Cited By ~ 59

Author(s):

V. Vié ◽

N. Van Mau ◽

P. Pomarède ◽

C. Dance ◽

J.L. Schwartz ◽

...

Keyword(s):

Bacillus Thuringiensis ◽

Pore Formation ◽

Insecticidal Toxin

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Two novel mutations of the calcium-sensing receptor gene affecting the same amino acid position lead to opposite phenotypes and reveal the importance of p.N802 on receptor activity

Acta Endocrinologica ◽

10.1530/eje-12-0714 ◽

2013 ◽

Vol 168 (2) ◽

pp. K27-K34 ◽

Cited By ~ 13

Author(s):

Anne-Sophie Lia-Baldini ◽

Corinne Magdelaine ◽

Angélique Nizou ◽

Coraline Airault ◽

Jean-Pierre Salles ◽

...

Keyword(s):

Amino Acid ◽

Transmembrane Domain ◽

Direct Sequencing ◽

Receptor Gene ◽

Amino Acid Position ◽

Receptor Activity ◽

Site Directed Mutagenesis ◽

Missense Mutations ◽

Calcium Sensing Receptor ◽

Calcium Sensing

ObjectiveGain-of-function mutations of the calcium-sensing receptor (CASR) gene have been identified in patients with sporadic or familial autosomal dominant hypocalcemia (ADH). Inactivating mutations of the CASR gene cause familial hypocalciuric hypercalcemia (FHH). Here, we report two novel CASR mutations affecting the same amino acid (p.N802); one causes ADH and the other atypical FHH.Patients and methodsThe first patient, an 11-year-old girl suffering from hypocalcemia, developed nephrocalcinosis when she was only 5 years old. The second patient is a 30-year-old woman who presented with mild hypercalcemia. PCR amplification of CASR coding exons and direct sequencing of PCR products were used to identify mutations. Site-directed mutagenesis was used to generate mutated CASR cDNAs in an expression plasmid. Using the MAPK assay system and transient transfection of Cos-7 cells with wild-type (WT) and mutated CASR, we studied the responses of these mutated receptors to extracellular Ca2+ and to the negative allosteric CASR modulator, NPS2143.ResultsTwo heterozygous missense mutations (p.N802I and p.N802S) affecting a residue in the sixth transmembrane domain of CASR were identified. In functional tests, the response of the p.N802S mutant to calcium was typical of an inactivating mutation. However, the p.N802I mutant had 70% of the maximally stimulated WT receptor activity even in the absence of extracellular calcium. This constitutive activity was only partially inhibited by the inhibitor, NPS2143.ConclusionsThe asparagine at amino acid position 802 appears to be essential for the activity of the CASR protein and is implicated in the mechanism of CASR signaling.

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