title: Bacillus thuringiensis Cry4Aa insecticidal protein: functional importance of the intrinsic stability of the unique α4-α5 loop comprising the Pro-rich sequence.
abstract: The long loop connecting transmembrane α4 and α5 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 (pH9.0), 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 α4-α5 loop is substantially stable as it showed low structural fluctuation with a 1.2-Å RMSF value. When the flexibility of the α4-α5 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 α4-α5 loop structure comprising the Pro-rich sequence plays an important role in toxin activity.
authors.affiliation: Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand.
authors.forename: Chompounoot
authors.initials: C
authors.lastname: Imtong
authors.affiliation: Laboratory of Theoretical and Computational Biophysics, Department of Physics, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.
authors.forename: Chalermpol
authors.initials: C
authors.lastname: Kanchanawarin
authors.affiliation: Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand.
authors.forename: Gerd
authors.initials: G
authors.lastname: Katzenmeier
authors.affiliation: Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand; Laboratory of Molecular Biophysics and Structural Biochemistry, Biophysics Institute for Research and Development (BIRD), Bangkok 10150, Thailand. Electronic address: chanan.ang@mahidol.ac.th.
authors.forename: Chanan
authors.initials: C
authors.lastname: Angsuthanasombat
mesh_terms: D000330:Aedes / Q000382:microbiology; D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004731:Endotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007814:Larva / Q000382:microbiology; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009154:Mutation; D011392:Proline / Q000235:genetics / Q000378:metabolism*; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D011392:Proline
keywords: Insecticidal protein; Larval toxicity; Loop rigidity; MD simulations; Pro-rich sequence; Site-specific mutagenesis