X - r a y C r y s t a l l o g r a p h i c A n a l y s i s o f X e n o b i o t i c E x p o r t e r P r o t e i n s o f E s c h e r i c h i a c o l i Fig. 1. Xenobiotic exporters world. A mammalian cell and a bacterial cell are illustrated. The xenobiotic exporter proteins, also known as multidrug efflux transporters, pump a wide variety of n o x i o u s c o m p o u n d s o u t o f a c e l l a c r o s s t h e c e l l m e m b r a n e . T h e y p l a y a n i m p o r t a n t r o l e i n m u l t i d r u g resistance of pathogenic bacteria and cancer cells [1]. Genomic sequence analysis has revealed the pr es en ce o f ma ny p ut at iv e xe no bi ot ic e xp or te r ge ne s i n c h r o m o s o m e s f r o m m i c r o o r g a n i s m s t o m a m m a l i a n ce ll s. Th er e ar e 19 tr an sp or te r ge ne s in Es ch er ic hi a c o l i , w h i c h a c t u a l l y m e d i a t e t h e e f f l u x o f s o m e drugs and toxic compounds. Among them, AcrB is co ns ti tu ti ve ly ex pr es se d an d is a ma jo r co nt ri bu to r f o r i n t r i n s i c d r u g r e s i s t a n c e o f E . c o l i . [ 2 ] . A c r B cooperates with a membrane fusion protein, AcrA, a n d a n o u t e r m e m b r a n e c h a n n e l , T o l C . T h i s A c r A B - T o l C s y s t e m e x p o r t s a n u n u s u a l l y w i d e va ri et y of no xi ou s co mp ou nd s, e. g. dy es , de te rg en ts and most lipophilic antibiotics directly out of the cell, by pa ss in g th e pe ri pl as mi c sp ac e dr iv en by pr ot on mo ti ve fo rc e. Ac rB is th e mo st im po rt an t co mp on en t o f t h i s s y s t e m ; i t m e d i a t e s e n e r g y c o u p l i n g a n d determines substrate specificity ( Fig. 1 ). Fir st of all we clo ned a his tid ine -ta gge d Acr B i n t o a m u l t i c o p y p l a s m i d a n d o v e r p r o d u c e d i t i n E . coli . Then, AcrB was solubilized and purified in the presence of a non-ionic detergent, n-dodecyl- β - D - m a l t o p y r a n o s i d e . U s i n g t h e v a p o r d i f f u s i o n method, we obtained crystals of AcrB (space group R 32), and we employed the multiple isomorphous replacement method to solve the phase problem. A mo le cu la r mo de l wa s bu il t an d re fi ne d at a re so lu ti on of 3.5 Å. All the diffraction data used for structure determination were collected at beamline BL44XU (Institute for Protein Research, Osaka Univ. ) [3]. A c r B c o m p r i s e s a t r i m e r o f 1 , 0 4 9 - r e s i d u e p r o t o m e r s ( F i g s . 2 ( a ) , 2 ( b ) ) , a n d i t s a p p e a r a n c e r e s e m b l e s t h a t o f a j e l l y f i s h w i t h a t h r e e - f o l d s y m m e t r y a x i s p e r p e n d i c u l a r t o t h e m e m b r a n e p l a n e . I t c o m p r i s e s a n e x t r a - m e m b r a n e ( p e r i p l a s m i c ) h e a d p i e c e a p p r o x i m a t e l y 5 0 Å × 1 0 0 Å , a n d a transmembrane region of dimensions 70 Å × 80 Å. T h e h e a d p i e c e i s d i v i d e d i n t o t w o s t a c k e d p a r t s : the upper and lower parts are 30-Å and 40-Å thick, respectively. The side view of the upper part has a tr ap ez oi da l ap pe ar an ce th at is ~ 70 -Å wi de at th e b o tt o m a n d ~ 4 0 - Å w i d e a t th e to p . Vi e w e d fr o m above, the upper part is open like a funnel, with an internal diameter of 30 Å. This funnel connected by cytoplasmic membrane MDR drug drug VMAT MRP conjugated drug intracellular vesicles Mammalian Cell Bacterial Cell drug drug LmrA TetA MacAB Bmr NorA cytoplasmic membrane drug periplasm outer membrane MexAB-OprM secondary transporter secondary transporter ABC transporter ATP ATP ATP ADP+Pi ADP+Pi ADP+Pi AcrAB-TolC H + H + ABC transporter H + 8 a p o r e , l o c a t e d b e t w e e n t h e h e a d p i e c e s o f t h e t h r e e p r o t o m e r s t o a l a r g e c e n t r a l c a v i t y a t t h e i n t e r f a c e o f t h e h e a d p i e c e a n d t r a n s m e m b r a n e region of the protomers. Within the membrane, the protomers are arranged like a ring with a 30-Å hole b e t w e e n t h e m , w h i c h m a y b e f i l l e d w i t h p h o s p h o l i p i d s . The protomers appear to be interlocked by three ha ir pi n- li ke st ru ct ur es ca . 35 Å lo ng , ea ch pr ot ru di ng f r o m o n e p r o t o m e r i n t o t h e n e x t p r o t o m e r . T o a c h i e v e t h i s m u t u a l i n s e r t i o n , t h e p r o t o m e r s a r e tightly packed as a trimer. Between every protomer in th e pe ri pl as mi c do ma in , th er e ar e th re e ve st ib ul es tha t ope n wid e int o the per ipl asm and lea d to the central cavity inside the headpiece, and a substrate l o c a t e d i n t h e p e r i p l a s m i c s p a c e o r o n t h e m e m b r a n e plane might gain access to the cavity through any of these vestibules ( Fig. 3 ). The funnel opened at the top of the AcrB trimer seems to be a perfect fit for the proximal end of the TolC protein ( Fig. 4(a) ). The crystal structure of TolC protrudes 100 Å into the periplasm [4], thus, the sum of the periplasmic l e n g t h o f A c r B a n d T o l C i s ~ 1 7 0 Å , w h i c h i s j u s t en ou gh to cr os s th e pe ri pl as mi c sp ac e, in di ca ti ng t h a t A c r B a n d T o l C m i g h t d i r e c t d o c k w i t h e a c h other ( Fig. 4(a) ). Fig. 2. Structure of AcrB. (a) Side view of a ribbon representation. Three protomers are individually c o l o u r e d . T h e N - t e r m i n a l a n d C - t e r m i n a l h a l v e s o f t h e p r o t o m e r s a r e d e p i c t e d b y d a r k a n d p a l e colours, respectively. The extra-membrane ( periplasmic ) headpieces are at the top. (b) Top view of a ribbon representation. The protomers are individually colored as in (a) . F i g . 3 . A c u t a w a y v i e w d i s p l a y i n g t h e s o l v e n t - a c c e s s i b l e s u r f a c e o f A c r B . T h e f r o n t p r o t o m e r i s r e m o v e d . T h e y e l l o w areas of the surface are colored according to residues belonging to the pore helix. 9 I n n e r M e m b r a n e T o l C D o c k i n g D o m a i n s ( ~ 3 0 Å ) P o r e D o m a i n s ( ~ 4 0 Å ) T r a n s m e m b r a n e D o m a i n s ( ~ 5 0 Å ) ( b ) ( a ) Fig. 4. (a) Proposed model of the AcrB-AcrA-TolC complex. (b) The schematic mechanism of xenobioic export mediated by AcrAB-TolC system. The TolC structure dock with the AcrB. (b) (a) References [1] Okusu et al. , J. Bacteriol. 178 (1996) 306. [ 2 ] N i k a i d o e t a l . , J . M o l . M i c r o b . B i o t e c h n o l . 3 (2001) 215. [ 3 ] S a t o s h i M u r a k a m i , R y o s u k e N a k a s h i m a , E i k i Y a m a s h i t a a n d A k i h i t o Y a m a g u c h i , N a t u r e 4 1 9 (2002) 587. [4] V. Koronakis et al. , Nature 405 (2000) 914. Satoshi Murakami a,b,c , Ryosuke Nakashima a and Akihito Yamaguchi a,c (a) Osaka University (b) Japan Science and Technology Corporation, PRESTO (c) Japan Science and Technology Corporation, CREST E-mail: mura @ sanken.osaka-u.ac.jp X e n o b i o t i c s u b s t r a t e s f r o m t h e c y t o p l a s m o r i n n e r l e a f l e t o f t h e m e m b r a n e a r e t r a n s p o r t e d through every protomer across the membrane and c o l l e c t i n t h e c e n t r a l c a v i t y . S u b s t r a t e s f r o m t h e peri plas mic surf ace of the inne r memb rane or the outer leaflet of the membrane also gain access in the cavity through the vestibules. It appears, then, th at su bs tr at es in th e ca vi ty ar e ac ti ve ly ex po rt ed th ro ug h th e po re in to th e fu nn el an d th en on in to the TolC tunnel. The energy for active transport is captured in th e tr an sm em br an e re gi on an d tr an sm it te d to th e ext ra- mem bra ne por e by a rem ote con for mat ion al coupling ( Fig. 4(b) ). This beautiful AcrB structure pro vid es a gre at dea l of imp orta nt ins igh t int o the f u n c t i o n o f m u l t i d r u g r e s i s t a n c e m e d i a t e d b y xenobiotic exporters. This structure is not only the first structure of a multidrug exporter, but also the f i r s t a t o m i c - l e v e l s t r u c t u r e o f a m e m b r a n e t r a n s p o r t e r t h a t c o u p l e s w i t h p r o t o n t r a n s l o c a t i o n a c r o s s t h e membrane. Therefore, we believe that this work is a r e a l m i l e s t o n e f o r u n d e r s t a n d i n g a c t i v e m e m b r a n e t r a n s p o r t m e c h a n i s m s b a s e d o n m o l e c u l a r s t r u c t u r e . AcrB Inner Membrane AcrA AcrA ToIC Outer Membrane AcrA AcrA AcrB Vestibule Vestibule Substrate Substrate Substrate Substrate Cytoplasm Central Cavity Inner Membrane Funnel Pore ToIC Periplasm + H + H Outer Membrane Medium Substrate 10
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