Mutagenic Analysis of a Conserved Region of Domain III in the Cry1Ac Toxin of Bacillus thuringiensis

Abstract

We used site-directed mutagenesis to probe the function of four alternating arginines located at amino acid positions 525, 527, 529, and 531 in a highly conserved region of domain III in the Cry1Ac toxin of Bacillus thuringiensis. We created 10 mutants: eight single mutants, with each arginine replaced by either glycine (G) or aspartic acid (D), and two double mutants (R525G/R527G and R529G/R531G). In lawn assays of the 10 mutants with a cultured Choristoneura fumiferana insect cell line (Cf1), replacement of a single arginine by either glycine or aspartic acid at position 525 or 529 decreased toxicity 4-to 12-fold relative to native Cry1Ac toxin, whereas replacement at position 527 or 531 decreased toxicity only 3-fold. The reduction in toxicity seen with double mutants was 8-fold for R525G/R527G and 25-fold for R529G/R531G. Five of the mutants (R525G, R525D, R527G, R529D, and R525G/R527G) were tested in bioassays with Plutella xylostella larvae and ion channel formation in planar lipid bilayers. In the bioassays, R525D, R529D, and R525G/R527G showed reduced toxicity. In planar lipid bilayers, the conductance and the selectivity of the mutants were similar to those of native Cry1Ac. Toxins with alteration at position 527 or 529 tended to remain in their subconducting states rather than the maximally conducting state. Our results suggest that the primary role of this conserved region is to maintain both the structural integrity of the native toxin and the full functionality of the formed membrane pore.Bacillus thuringiensis is a gram-positive bacterium that produces one or more insecticidal crystal (Cry) proteins deposited in the form of an intracellular parasporal crystal during sporulation (20). Shortly after ingestion by a susceptible insect, most Cry proteins dissolve in the insect midgut, are activated by midgut proteases, and bind to a specific midgut epithelial cell receptor. After binding to specific docking proteins on the microvillous surface of susceptible epithelial cells in the larval midgut (12, 27), the toxin undergoes a conformational change in which the helix-rich domain I (DI) separates from the other two domains (23). A hairpin, composed of helices ␣4 and ␣5, subsequently inserts into the membrane with the other five helices spreading, in an umbrella-like fashion, over the membrane surface (8), with ␣4 lining the lumen of the pore to create a functional ion channel (19). The toxin-exposed midgut epithelial cells eventually die by a colloid-osmotic lysis mechanism (14).The atomic structures of two activated Cry proteins, Cry1Aa and Cry3A, have been reported (11, 16). The two proteins have similar three-domain structures. DI is directly involved in ion channel formation (8,23,28). The second domain, DII, is involved in binding (9, 17, 21), whereas DIII is thought to play both a structural role and a toxin recognition role (2,5,15,17).A multiple sequence alignment of cry genes shows five conserved blocks (13), with the fourth conserved block (in DIII) consisting of an intriguing series of four al...

Mosquito Resistance to Bacterial Larvicidal Toxins

“…Although an overall topology of this domain is rather similar to some carbohydrate-binding protein domains such as the cellulosebinding domain of a 1,4--glucanase enzyme (Johnson Biochemistry 1999) [28], its functional role is still not clearly elucidated. Nevertheless, it has been implicated in membrane permeabilisation (Masson AEM 2002) [29] or receptor recognition and specificity determination (Burton JMB 1999) [30] (De Maagd AEM 2000) [31] (Chayaratanasin JBMB 2007) [32]. This domain could be also critical for the structural integrity of the toxin molecule as the position of the C-terminus (i.e.…”

Bacillus thuringiensis Cry4A and Cry4B Mosquito-larvicidal Proteins: Homology-based 3D Model and Implications for Toxin Activity

“…Domain III is a β-sandwich that is comprised of two anti-parallel β-sheets. The role of this C-terminal domain is still unclear although it has been implicated in the structural integrity of toxin molecules (Li et al, 1991), membrane permeabilisation (Schwartz et al, 1997;Masson et al, 2002), or receptor binding that can influence insect specificity (Lee et al, 1995;de Maagd et al, 1996;Burton et al, 1999).…”

The role of β18–β19 loop structure in insecticidal activity of Cry1Ac toxin from Bacillus thuringiensis

“…The toxins subsequently undergo a conformational change and insert into the membrane, forming a pore to create a functional ion channel. The toxin-exposed midgut epithelial cells eventually die by a colloid-osmotic lysis mechanism [2][3][4] .X-ray crystallography structures of 6 different Bt Cry δ-endotoxins were reported as Cry1Aa [5] , Cry2Aa [6] , Cry3Aa [7] , Cry3Bb [8] , Cry4Ba [9] and Cry4Aa [10] , which revealed a high degree of overall structural similarity, comprising 3 distinct domains. The N-terminal Domain I is directly involved in penetration of target membrane and ion channel formation [1,11] .…”

“…The toxins subsequently undergo a conformational change and insert into the membrane, forming a pore to create a functional ion channel. The toxin-exposed midgut epithelial cells eventually die by a colloid-osmotic lysis mechanism [2][3][4] .…”