Insilico structural analysis of parasporin 2 protein sequences of non-toxic bacillus thuringiensis

Abstract

The unusual and remarkable property of parasporin 2 of non-insecticidal Bacillus thuringiensis is specifically recognizing and selectively targeting human leukemic cell lines. The 37-kDa inactive nascent protein is proteolytically cleaved to the 30-kDa active form that loses both the N-terminal and the C-terminal segments. Accumulated cytological and biochemical observations on parasporin-2 imply that the protein is a pore-forming toxin. To confirm the hypothesis, insilico analysis was performed using homology modeling. The resulting model of parasporin 2 protein is unusually elongated and mainly comprises long β-strands aligned with its long axis. It is similar to aerolysin-type β-pore-forming toxins, which strongly reinforce the pore-forming hypothesis. The molecule can be divided into three domains. Domain 1, comprising a small β-sheet sandwiched by short α-helices, is probably the target-binding module. Two other domains are both β-sandwiches and thought to be involved in oligomerization and pore formation. Domain 2 has a putative channel-forming β-hairpin characteristic of aerolysin-type toxins. The surface of the protein has an extensive track of exposed side chains of serine and threonine residues. The track might orient the molecule on the cell membrane when domain 1 binds to the target until oligomerization and pore formation are initiated. The β-hairpin has such a tight structure that it seems unlikely to reform as postulated in a recent model of pore formation developed for aerolysin-type toxins. Parasporin 2 (Accession no: BAD35170) protein sequence analysis indicated two different domains namely, aerolysin toxin and clostridium toxin domain based on different database searches (CDD and Pfam). It showed a close similarity with the available PDB template (PDB id: 2ZTB) of parasporin which has cytocidal activity against MOLT-4, HL60 and Jurkat cell lines. Based on the PSI Blast analysis, 3D structures of the domains were predicted by using Swiss model server. Accuracy of the prediction of 3D structure of different domains of parasporin protein was further validated by Ramachandran plot and PROCHECK (G-value). The structure is dominated by β-strands (67%, S1-12), most of which are remarkably extensive, running all or most of the longer axis of the molecule. This study helped to elucidate the 3D structure of parasporin 2 (Acc. No. BAD35170) which might enable to probe further its specific mechanism of action. Though the similarity is observed in the domain architecture, there is variation in the regions of the domains even among the same group of parasporin 2. Docking of this model structure and experimental structure with specific receptors of the cancer cells will facilitate to explore mechanism of parasporin 2 action and also provide information about its evolutionary relationship with toxic Cry proteins.