Abstract
Vip3 proteins are increasingly used in insect control in transgenic crops. To shed light on the structure of these proteins, we used the approach of the trypsin fragmentation of mutants altering the conformation of the Vip3Af protein. From an alanine scanning of Vip3Af, we selected mutants with an altered proteolytic pattern. Based on protease digestion patterns, their effect on oligomer formation, and theoretical cleavage sites, we generated a map of the Vip3Af protein with five domains which match some of the domains proposed independently by two in silico models. Domain I ranges amino acids (aa) 12–198, domain II aa199–313, domain III aa314–526, domain IV aa527–668, and domain V aa669–788. The effect of some mutations on the ability to form a tetrameric molecule revealed that domains I–II are required for tetramerization, while domain V is not. The involvement of domain IV in the tetramer formation is not clear. Some mutations distributed from near the end of domain I up to the end of domain II affect the stability of the first three domains of the protein and destroy the tetrameric form upon trypsin treatment. Because of the high sequence similarity among Vip3 proteins, we propose that our domain map can be extended to the Vip3 family of proteins.
Highlights
Vip3A proteins are produced during the vegetative phase of growth of Bacillus thuringiensis and are of practical interest because of their insecticidal activity against Lepidoptera [1]
Changes in protein conformation may expose potential cleavage sites otherwise buried inside the protein which, when exposed to proteases, give rise to altered patterns of fragments compared with that of the wild type protein (WT)
Vip3Af protein, we have defined five domains in the structure of Vip3Af which match some of the domains proposed independently by two in silico models
Summary
Vip3A proteins are produced during the vegetative phase of growth of Bacillus thuringiensis and are of practical interest because of their insecticidal activity against Lepidoptera [1]. Vip3A proteins share no sequence and structural homology with B. thuringiensis Cry proteins, they are considered an excellent complement to Cry proteins in crop protection and resistance management. Some commercial Bt-crops (crops protected from insect attacks by expressing insecticidal proteins from B. thuringiensis) combine Cry and Vip proteins, and this strategy of pyramiding proteins with different modes of action is expected to continue in the future [2]. Despite the increasing interest in Vip proteins, their mode of action is not completely understood, and their 3D structure still remains unknown. Multiple-sequence alignments of Vip proteins have shown that they contain between 786 and 803 amino acids (corresponding to a molecular weight of around 89 kDa), with a highly conserved N-terminal part (up to residue 334) and a highly variable
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