(a) (b) 118.7 51.9 D313Y D107E A414V G426A A427V R124A N433D A449V 51.9 T h e b a c t e r i a l f l a g e l l u m c o n s i s t s o f a r o t a r y m o t o r a n d a h e l i c a l p r o p e l l e r b y m e a n s o f w h i c h bacteria swim. The flagellar motor is at the base of the flagellum and drives its rotation at around 300 Hz. The long helical propeller is called the flagellar f i l a m e n t , a n d i t g r o w s u p t o a r o u n d 1 5 μ m b y polymerization of a single kind of protein, flagellin. The fil ame nt is not sim ply a rig id hel ica l pro pel ler but switc hes its helic al form from a left- hande d to right-handed ones upon quick reversal of the motor r o t a t i o n , b y w h i c h a b u n d l e o f s e v e r a l f i l a m e n t s formed during a straight run falls apart quickly, and this makes the bacterial cell tumble and change its swimming direction for chemotaxis or thermotaxis. I t i s n o t a s i m p l e q u e s t i o n t o a n s w e r h o w chemically identical molecules can build curve and t w i s t e d t u b u l a r s t r u c t u r e s . I t w a s p r o p o s e d b y A s a k u r a [ 1 ] t h a t v a r i o u s h e l i c a l f o r m s o f t h e f l a g e l l a r f i l a m e n t a r e p r o d u c e d b y t w o t y p e s o f p r o t o f i l a m e n t s w i t h d i s t i n c t r e p e a t d i s t a n c e s a n d l a t e r a l p a c k i n g m o d e s i n e a c h o f t h e 1 1 p r o t o f i l a m e n t s t h a t f o r m t h e t u b u l a r s t r u c t u r e . E l e c t r o n cryom icros copic struc ture analy sis of two types of straight flagellar filaments, the L- and R-type, each made of either L- or R-type protofilaments, showed t h a t t h e o v e r a l l s t r u c t u r e o f t h e f l a g e l l i n s u b u n i t does not chan ge much in the two pack ing mode s [2]. X-ray fiber diffraction revealed that the L- and R - t y p e p r o t o f i l a m e n t s h a v e a s u b u n i t r e p e a t distance of 52.7 Å and 51.9 Å, respectively [3]. By simple mechanical simulation to build a tube with a m i x t u r e o f t w o t y p e s o f p r o t o f i l a m e n t s , t h i s difference of 0.8 Å and a small change in the lateral p a c k i n g e x p l a i n e d t h e c u r v a t u r e a n d t w i s t o f v a r i o u s h e l i c a l f o r m s o f t h e f i l a m e n t o b s e r v e d b y d a r k - f i e l d o p t i c a l m i c r o s c o p y , d e m o n s t r a t i n g t h a t th is su bt le di ff er en ce is ph ys ic al ly me an in gf ul [3 ]. A Mechanical Switch Found in the Bacterial Flagellar Protofilament Structure Fig. 1. Crystal structure of the flagellin fragment F41 (a) and docking of the protofilament model into an EM density map (b) . We then carried out X-ray crystallographic structure a n a l y s i s o f m o n o m e r i c f l a g e l l i n t o f i n d o u t w h a t structural changes would be responsible. C r y s t a l l i z a t i o n o f f l a g e l l i n , h o w e v e r , w a s impossible because flagellin has a strong tendency to polymerize into filaments. We therefore cleaved off 52 NH 2 - and 44 COO H-t erm ina l res idu es fro m 494 amino acid residues of Salmonella flagellin to suppress the strong polymerization ability. This 41 kDa fragment named F41 formed crystals but they we re on ly ab ou t 5 μ m th ic k. It wo ul d ha ve be en i m p o s s i b l e t o c o l l e c t d i f f r a c t i o n d a t a f r o m s u c h a t h i n c r y s t a l w i t h o u t a h i g h l y b r i l l i a n t X - r a y b e a m 21 N150 Q146 I155 L143 N132 K160 Q128 R124 K135 (a) (b) Fadel A. Samatey a , Katsumi Imada a,b and Keiichi Namba a,b (a) Protonic NanoMachine Project, ERATO, JST (b) Osaka University E-mail: keiichi @ fbs.osaka-u.ac.jp from SPrin g-8 beam line s. Annea ling of froz en c r y s t a l s w a s a l s o e s s e n t i a l f o r c o l l e c t i n g h i g h - resolution data. We collected a set of multiple anomalous diffraction data to 2.0 Å resolution at one of the RIKEN beamline BL45XU , which was designed based on the trichromatic concept [4]. T h e s t r u c t u r e o f F 4 1 h a d t h r e e d o m a i n s . The one with an elongated shape and made of three α -helices and a β -hairpin formed an axial a r r a y w i t h v e r y i n t i m a t e i n t e r a c t i o n s i n t h e cr ys ta l pa ck in g. Th e re pe at d is ta nc e wa s 51 .9 Å , e x a c t l y t h e s a m e v a l u e a s t h a t o f t h e R - t y p e p r o t o f i l a m e n t ( F i g . 1 a ) . T h e a t o m i c m o d e l o f th is ar ra y of F4 1 fi tt ed ve ry we ll wh en do ck ed i n t o a d e n s i t y m a p o f t h e R - t y p e f i l a m e n t ded uce d by ele ctr on cry omi cro sco py ( Fig . 1b ), i n d i c a t i n g t h a t t h i s i s t h e a t o m i c m o d e l o f t h e protofilament [4]. Because the flagellar protofilament is a kind of me ch an ic al sw it ch th at sw it ch es be tw ee n th e tw o states with distinct repeat distances, we carried out a c o m p u t a t i o n a l s i m u l a t i o n o f e x t e n d i n g t h e p r o t o f i l a m e n t m o d e l t o i d e n t i f y t h e s w i t c h r e g i o n w i t h i n t h e m o l e c u l a r s t r u c t u r e . W e t r e a t e d t h r e e ax ia ll y al ig ne d su bu ni ts as a pr ot of il am en t mo de l, a n d w h i l e f i x i n g C α a t o m s o f t h e t o p s u b u n i t , w e t r a n s l a t e d t h e b o t t o m s u b u n i t b y 0 . 1 i n t h e direction extending the protofilament model and did e n e r g y m i n i m i z a t i o n o f t h e w h o l e m o d e l w i t h C α atoms of the top and bottom subunits all fixed. We repeated this procedure up to an extension of 6 Å Å Å . U p t o a n e x t e n s i o n o f 4 . 5 Å , t h e m i d d l e s u b u n i t showed only gradual elongation in every portion of domain D1, but from 4.5 to 4.7 Å , the β -hairpin in d o m a i n D 1 s h o w e d a n a b r u p t j u m p i n i t s c o n f o r m a t i o n ( F i g . 2 ) . W e t h u s i d e n t i f i e d t h a t t h i s β - h a i r p i n st ru ct ur e, wh ic h pa ir s wi th th re e lo ng α -h el ic es to f o r m d o m a i n D 1 , i s t h e m e c h a n i c a l s w i t c h i n f l a g e l l i n t o p r o d u c e t h e t w o d i s t i n c t p r o t o f i l a m e n t states with slightly different repeat distances [4]. F i g . 2 . S i m u l a t e d e x t e n s i o n o f t h e protofilament model. (a) Superposition of diff eren t stag es at ever y 0.5 exte nsio n. ( b ) M a g n i f i e d i m a g e o f t h e β - h a i r p i n por tio n of dom ain D1. Col ors rep res ent 4.5 Å (cyan) and 4.7 Å (pink) extension. References [1] S. Asakura, Adv. Biophs. 1 (1970) 99. [2] Y. Mimori-Kiyosue et al. , Proc. Nat. Acad. Sci. USA 93 (1996) 15108. [3] I. Yamashita et al. , Nature Struct. Biol. 5 ( 1998) 125. [ 4 ] F . A . S a m a t e y , K . I m a d a , S . N a g a s h i m a , F . Vo nd er vi sz t, T. Ku ma sa ka , M. Ya ma mo to an d K. Namba, Nature 410 (2001) 331. 22
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