Paper Citing NAMD - Abstract
Imtong, Chompounoot; Kanchanawarin, Chalermpol; Katzenmeier, Gerd; Angsuthanasombat, Chanan
Bacillus thuringiensis Cry4Aa insecticidal protein: Functional importance of the intrinsic stability of the unique alpha 4-alpha 5 loop comprising the Pro-rich sequence
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, 1844:1111-1118, JUN 2014
The long loop connecting transmembrane alpha 4 and alpha 5 of the Bacillus thuringiensis Cry4Aa toxin possesses a unique feature with Pro-rich sequence (Pro(193)Pro(194)_Pro(196)) which was shown to be crucial for toxicity. Here, the structural role in the intrinsic stability of the Pro-rich sequence toward toxin activity was investigated. Three Val-substituted mutants (P193V, P194V and P196V) and one Phe-substituted mutant (P193F) were generated and over-expressed in Escherichia coli as inclusions at levels equal to the wild-type. Bioassays demonstrated that all mutants, particularly P193V and P193F whose inclusions were hardly soluble in carbonate buffer (pH 9.0), exhibited reduced toxicity, suggesting an essential role in toxin function by the specific cyclic structure of individual Pro residues. Analysis of the 65-kDa Cry4Aa structure from 10-ns molecular dynamics (MD) simulations revealed that the alpha 4-alpha 5 loop is substantially stable as it showed low structural fluctuation with a 1.2-angstrom RMSF value. When the flexibility of the a4-a5 loop was increased through P193G, P194G and P196G substitutions, decreased toxicity was also observed for all mutants, mostly for the P193G mutant with low alkali-solubility, suggesting a functional importance of loop-rigidity attributed by individual Pro-cyclic side-chains, particularly Pro(193). Further MD simulations revealed that the most critical residue-Pro(193) for which mutations vastly affect toxin solubility and larval toxicity is in close contact with several surrounding residues, thus playing an additional role in the structural arrangement of the Cry4Aa toxin molecule. Altogether, our data signify that the intrinsic stability of the unique Cry4Aa alpha 4-alpha 5 loop structure comprising the Pro-rich sequence plays an important role in toxin activity. (C) 2014 Elsevier B.V. All rights reserved.
