ATOMIC STRUCTURE OF BOVINE RHODOPSIN: A SEVEN TRANSMEMBRANE RECEPTOR

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ATOMIC STRUCTURE OF BOVINE RHODOPSIN: A SEVEN TRANSMEMBRANE RECEPTOR S e v e n t r a n s m e m b r a n e ( 7 T M ) r e c e p t o r i s a major protein family. The interest in 7TM receptor is growing not only due to its biological significance but also because of its practical importance in the post-genomic era. It could be a potential target for devel opin g thera peuti cs for diver se ailm ents, such as gen era l pai n, all erg y, cir cul ati on dis ord er, CNS ( c e n t r a l n e u r a l s y s t e m ) d i s o r d e r a n d A I D S . I n vertebrates, the 7TM receptor family is believed to contain at least 600 members, wherein 370 odorant r e c e p t o r s h a v e b e e n i d e n t i f i e d i n t h e h u m a n g e n o m e . 7 T M r e c e p t o r s b i n d a v a r i e t y o f l i g a n d c l a s s e s : r e t i n a l s , b i o - o r g a n i c a m i n e s , o d o r a n t s , a m i n o a c i d s a n d t h e i r d e r i v a t i v e s , p e p t i d e s , pro tei ns, nuc lei c aci ds and the ir der iva tiv es, li pid s an d th ei r de ri va ti ve s, an d gl yc op ro te in s. Ma ny of these 7TM receptors are primary cellular acceptors of environmental stimuli for many processes, such a s v i s i o n , o d o r a n t s e n s i n g , n e u r o - t r a n s m i s s i o n , hormone regulations and chemotaxis. F i g . 1 . S t r u c t u r e o f b o v i n e r h o d o p s i n . C a r t o o n m o d e l o f b o v i n e r h o d o p s i n s t r u c t u r e w i t h t r a n s p a r e n t m o l e c u l a r s u r f a c e a c c o m p a n i e d w i t h p u t a t i v e l i p i d b i - l a y e r m o d e l . S e v e n t r a n s - me mb ra ne he li ce s ar e co lo re d in rainbow from α -helix I to VII with additional short helix VIII in red. B o u n d 1 1 - c i s - r e t i n a l s h o w s b a l l m o d e l i n y e l l o w . F o u r s h o r t β - strands of N-terminal domain are in sky-blue. All loops are in pink wire with blue colored sugars. I n c e l l u l a r s i g n a l t r a n s d u c t i o n , t h e m o l e c u l a r m e c h a n i s m o f t h e 7 T M r e c e p t o r i s c o m m o n l y tri gge red via the act iva tio n of a lim ite d num ber of G T P - b i d i n g p r o t e i n s . F o r t h i s r e a s o n , t h e 7 T M r e c e p t o r i s o f t e n c a l l e d a s G - p r o t e i n c o u p l e d r e c e p t o r ( G P C R ) . T h e m o l e c u l a r s t r u c t u r e o f t h e 7 T M receptor is similar in its seven transmembrane helix b u n d l e ; f u r t h e r m o r e i t s d o m i n a n t s u b - f a m i l y , rh od op si n fa mi ly me mb er s, sh ar es hi gh ly co ns er ve d residues in amino-acid sequences. Thus, most of the 7TM receptors are thought to be activated via a co mm on me ch an is m. To el uc id at e th e mo le cu la r mechanism of the 7TM receptor, the experimental atomic model of a visual pigment, rhodopsin in the r e t i n a , r e c e n t l y h a s b e e n d e t e r m i n e d u s i n g s y n c h r o t r o n X - r a y c r y s t a l l o g r a p h y a t b e a m l i n e BL45XU by the MAD method, while low-resolution m o d e l s h a d b e e n p r e v i o u s l y o b t a i n e d u s i n g e l e c t r o n d i f f r a c t i o n m e t h o d s f o r f r o g o r b o v i n e rhodopsin [ Schertler et al. ]. The first structure of the 7TM receptor d e t e r m i n e d f o r b o v i n e r h o d o p s i n w h i c h p r e s e r v e d 1 1 - c i s - r e t i n a l , a v i t a m i n A d e r i v a t i v e , r e v e a l e d s e v e r a l i n t e r e s t i n g fe at ur es wi th th e co mm on 7T M bu nd le in a n i n a c t i v e s t a t e ( F i g s . 1 a n d 2 ) [ 1 ] . A d d i t i o n a l a m p h i p h i l i c s h o r t h e l i x V I I I i s located directly adjacent to the 7 th TM helix on the putative membrane surface ( Fig. 3 ). The helices VII and VIII nearly form a right a n g l e , s u p p o r t e d b y t h e a r o m a t i c r i n g s t a c k i n g o f t h e c o n s e r v a t i v e T y r 3 0 6 a n d P h e 3 1 3 ( F i g . 4 D ) [ 2 ] . T h e N - t e r m i n a l d o m a i n f o l d s c o m p a c t l y i n a t w o - l a y e r e d s t r u c t u r e ( F i g . 3 ) . T h e t w o l a y e r s a r e composed of an N-terminal region and the long loop between helices IV and V with an S - S b r i d g e o f t h e c o n s e r v e d C y s 1 8 7 a n d C y s 1 1 0 a t t h e e n d o f h e l i x I I I ( F i g . 4 A ) . F i g . 2 . C r y s t a l p a c k i n g o f b o v i n e r h o d o p s i n . A ) P r o j e c t i o n i m a g e o f crystal packing onto b - c plane. Unit cell is indicated by square with the origin ( 0 ) a n d l a t t i c e ( b a n d c ) . B ) D i m e r i n a c r y s t a l l o g r a p h i c a s y m m e t r i c u n i t w i t h w a t e r a c c e s s i b l e s u r f a c e c o l o r e d b y electrostatic potential, positive in blue and n e g a t i v e i n r e d . L a b e l s o f N a n d C indicate the direction of N - and C -termini, respectively. Each molecule in the dimer packs in up side down artificially. Conversely, cytoplasmic loops almost open to form the putative interface with the G-protein, transducin [ 3 ] . M o s t o f t h e h i g h l y c o n s e r v e d a m i n o - a c i d r e s i d u e s a m o n g r h o d o p s i n f a m i l y m e m b e r s f o r m n o n - c o v a l e n t b o n d s t o s u p p o r t t h e i n a c t i v e f o r m . Th e GP CR mo ti f of Gl u 134 -A rg 135 -T yr 136 in te ra ct s with Glu 247 and Thr 251 of helix VI at the cytoplasmic end of helix III ( Fig. 4B ). Asn 55 , Asp 83 , Asn 78 and T r p 1 6 1 a r e s h o w n t o c o n s t i t u t e h y d r o g e n - b o n d n e t w o r k s ( F i g . 4 C ) [ 4 ] . M a n y p a r t s o f t h e s e v e n transmembrane helices are irregular, due to many e x i s t i n g α - h e l i x - d e s t r u c t i v e G l y a n d P r o r e s i d u e s Fig. 3. Stereo view of rhodopsin cartoon model. A B c o n t a i n e d i n t h e 7 T M h e l i c e s ( F i g . 3 ) . I n f a c t , α - helices II, VI and VII are clearly kinked, and, in helix VI I, se ve ra l re si du es ar ou nd Ly s 29 6 th at bi nd s 11 - c i s - r e t i n a l v i a t h e S c h i f f b a s e f o r m a 3 1 0 h e l i x . Th es e be nt -h el ic es fo rm a sp ac io us ca vi ty th at is l a r g e r th a n w h i c h i s n e e d e d to a c c o m m o d a te th e b o u n d r e t i n a l , w h i l e t h e c y t o p l a s m i c s i d e o f t h e he li ce s is ti gh tl y bu nd le d ( Fi g. 3 an d Fi g. 4A ) [6 ] . T h e c o n f i g u r a t i o n o f t h e r e v e r s e a g o n i s t , 1 1 - c i s - retinal, is bow-shaped in 6s- cis , 12s- trans, and anti- C=N of the Schiff base with a direct salt bridge of Asp 113 ( Fig. 5 ). Fig. 5. Closed up structure of bound retinal vicinity. A ) E x p e r i m e n t a l e l e c t r o n d e n s i t y m a p w i t h t h e c u r r e n t model at 3.3 Å. B) 2FoFc and omit FoFc map c a l c u l a t e d u s i n g m o d e l phasing at 2.8 Å. C ) C a r t o o n m o d e l o f r e t i n a l vicinity in stereo. T h e a t o m i c s t r u c t u r e o f i n a c t i v e r h o d o p s i n c o n s i s t e n t l y r e i n f o r c e s e x p e r i m e n t a l r e s u l t s o b t a i n e d t o t h i s p o i n t , i n c l u d i n g v i s u a l d i s e a s e s , y i e l d i n g m a n y c l u e s f o r f u r t h e r s t u d y o f t h e mo le cu la r me ch an is m of 7T M re ce pt or ac ti va ti on , a s w e l l a s t h e m o l e c u l a r b a s i s o f c o l o r v i s i o n . Further studies on the activated form of rhodopsin will elucidate novel features of the molecular basis o f t h e 7 T M r e c e p t o r , w h i l e t h e s t r u c t u r e o f t h e i n a c t i v e f o r m i s w e l l - s u i t e d t o t h e d e s i g n o f antagonists. Masashi Miyano SPring-8 / RIKEN E-mail: miyano @ spring8.or.jp References [1] J. Bockaert and J. P. Pin, EMBO J. 18 (1999) 1723. [2] V. M. Unger et al. , Nature 389 (1997) 203. [3] T. Okada et al. , J. Struct. Biol. 130 (2000) 73. [ 4 ] K r z y s z t o f P a l c z e w s k i , T a k a s h i K u m a s a k a , T e t s u y a H o r i , C r a i g A . B e h n k e , H i r o y u k i M o t o s h i m a , B r i a n A . F o x , I s o l d e L e T r o n g , D a v i d C . T e l l e r , T e t s u j i Oka da, R o n a l d E . S t e n k a m p , M a s a k i Y a m a m o t o and Masashi Miyano, Science 289 (2000) 739. A B C B A C Fig. 4. Structure of highly conserved residues among 7TM receptors.