Investigation of the possible relationship between mutation at three dimensianal structural level and resistance of culex pipiens s esterases to insecticides

V N U vlOURNAL O F SC IEN C E N Ít Sci & Tech T XIX N 2003 IN V E S T K A T IO N O F T H E P O S S I B L E R E L A T IO N S H I p BETVVEKN M U T A T IO N A T TH K E E -D IM E N S IO N A L S T R U C T U R A L L E V E L A N D KKSISTANCK O F C V L E X P I P I E N S ’S E S T E R A S E S TO IN S E C T IC ID E S D a m lỉa c h U u o n g Center Ị } f lìiotvchnology, Yictnam National Universitv, líanoi C h ic h e L a u r e n t ƯMR 504S Dlỉ /.? MOXTPKLLIER Center of Siructural ỉìiochinìiv, CNHS Framv I n tm d u c tio n P ro te in m o d e llin g was p ctice d on a specific proble m vvith h ig h h e a lth im p a ct: the a c q u ire d re sista n cv o f m osquitoes to in se cticid e s It is k n o w n th a t the ta rg e t o f coram on or^an oph osph atí* in se cticid e s is esterase One w a y rrs is ta n c e is o h ta in e d is tlm n itfh ostorase ovo rexp ressio n (1) H o w cver p o in t m u ta tio n s have been noticed in e s te s i1 A and B loci o f re s is ta n t cu lcx p ip ie n s vvhich m ig h t p la v somo ro le in th e re sista n ce (2) M aterials and m eth o d s Tho esterases fro m v a rio u s in se c tic id e re s is ta n t and in s e c tirid e se n s ih le culex p ip ie n s p o p u la tio n s vvrre s tu d ird at th e In s titu t des Sciences de 'K v o lu tio n (IS K M ), U n iv e rs ite des Sciences et T e ch n iq u e s du Languedoc (P rance) O v o ll, 28 p ro te in s have been sequcnced fo r loci A and ỉ i as shovvn belovv A lo ci B_loci re s is ta n t and sensible esterases í> re sista n t and H sensiblt* esterases Resistant Sensible Resistant Sen sih le SA 1prot S A 2prot SA Iprot SAõprot S A7prot SAHprot S la b lp ro t S lab2prot lỉru g e À R ru g e lỉ T n sp ro t Crisse 1ỉeteren Bleuet BI B2 B4 Bf> B7 B8 S la b l B rugeA B rugeB T n s BSAl B leutìt H ete ren Slab2 11 D a m B a c h Duongy C h ic h e L a u r e n t 12 R e s u lts A l i f í n m e n t 3.1.1 A lig n m e n t T h e a lig n m e n t o f 28 E ste se vvas p e ríb rm e d u s in g C L U S T A L W (H ig g in s I) et a l., 1994, N u c le ic A c id s Res 22:4673-4680, a n a ly z e d u s in g B E L V U h ttp ://w w w e b i.a c u k /c lu s ta lw /) and (h ttp ://w w w c g r.k i.s e /c g r/g ro u p s /s o n n h a m m e r/B e lv u h tm l) an d S E A V IE W (h ttp ://p b il.u n iv - ly o n l.fr /s o ftw a r e /s e a v ie w h tm l) T h re e a lig n m e n t file s in F A S T A fo rm a t w ere p re p a re d C o E s.íầ sta fo r a ll E ste se ; A _ o n ly fa s ta fo r A lo ci a n d B _ o n ly fa s ta fo r B_loci 3.1 S electỉo n o f one reference sequence f o r D m o d e lin g T h e sequence o f S A S p ro t (re s is ta n t) vvas selected because it is th e closest to se n sib le sequences (o n ly d iffe r e n t re s id u e s in c o m p a ris o n vvith s e n s ib le S la b lp ro t) 3.2 M o d e lin g o f S A p r o t (A _ lo c i) 2.1 S earch f o r a s tru c tu rc il te m p la te The sequence S A p ro t vvas s u b m itte d to th e In te r n e t s e rv e rs B IO S E R V (h ttp ://b io s e rv c b s c n rs fr/) an d 3D -P S S M (h ttp ://w w w s b g b io ic a c u k /^ d p s s m /) to fin d th e b e st s tr u c tu r a l te m p la te s fo r m odeling T h e fo llo w in g te m p la te s w e re o b ta in e d fo r S A B p ro t Top 3D -PSSM 1: lm a a , 2: lq id 3: le q h 4: lđ x 5: lk y Top P D B -B L A S T 1: lm a a 2: lm a h , 3: le b 4: lc2o, 5; lf8 u Top T IT O score 1: lm a a , 2: Iq id , 3: le q h 4: k4y 2.2 B u ild i n i t i a l S A S p ro t D m odels Each te m p la te w as used to b u ilt m odels fo r S A B p ro t (M o d e Ile r4 th ro u g h B I()S F ]R V ) T h e scores o f the se m odels are as fo llo w s : P R O S A -II V E R IF Y -3 D M o d e ll M odel2 M odel3 M odel M odel2 M odel3 le q h - 0,503 • 0,412 - 0,352 0,350 0,348 0.340 1maa ■ 0.377 - 0,232 - ,3 28 0,316 0,331 0,369 Iq id - 0.482 - 0,500 ■ 0,438 0,363 0,331 0.370 lk y ■ 0.420 • 0,300 - 0,392 0,263 0,310 0,310 h ìv e s tig a tio n o f t ỉìc ỊìtiS H Ìb ic r c l a t i o t i s h i p b e tiveen Vrom nhovi* tiih lr s t h r h rs t tiio íli‘1 fo r lq e h is M o đ c ll fo r 1m an is M o di*l3 fo r 1q id is M o d c l fo r l k ‘1v is M o d e ltỉ and am ong th e s r m ocỉrls M o d rl Ị (rom lq id d is p líìy s t h r l)i\st scores Is w as nam ed M odel l 3.2.3 C vsteìnvs a n d (iis u lfid e b rid g e s C vsteines can m ake d isu lp h ick* b rid g e w ith o th e r c y s te in e s a n d d is u lfid e briclges are p re se n t in a ll s r liT t r d s tr u c tu r a l te m p la te s A fre e c y s te in e is aỉso ohsi*rvi*d in th(* lq id trm p h m * I)is u lfid e b rid g e s a n d fre e c y s te in e s ob served in th t' fo u r te m p la te s a n d in th e re s u ltin g m odels are s u n im a riz e d be low : 1maa tem plate lm a a model ;ỉ bridges (6 CYS) : 69 - 96 257 - 272 409 - 529 freo ( YS 61 ‘2 96 306 328, 362 436 508 and b riđ g e 195 • 219 I iịk I tem plate lqicl model bridges 67 * 94, 254 • 265, 402 • 521 and free CYS 231 free CYS 61 296 306, 328 362 436, 508 and b rid g e 195 - 219 1k i V tem plate 1k-ly model hriclges 87 - 116 and 273 - 284 ừee CYS 61 296, 306, 328 362 436, 508 and brid ge 195 - 219 IimịIì template briđ g e 36 • 47 41 * 57, 09 - 69 and 37 - 159 free ỎYS 61 296, 306, 328, 362, 436 508 and b rid g e -2 li*(jh model It can ỈH* observocl th a t (i) (lis u líid e b rid g e s are n o t f u lly c o n s c rv e d betw een tc m p la te s and, ( ii) thí* n u m b e r and p o s itio n o f b rid g e s a n d fre e c y s te in e s in the models is id e n tir n l vvhatever th e tc m p lrite used Tho n u m b e r o f fre e cyste in e s in th e m odels is la rg e in c o m p a ris o n to th e tc m p la te s A t th is tim < \ we h a vc no e x p la n a tio n fo r th is I t m a y h a p p e n th a t the protein is multimuric and that so me disulíide bridges are íormed between monom ers 2.4 A n a ly s is a n d o p tim u a tio n o f i n i t i a l S A S p ro í M o d e ll We used I n s ig h t ll to vievv a n d a n a ly z e M o d e ll, in c o m p a ris o n w ith the s tru c tu l te m p la ti* ( I •I >Ii ID: lq id ) VVe observed an u n u s u a l loop c o n fo rm a tio n fo r re s id u e s ‘2 70 to 290 w as fo u n d loop The reason lo r th is is a p p a re n tly due to a too s t r ic t a lig n m e n t T o get b e tte r c o n íb rm a tio n s, WI* rrla x i-d a lig n m e n t " u iìa lig n in g " few re s id u e s a t th e C -te rm in a ỉ loop end as in d ic a tr d bí*low: D a m B a c h D u o ìig , C h ic h c L a u r e n t 14 M o d e ll L L N E N E 1QI D VL p - • I E N R I L L L N E N E - - - I E N R I L F D s I F R V L p - - - F D s • - -I F R T h is changes in file m o d e ll.a li was save in to new file : m o d e l2 a li F ile s m o d e l2 a li and m odel2 top were used to b u ild M o d e l‘2 th ro u g h a s ta n d a lo n e ve rsio n o f M o d e lle r4 ru n n in g on a local SG I w o rk s ta tio n The tw o m odels w ere (h ttp ://b io s e rv c b s c n rs fr/v a lid h tm l) sent The to th e e v a lu a tio n E v a ll2 D scores s e rv e r p ro v id e d e ta ile d in íb rm a tio n fo r each re sid u e and each íra g m e n t o f the m odel From th e E v a lu a tio n scores we can see th a t, g e n e lly the s tru c tu re o f m odel2 is b e tte r th a n m o d e ll, except fo r re sid ue s 414 * 434 In th is p a rt, e v a lu a tio n scores o f m o d e ll are bette^r T h is p a rt o f M odel2 was m o d ifie d according to M o d e ll T h e changes in file m o d e l2 a li w ere saved in to a new file : m o d e l3 a li F ile m o d e l3 a li an d m odel3.top w ere usecỉ to b u ild M odel3 M odel4 was th e o b ta in e d fro m M odel3 by m o đ iíy in g íra g m e n t 62 - 73 E v a lu a tio n scores o f m odels and te m p la te 1Q ID are show n in th e tablo: E v a l2 D V e r if y 3D P ro s a I I E v T re e SFE M odel 0,052 0,236 0,037 - 0,365 - 540,6 Model 0.051 0,219 0,008 - 0,405 - 529,5 Model 0,047 0,219 0,021 - 0,385 - 522,2 M odel 0,063 0,210 0,026 - 0,403 - 526,0 1QID 0,111 0,321 0,192 0,290 - 569,2 From th e above ta b le , vve note th a t M odel d is p la y s a good E va l2 D score, but th a t M o đ e ll is th e b e tte r acco rdin g to a ll o th e r scores We th e re ib re selected M odel as%our M odel and m o d iỉie d i t in tw o regions, v\4iere M odel4 has b e tte r scores (re g io n s - and 85 - 95) T h e changes in file m o d e ll.a li w ere saved in to new file : m o delõ ali Piles m o d e is a li; m odelo.top were used to b u ild M odel5 E v a l 23D V e r if y 3D P ro s a I I E v T re e SFE Mođel 0.052 0.236 0,037 - 0,365 - 540,6 M odel 0.063 0,210 0.026 - 0,403 • 526,0 M o d e l 5/1 0,072 0,241 0,047 - 0,363 - 551,3 Modtj l Õ/2 0,066 0,239 0,036 - 0,366 - 542,4 M ođel 5/3 0,070 0.257 0,045 - 0,369 - 536,1 I m c s t iịỊ o t io n o f t h ỉ ’ po s s ib lc r c i a t io n s h ip betivcen 15 lí Cỉin he S(*(*n th it a ll nl v ;m ;itio n s o f mođel have h e tte r scorivs th a n M odel ỉin ri M odel Thi* hest is M n d rl 5/1 K x ỉim in a tio n n f M ođcl Ít was nam ed M odel 5 (use In s ig h t II) show s th a t a v e ry bad loop eo n ío rm ation (knot) IS p res rn t íor í r a g m e n t 421 • 440 To relax th is region we "u n.iliự iK H r tw o r r s u lu e s Mocli-I r> Q II) F s V D s I) T Y N H Y R I V F c D F N H R A S - - N L V w Mock-I