DSpace at VNU: Investigation of the possible relationship between mutation at three-dimensional structural level and resistance of culex pipien''s esterases to insecticides

DSpace at VNU: Investigation of the possible relationship between mutation at three-dimensional structural level and res...

V N U vlO U RN AL O F S C IE N C E N Ít Sci & Tech T XIX N 4 2003

I N V E S T K 1 A T I O 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 I O N A T TH K E E - D I M E N S I O N A L S T R U C T U R A L L E V E L A N D

I N S E C T I C I D E S

D a m lỉa c h U u o n g

C h ic h e L a u r e n t

1 I n t m d u c t i o n

P ro te in m o d e llin g was p ra c tic e d on a sp e cific p ro b le m vvith h ig h h e a lth

im p a c t: the a c q u ire d re s is ta n c v o f m o squitoes to in s e c tic id e s It is k n o w n th a t th e

ta rg e t o f coram on o r^a n o p h o sp h a tí* 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 o vo re xp re ssio n (1) H o w cve r p o in t m u ta tio n s have

been no tice d in e s te ra s i1 A and B lo ci o f re s is ta n t c u lcx p ip ie n s vvhich m ig h t p la v

somo ro le in th e re s is ta n c e (2)

2 M a ter ia ls and m e th o d s

T h o esterases fro m v a rio u s in s e c tic id e re s is ta n t an d in s e c tirid e s e n s ih le culex

p ip ie n s p o p u la tio n s vvrre s tu d ir d at th e In s titu t des Sciences de 1 'K v o lu tio n (IS K M ),

U n iv e rs ite des Sciences et T e c h n iq u e s du Languedoc (P ra nce) O v o r a ll, 28 p ro te in s have been sequcnced fo r 2 loci A and ỉ i as shovvn belovv

A lo c i

í> re s is ta n t and H sensiblt* esterases

B_loci

6 re s is ta n t and 8 sensible esterases

1 1

12 D a m B a c h Duongy C h ic h e L a u r 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 s te ra s e 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) e t

a l., 1994, N u c le ic A c id s Res 22 :4 6 7 3 -4 6 8 0 , h ttp ://w w w e b i.a c u k /c lu s ta lw /) and

a n a ly z e d u s in g B E L V U (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)

a n d S E A V IE W (h tt p ://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 s ta fo r a ll E s te ra s e ; A _ o n ly fa s ta fo r A lo c i a n d B _ o n ly fa s ta fo r B_loci

3.1 2 S e le ctỉo n o f one reference sequence f o r 3 D m o d e lin g

T h e sequence o f S A S p ro t (r e s is ta n t) vvas sele cted because it is th e close st to

se n sib le sequences (o n ly 3 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 r o t)

3.2 M o d e lin g o f S A 8 p r o t ( A _ lo c i)

3 2.1 S earch f o r a s tr u c tu r c il te m p la te

T h e sequence S A 8 p ro t vvas s u b m itte d to th e I n t e r n e t s e rv e rs B IO S E R V (h ttp ://b io s e r v c b s c n r s fr/) a n d 3 D -P S S M (h ttp ://w w w s b g b io ic a c u k /^ 3 d p s s m /) to fin d th e b e s t s tr u c tu r a l te m p la te s fo r m o d e lin g

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

3 R e s u lts

3 1 A l i f í n m e n t

T op 3D -P S S M 1: lm a a , 2: lq id 3: le q h 4: lđ x 4 5: lk 4 y Top P D B -B L A S T 1: lm a a 2: lm a h , 3: le 2 b 4: lc2o , 5; lf8 u Top T IT O score 1: lm a a , 2: Iq id , 3: le q h 4: k4 y

3 2.2 B u ild i n i t i a l S A S p ro t 3 D m odels

Each te m p la te w as used to b u ilt 3 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 th e se m o d e ls a re as fo llo w s :

h ì v e s t ig a t io n o f t ỉì c Ị ì t i S H Ì b i c r c l a t i o t i s h i p b e tiv e e n.

Vrom nhovi* t iih lr s t h r h rs t tiio íli‘1 fo r lq e h is M o đ c ll

fo r 1 m an is M o d i*l3

fo r 1 q id is M o d c l 1

fo r l k ‘1v is M o d e ltỉ and am ong th e s r m o cỉrls 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 o del l

3.2.3 C vsteìnvs a n d ( iis u lfid e b rid g e s

C vste in e s can m ake d is u lp h ic k * 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 s e 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 o b serve d in th t'

fo u r te m p la te s a n d in th e r e s u ltin g m o dels a re s u n im a riz e d b e lo w :

1 maa te m p la te

lm a a model

;ỉ bridges (6 CYS) : 69 - 96 257 - 272 409 - 529

7 freo ( YS 61 ‘296 306 328, 362 436 508 an d 1 b riđ g e 195 • 219

IiịkI te m p la te

lqicl model

3 brid ge s 67 * 94, 254 • 265, 402 • 521 and 1 fre e CYS 231

7 free CYS 61 296 306, 328 362 436, 508 and 1 b rid g e 195 - 219

1 k i V tem pl at e

1 k-ly model

2 hriclges 87 - 116 and 273 - 284

7 ừee CYS 61 296, 306, 328 362 436, 508 and 1 b rid g e 195 - 219 IimịIì tem plate

li*(jh model

1 b riđ g e 36 • 47 41 * 57, 09 - 69 and 37 - 159

7 free ỎYS 61 296, 306, 328, 362, 436 508 and 1 b rid g e 1 9 5 -2 1 9

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 an d, ( ii) thí* n u m b e r an d 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 th e

m odels is id e n tir n l vvhatever th e tc m p lrite used

T h o n u m b e r o f fre e c y s te in e s in th e m o dels 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 v c 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 th e

m onom ers

3 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 th e

s tru c tu ra 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 ‘270 to 290 w as fo u n d loop T h e 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* rr la x i-d a lig n m e n t " u iìa lig n in g " fe w 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:

14 D a m B a c h D u o ìig , C h ic h c L a u r e n t

M o d e ll L L N E N E I E N R I L L L N E N E - - - I E N R I L

1 Q I D V L p - • 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 o d e l2 to p 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 v e rs io n o f M o d e lle r4 r u n n in g on a local S G I w o rk s ta tio n

T h e tw o m odels w ere sent to th e E v a l l 2 3 D s e r v e r

(h ttp ://b io s e rv c b s c n rs fr/v a lid h tm l) The e v a lu a tio n scores p ro v id e d e ta ile d

in íb rm a tio n fo r each re s id u e and each íra g m e n t o f th e m odel

F ro m th e E v a lu a tio n scores we can see th a t, g e n e ra lly th e s tr u c tu r e o f m odel2

is b e tte r th a n m o d e ll, except fo r re sid u e s 414 * 434 In th is p a r t, 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 o d e l2 was m o d ifie d a cco rdin g 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 a n d m odel3.top

w ere usecỉ to b u ild M o del3

M odel4 was th e o b ta in e d fro m M o del3 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 4 m odels and te m p la te 1Q ID are sho w n in th e tablo:

E v a l2 3 D V e r if y 3D P ro s a I I E v T r e e S F E

From th e above ta b le , vve no te th a t M odel 4 d is p la y s a good E v a l2 3 D score, but th a t M o đ e ll is th e b e tte r a cco rd in g to a ll o th e r scores

We th e re ib re selected M o d e l 1 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 7 1 - 7 8 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 d e lõ a li Piles m o d e is a li; m o d e lo to p were used to b u ild M o d e l5

E v a l 23D V e r i f y 3D P ro s a I I E v T r e e S F E

I m c s t i ị Ị o t i o n o f t h ỉ ’ p o s s ib lc r c i a t i o n s h i p betivcen. 1 5

lí Cỉin he S(*(*n th it a ll nl 3 v ;m ;itio n s o f m ođel 5 have h e tte r scorivs th a n M odel

1 ỉin ri M o d e l 4 Thi* hest is M n d rl 5/1 Ít was nam ed M odel 5

K x ỉim in a tio n n f M ođcl 5 (use In s ig h t II) show s th a t a v e ry bad loop

e o n ío r m a t i o n (knot) IS p r e s r n t íor í r a g m e n t 421 • 440 To rela x th is region we

" u n i l i ự i K H r t w o r r s u l u e s

Mocli-I r> F s V D s I) T Y N H Y R I V F c D

1 Q II) F N H R A S - - N L V w

1QID F N H R A S N L - v w

T h e changes in file m o đ e ll.a li saved in to new file : m o d e l6 a li K ile s m o d e l6 a li:

m o ck'l6 ỉo p werc* u s rđ to In n ld M odel 6

E v a l2 3 D V e r if y 3D P ro s a I I E v T re e S F E

U s in g I n s ig h t ll we can see th a i in M odel 6 th e s tra n g e loop c o n fo rm a tio n has

đ isa p p e a re đ T h is m eans th a t o u r loop re la x a tio n vvas w e ll done Hovvever, re s u lts

in ta b le show th a t a ll v a r ia tio n s o f M o d e l 6 are w orse th a n M o d e l 5 O n ly th e best o f the m , M o d c l 6/2, is n e a rly close to M o d e l 5 (e sp e cia lly by th e m o st im p o rta n t score, Prosa II) T h is in d ic a te s th a t we co u ld t r y to im p ro v e a g a in M o del 6/2 Hovvever, (hu* to tim e lim it a t io n th e m o d c lin g o f S A B prot was stopped

Krom a ll o f th is we chose M o del6 /2 as o u r M odel 6 fo r S A S p ro t (re s is ta n t in

A _loci) T h is m odel has c o rre c t backbone c o n fo rm a tio n and r e la tiv e ly good scores (P ic l)

3 3 M o d e llin Ị Ị o f o P r o te in f r o m B _ lo c i:

A s im ila r s tra te g v was used to b u ild a re fere nce 3D m o d e ỉ fo r B_loci esterases

U s in g b e lv u and seaview we chose fro m B_loci B8 (r e s is ta n t) fo r m o d e lin g ( It has h ig h e s t id e n tity score to S A S prot)

The B8 sequence was sent to BIOSERV and 3D-PSSM and the 1QID

s tru c tu ra l te m p la te was selected as p re v io u s ly done fo r A _loci

B8 w e re b u ilt w ith e ith e r one o f ‘2 ways:

1(3 D a m B a c h D u o n g , C h i chi* L a u r e n t

S ta n d a lo n e M o d e lle r4 : O u tp u t M odel M l/1 M l/2 , M l/.‘ỉ

B 10S E R V M o d e lle r4 : O u tp u t M o del M 2/1, M 2/2, M 2/3, M 2 /4

A ll m odels w ere se n t to th e E v a ll2 3 D se rve r

E v a l 23 D V e r if y 3D P ro s a I I E v T r e e S F E

1

i

From re s u lts in ta b le , th e b e st m odel is M odel 1/1 U s in g I n s i^ h t II vve can see

th a t the m odel has no a p p a re n t e rro r F rom a ll o f th is , we chose M o d e l 1/1 as o u r

M o del 1 fo r B8 (re s is ta n t in B _loci) T h is m odel has a e o rre c t backbone

c o n fo rm a tio n and r e la tiv e ly good scores

3.4 M o d e l l i n g a n d a n a l y s i s o f a l l P r o t e i n s i n A J o c i

U sin g seavievv we checked a ll diíTerences betvveen S A S p rot and cach IV o tc in

in A _loci T h e n , u s in g S P D lỉV ie v v e r, we m utatecỉ th e 31) modc*l o f S A S p rot to o h ta in

m odels o f a ll o th e r A _ lo ci p ro te in s

S A 8 p r o t

S A l p r o t 43 76 95 175 303 364 504 505 528

X S A 2 p ro t 81 295 493, 504

£ SA4prot 21 182 364 492 504 528 540

S A ỗ p r o t 39 95, 175, 227 250, 269 292 364 500 50-1, 52« 540

S A 7 p ro t 9 44 262 356 503, 504

B ru g e A 98 364 504 528

B ru g e B 98, 269, 292 364, 504 528

S la b l 177, 504, 540

■»«*

■c S la b 2 21, 98 269, 361, 364, 439 492 504, 528, 540

A

ị C ris s e 95, 152 175 250 292 337, :i64 492 493 504, 528 1

05 He te re n 21 98 269 361 364 492 504 528

T r a n s p r o t 56 261 500, 512

B le u e t 21 98 492 504

Ị n v e s t ig a t io n <ìf th c p o s s ib lc r c l a t io n s h ip betiveen. 17

S in c r th e a im o f w o rk is "T o in v e s tig a te the p o s s ib lc re ìa tio n betỉveen

s tr u c tu r a l m u ta tio n a n d resistance (>f Esterase to in s e c tic id e s ", so o u r in te re s t is

fo c u s in tf in m u ta tio n s , vvhich are observed o n ly in re s is ta n t p ro te in s F o r A_loci the.se m u tỉilio n s ;ire rc»si(liK*s 39» 4 ^ 44, 76, 81, 182, 227, 295, »303, 356 503 and 505

A ll m u ta tio n s wt»ro c a re fu lly analyzeci u s in g In s ig h t II AU m u ta tio n s are loca ted on t h i' su ría ce ot thí* p ro te in , and re a so n a b ly fa r fro m th e a c tiv e site None

o f th e m u ta tio n ap pea rs to have a s ig n iíìc a n t im p a c t on s tr u c tu r e and s ta b ilit y o f

th e p ro te in It IS likely that the mutations observed in the sequences o f the A loci esterases o f

resistani (ttlcx piptcns arc not responsible for the resistance.

O rig in al a;i p

3.5 M o d e llin ịi a n d a n a ly s is o f a l l P r o te in s in B lo c ì

U sin g st?aviuw Wí! check a ll d iffe rc n c e s hetw een B8 a n d each o f P ro te in in B_loci T h e n , u s in g S P D B V ievver, we m u ta te d the 3D m odel o f B8 t 0 o b ta in models

o f a ll o th e r B J o c i p ro te in s

B8

H B I 20 38 98 188 213 222 257 301 335 376 461 476

.8 B2 38, 62 256 26(5 461

B4 20 38 51 98 100 127 188 222 297 376 461 476

B5 20 38 54 98 127 176 188 222 376 461 476 511

B7 25 38 98 151 176 202 246 461

B r u g c A 20 26 38 93 98, 176, 188, 266, 297, 303, 407 461 476, 517

B r u g e B 20, 38 98 176, 222 266, 288, 297, 303, 376, 461 476 481 517

S la b l 25, 38 169 176 262, 459, 461, 500

S la b 2 20 38 98, 176 222 266, 297, 303, 376 460, 461 476

«9

e

c o

BSA1 20 38 54 93, 98 188 239, 266 297, 376, 461 476, 492 497

H e te re n 3 20, 38, 54 95, 98 176, 222 266, 297 303, 376, 460, 461, 476, 517

T r a n s 38, 256 461

B le u e t 25, 38 US9 176 297, 320 375, 461, 476 517

W ith th e sam e a im o f w o rk as in A _loci, o u r in te re s t is fo c u s in g in m u ta tio n s vvhich are h a p p e n in g o n ly in re s is ta n t p ro tc in s F o r B _loci these m u ta tio n s are residues 62 100 127 151 202 213 246 257, 301 335 an d 511

18 D a m B a c h I ) u o n g 9 C h ic h e L a u r e n t

A ll m u tn tio n s w ere c a r e fu lly a n n lyze d ư sing ỉn s ig h t I I

Pic 1 3D M odel o f te m p la te

As shovvn below stĩveral mutations are observgd f'or buried residues One of

th e m , p o s itio n 213 is p ro x im a l to a c tiv e site M o s t m u ta tio n s a re c o n se rva tive Hovvever, in p o s itio n 151, an exposed p o la r re s id u e is m u ta te d to a v a lin e In

p o s itio n 213, a s m a ll b u rie d a la n in e is m u ta te d to a rg in in e T h e c o rre s p o iu iin g

re s id u e in te m p la te is an a la n in e an d s ig n iíic a n t s te ric h in d ra n c e a p pea rs in the

a r g in in e m u ta n t T h is p o in t s h o u ld deserve fu r th e r s tu d ie s (Pic 2) S im ila r lv the

m u ta tio n to h is tid in e a t h u rie d p o s itio n 335 deserves f u r t h o r s tu đ ie s re ^ a rd in g

charge complementarities This position is moreover in close proximity to a loop

re g io n in w h ic h a lig n m e n t be tw e e n th e esterase sequence a n d th e te m p la te is

q u e s tio n a b le (P ic 3)

P ic 2 3D M o d e l o f B8 Pic 3 3D M«dt*l ()f B l w ith m u ta tio n s

I n r c s t i g a t i o n o f the po ssitìle r c l a t i o n s h i p betu ccn.

T h r i v l n r v ' i l t h o u ^ h m o s t m u t ỉ t i o n s o b s e r v i » ( l i n t h e s r < | u t * r u ' í as C ) f t h e |{ l o c i

r s t r r a s t s u f n s is ia n t ( u l r \ pipim N ỉip p e a r u n r e la tr d to the ri/s is ta n c e p h cn o m e n o n

t l m - r n i u t ỉ i t Ì I H Ì S ;it p o s i t i n n l ‘) l 2 1 M a n d 3 Í Ỉ 5 n i i v h i v c s t r u c t u r n l

im p a c t 1 'iir th iT m v o s ti^ ;n in n w ill tu* necessỉirv to mon* c*arc*fully ỉtn iilv /.c the

p o s s i M r i m p í ỉ c t o l t h r s c t l u v r m u t ỉ i t i o n s

N u m b c *r O r ií í in a l aa M u ta te d aa aa in t e m p la te C o m m e n t

—

4 C o n c lu sio n

1 28 th re e -d im e n s io n a l m odels o f in s e c tic id e se n sihle a n d in s e c tic id e

rc s is ta n t c u le x p ip ie n s esterases vvere b u ilt (11 fo r A _ lo ci and 14 fo r

B _loci)

2 A ll o f m u ta tio n s in A _ lo ci w h ic h are speciíìc to re s is ta n t p o p u la tio n s

a p p e a re d in s itfn ific a n t and p ro b a b ly do not p la y a n v rolí* in re s is ta n c e o f

Culex pipiens to insecticides.

• i M o st m u ta t io n s in IV loci also appeared u n re la te d to inse cti ci de re s is ta n c c , except m u ta tio n s in p o s itio n s 151, 213 and ‘M õ ( B l) T h e se m u tỉitio n s

m ig h t havt* s ig n iíìc a n t im p a ct on s tru c tu re o r s t a b ilit v o f th e e ste ra se in (he re s is ta n t p o p u lỉitio n M o re d e ta ile d d iscu ssio n o f th e p o ssib le ro le o f

theso m u ta tio n s in re s is tíin c e o f C u le x p ip ie n s to in s e c tic id e s s h o u ld avvait

f u r t h e r in v e s tig a tio n s

T h is w o rk vvas s u p p o rte d b y P r o je c t 2002 N ° 12735 C N R S - C N S T ( E n z y m e a n d P r o t e in T e c h n o lo g y L a b C B T , V N U )

R e fe r e n c e s

Culex p i p i e n s P h il T r a n s R Soc Lond B, 353(1998), pp 1707* 1711

2 VValsh et al ld ikn tific;itio n and <*haracterization of m utations m housíly (Musca domustica) achetvlcholinosterase involved in insecticide resistance Btochem J

3 5 9 ( 2 0 0 1 ) p p 175* 1 S I

2 0 D a m B a c h D u o n g , C h ic h e L a u r e n t

N G H IÊ N CỬ U S ơ BỘ K H Ả N Ă N G L IÊ N Q U A N G IỬ A Đ Ộ T B IÊ N

Đ à m B ạ c h D ư ơ n g

T ru n g tá m Công nghệ S in h học, D ạ i học Quốc g ia H à N ộ i

C h ic h e L a u r e n t

T ru n g tá m cấu trú c s in h hoá, U M R 5048, D R 13, M o n tp e llie r, C NRS, France

Loài m u ỗ i C u le x p ip ie n s có kh à nãng kh á n g th u ô c tr ừ côn tr ù n g nhò vào sự

th a v đổi tín h c h ấ t của p ro te in E ste ra se Việc n g h iê n cứu k h ả n ă ng liê n qu a n giữa các đột biến cấu trú c kh ô n g ia n của phân tử này và tín h k h á n g th u ố c đã được tiế n

h à n h tạ i T ru n g tâ m cấu tr ú c s in h hóa M o n tp e llie r (P há p)

M ô th ìn h phân tử 3D của 28 p ro te in th u ộ c nh óm E ste ra se đã được xây dựng

Q ua phân tíc h các đ ộ t biến cấu trú c , có th ể k ế t lu ậ n rằ n g m ọi đ ộ t b iế n tạ i nhóm A_loei đều k h ô n g gây ản h hương đến tín h c h ấ t của p ro te in Đ iê u n à y có ng hía là

nhữ ng đột biế n này k h ô n g liê n q u a n đến tín h k h á n g th u ố c của C u le x p ỉp ie n s

Đôi với nhóm B J o c i, có 3 đ ộ t biến cấu trú c tạ i các điế m 151, 213 và 335 có th ể gây ra sự th a y (tối tin h c h ấ t cua p ro te in T u y vậy, việ c tìm ra câu trả lới cu (Vi cùn g

vể sự liê n quan giữa nh ừ n g đột biên cấu trú c n à y với tín h k h a n g s th u ố c trừ côn

tr ù n g đòi hỏi p h ả i tiế n h à n h th ê m các n g h iê n cứu kh á c nữa

Ĩ A P C H ) K H O A H O C O H Q G H N , K H T N & C N , T X IX , N 04 2 0 0 3