C r y s t a l S t r u c t u r e A n a l y s i s o f C a l c i u m P u m p o f S a r c o p l a s m i c R e t i c u l u m Na tu re us es io n g ra di en ts ac ro ss ce ll me mb ra ne s very efficiently. When cell membrane excites, ions c o m e i n t o c y t o p l a s m r a p i d l y f o l l o w i n g t h e i o n gradients. To restore the original resting state, the io ns mu st be pu mp ed ba ck . P- ty pe AT Pa se is a f a m i l y o f i o n t r a n s p o r t i n g A T P a s e s t h a t a r e r e s p o n s i b l e f o r e s t a b l i s h i n g s u c h i o n g r a d i e n t s . T h e y i n c l u d e N a + K + - A T P a s e , s a r c o p l a s m i c r e t i c u l u m ( S R ) C a 2 + - A T P a s e a n d g a s t r i c H + K + - ATPase among others. Of these SR Ca 2 + -ATPase is the simplest and the best studied member. The transport is thought to be achieved by changing the binding site from high affinity and facing cytoplasm (E1 form) to low affinity and facing the extracellular s i d e ( E 2 f o r m ) . T h e r e a c t i o n c y c l e c o m p l e t e s b y t r a n s p o r t i n g a n o t h e r t y p e o f i o n ( H + f o r C a 2 + - ATPase ) in the opposite direction. This process is called counter transport. When muscle contracts, large amounts of Ca 2 + stored in SR are released into muscle cells. Ca 2 + - ATPase in the SR membrane pumps Ca 2+ back into SR to relax muscle cells. Compared to channels, w h i c h c a n t r a n s f e r m i l l i o n s o f i o n s p e r s e c o n d , pumps work much more slowly. Ca 2 + -ATPase can transfer only 2 Ca 2 + per ATP-hydrolysis or 60 Ca 2 + p e r s e c o n d . T o m a k e t h e r e l a x a t i o n p r o c e s s e f f i c i e n t , S R m e m b r a n e i s f u l l o f C a 2 + - A T P a s e (more than 60% of the proteins in SR membrane). Therefore, Ca 2+ -ATPase is one of the most suitable m e m b r a n e p r o t e i n s f o r s t r u c t u r a l s t u d i e s . A l s o , b e c a u s e C a 2 + i s t h e m o s t u b i q u i t o u s f a c t o r f o r reg ul ati on of ce ll ul ar pro ce sse s, el uc id ati on of the m e c h a n i s m o f C a 2 + - A T P a s e h a s t r e m e n d o u s importance in both biological and medical aspects. We have been working on this ATPase using X- ray crystallography and determined its structure to F i g . 1 . S t r u c t u r a l c h a n g e s i n s a r c o p l a s m i c r e t i c u l u m C a 2 + - A T P a s e a c c o m p a n y i n g t h e disso ciatio n of Ca 2+ . Colou r chang es gradu ally from the N termi nus (blue ) to the C termi nus ( r e d ) . T w o p u r p l e s p h e r e s i n t h e m e m b r a n e d o m a i n r e p r e s e n t b o u n d C a 2+ i o n s . A d e n o s i n e moiety of ATP binds to the N-domain, whereas the catalytic site (phosphorylation site) Asp351 is located in the P-domain. The binding sites for thapsigargin ( TG, a potent inhibitor) is also shown. 11 Ca + 2 + Ca - 2 + A A N P N P 2Ca 2 + TG D351 N N C C ATP D351 lumen SR membrane cytoplasm F i g . 1 Fig. 1 Fig. 2 F i g . 2 . R e a r r a n g e m e n t o f t r a n s m e m b r a n e h e l i c e s v i e w e d f r o m t h e s i d e o p p o s i t e t o F i g . 1 . Cylinder s represe nt α -helices . Double circles show pivot positio ns for the M2 and M5 helices . Arrows indicate the directions of movements during the change from Ca 2+ -bound to unbound form. F i g . 3 F i g . 3 2.6 Å resolution for the Ca 2 + -bound (E1 Ca 2 + ) form [ 1 ] a n d t o 3 . 1 Å r e s o l u t i o n f o r a C a 2 + - u n b o u n d ( E 2 ( T G ) ) f o r m s t a b i l i z e d b y t h a p s i g a r g i n , a v e r y p o t e n t i n h i b i t o r [ 2 ] ( ) . D i f f r a c t i o n d a t a f r o m t h e E 2 ( T G ) c r y s t a l s w e r e c o l l e c t e d a t b e a m l i n e B L 4 4 X U u s i n g i m a g i n g p l a t e s o f 4 0 0 0 × 4 0 0 0 pixels. This was essential because one dimension o f t h e u n i t c e l l w a s a s l a r g e a s 6 0 0 Å , y e t t h e diffraction spots went out to 3.0 Å resolution. S R C a 2 + - A T P a s e c o n s i s t s o f a s i n g l e polypeptide chain of 110 kDa. It comprises 3 (A, N an d P) cy to pl as mi c do ma in s an d 10 tr an sm em br an e α -helices. The differences in structure between the C a 2 + - b o u n d a n d u n b o u n d f o r m s a r e g l o b a l a n d very large ( ). In the trans membr ane regio n, complicated movements of transmembrane helices are obs erv ed. In par tic ula r, it is sur pri sin g to see l a r g e ( ~ 5 . 5 Å ) m o v e m e n t s o f t h e M 3 a n d M 4 helices normal to the membrane ( ). Because t h e M 4 h e l i x i s a k e y c o m p o n e n t o f t h e t r a n s m e m b r a n e C a 2 + - b i n d i n g s i t e s ( ) , i t i s cl ea r th at su ch pi st on -l ik e mo ve me nt s wi ll ab ol is h th e bi nd in g of Ca 2+ at si te II . Al so , th e un wo un d part of M6, another key helix in Ca 2 + coordination, r o t a t e s n e a r l y 9 0 t o d e s t r o y s i t e I ( ) . I t i s n o t o b v i o u s w h y s u c h l a r g e m o v e m e n t s o f t h e transmembrane helices are necessary. Homology m o d e l l i n g o f t h e c a t i o n b i n d i n g s i t e s o f a r e l a t e d p u m p , N a + K + - A T P a s e , s u g g e s t s t h a t s u c h m o v e m e n t s a r e r e q u i r e d f o r a s s u r i n g t h e r e l e a s e of one type of ion and binding of the other type of ions that are counter-transported at the same time [3]. All three cytoplasmic domains show very large domain movements, keeping the structure of each d o m a i n v i r t u a l l y u n c h a n g e d . I n t h e C a 2 + - b o u n d form, they are widely separated but gather to form a c o m p a c t h e a d p i e c e i n t h e C a 2 + - u n b o u n d f o r m . 12 Obviously these movements are linked with those of th e tr an sm em br an e he li ce s. Si nc e no AT P or ph os ph ory la tio n is req ui red for tra ns iti on be twe en t h e s e t w o s t a t e s , t h e r m a l e n e r g y m u s t b e a b l e t o c a u s e s u c h l a r g e m o v e m e n t s . T h e c l o s e References [1] C. Toyoshima, M. Nakasako, H. Nomura and H. Ogawa, Nature 405 (2000) 647. [ 2 ] C . T o y o s h i m a a n d H . N o m u r a , N a t u r e 4 1 8 (2002) 605. [3] H. Ogawa and C. Toyoshima, Proc. Natl. Acad. Sci. USA. 99 (2002) 15977. Fig. 3. Schematic diagram of the Ca 2 + binding sites of the Ca 2 + ATPase in the Ca 2 + - - - bound and unbound forms. The arrows in the left panel (+ Ca 2 + ) show the directions of move ment s acco mpa nyin g the diss ocia tion of Ca 2+ . Oxyg en atom s are sho wn in red, nitrogen in cyan, carbon in orange. Ca 2+ ions are thought to enter into the binding sites through E309. First Ca 2 + binds to site I and the second to site II. The binding of the second Ca 2+ causes conformation changes that result in the hydrolysis of ATP. Chikashi Toyoshima and Hiromi Nomura The University of Tokyo E-mail: ct@iam.u-tokyo.ac.jp a s s o c i a t i o n o f t h e 3 c y t o p l a s m i c d o m a i n s w i l l r e s t r i c t t h e t h e r m a l m o v e m e n t s o f t h e t r a n s m e m b r a n e h e l i c e s , a n d w i l l l i m i t t h e d e l i v e r y o f A T P t o t h e c a t a l y t i c s i t e i n t h e P - domain ( D351 in Fig. 1 ). 13
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