S tructural Analyses of Cry 1Ac Protein from Bacillus thuringiensis

C ry p ro tein is a d elta e n d o to x in o f the B acillus fam ily that p ro v id es an e n to m o p ato g e n ic ac tiv ity to B acillus thuringiensis. T h ese p ro te in s h av e a sp ec ific toxic activity against three types o f insect larva: L ep d o p th e ra, D ip tera and C oleopthera. T he in secticid a l to x in s arc produced d u rin g spore form ation. W hen an in sect in g ests these proteins they are activated by p ro teo ly tic c le av ag e . T h e toxin, afte r the ingestion, is solubilized by th e alc a lin pH in the d ig estiv e trac t o f the target insect. O nce activ ated th e en d o to x in b in d s to the gut epithelium and causes cell lysis lea d in g to d eath . T h ese p roteins arc the active agents used in th e m ajo rity o f bio ratio n al pesticid es and insect-resistant tran sg e n ic crops. T h is a c tiv a ted re g io n o f the d elta endo to x in is com posed o f three structural d o m a in s. D om ain I is involved in m em brane insertion, pore fo rm a tio n a n d toxicity. T h e second and third dom ains are in volved in re c e p to r b inding and specifically dom ain III is im p o rtan t in in sect specificity. T h ere are a ro u n d 120 seq u e n ces o f C ry to x in s, and on ly five s tru ctu re s w e re d e p o sited on the P ro tein D ata B ank (P D B ). There is a large in tere st in the toxin C ry lA c because it is com m only u sed to c rc a te tran sg e n ic plants w ith insect resistance. A th eo re tic al m odel o f the C iy lA c toxin w as obtained on the basis o f th e c o o rd in a tes o f the in secticidal protein C ry lA a (PDB c o d e :lc iy .p d b ) [1] as a tem p late. T he high sequence identity (7 3 % ) and a g o o d co rre la tio n c o e ffic ie n t ob tain ed from the elctron den sity s e rv e r [2] in d icate s that lc iy structure could be used as a tem p late.. T he m o d el co rresp o n d s to residues 33-618 o f the p rim ary stru ctu re an d co n sists o f dom ains I, II and III. The seq u e n ce o f d o m a in I is highly co n se rv ed , w hile dom ain II and III have the lo w er s e q u e n c e s im ilarity . T he 3D m odel w as construct u sin g M o d eller v 7 .7 . |3 ]. A im ed at v erify in g w h e th e r th e a m in o acid d iffe ren c es in dom ains II and III could be re sp o n sib le fo r insect sp ec ificity , this tw o structures w e re stru ctu ra lly alig n ed a n d co m p a red w ith th e S ting protein s tru ctu re an a ly sis pro g ram . K n o w in g th at ea ch am in o acid substitution co u ld p o ten tially affe c t the p r o te in s tru ctu re and function, th ese am in o ac id s w e re m ap p e d an d e v a lu ated a c co rd in g to: co n servation, ch a n g e in so lv en t ac ce ssib ility , sid e-c h ain volum e ch a n g e, effec t o n a m in o -a cid in teractions, protein electrostatics, a n d p h y sico ch e m ic al p ro p e rties o f a m in o acids. Such approach can open a new insight into understanding of specificity that Cry protein has for receptors in plant pests and can help in the design o f mutagenesis experiments aimed to elucidate the m echanism o f action o f the Cry 1 Ac toxin.