Motion of Activated Myosin Heads as Detected by Fiber X-ray Diffraction

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(b) (c) myosin filament myosin molecule contraction (d) (e) tail head lever arm motor domain (a) actin filament Motion of Activated Myosin Heads as Detected by Fiber X-ray Diffraction Muscle contraction is caused by the interaction b e t w e e n t h e t w o c o n t r a c t i l e p r o t e i n s , a c t i n a n d myosin. Each of the proteins polymerizes to form filaments, and the contractile force is generated as t h e m y o s i n h e a d , w h i c h s t i c k s o u t o f t h e m y o s i n f i l a m e n t b a c k b o n e , e x e r t s a p u l l o n t h e a c t i n filam ent ( Fig. 1a , 1b ). The myosi n head conta ins all the components needed to exert force, including the actin-binding site and the ATP-binding pocket, while the rest of the myosin molecule (myosin tails) forms the backbone of the filament ( Fig. 1c ). The recen t cryst allog raphi c resul ts [1] showe d that the head is further divided into two parts, i.e. , the motor a n d t h e l e v e r a r m d o m a i n s ( F i g . 1 d ) . T h e c o n v e n t i o n a l t h e o r y f o r f o r c e g e n e r a t i o n m e c h a n i s m a s s u m e s t h a t t h e m o t o r d o m a i n g r a b s t h e a c t i n f i l a m e n t firmly (by making stereospecific interactions at the actin-myosin interface) and the lever arm makes a swing on it ( Fig. 1e ). I n t h e c o n v e n t i o n a l t h e o r y , t h e r e f o r e , t h e s t e r e o s p e c i f i c i n t e r a c t i o n s p l a y a c r u c i a l r o l e i n f o r c e g e n e r a t i o n . T h e q u e s t i o n i s h o w s u c h interactions can be detected. A potent method to ach iev e thi s is to rec ord X-r ay dif fra cti on pat ter ns f r o m a m u s c l e o r m u s c l e f i b e r s u n d e r v a r i o u s conditions. A regular array of molecules, such as that in a protein crystal, gives rise to a number of b r i g h t s p o t s o r l i n e s a t s p e c i f i c p o s i t i o n s i n t h e diffraction pattern. They are called reflections, and t h e i r p o s i t i o n s a n d i n t e n s i t i e s c a r r y i n f o r m a t i o n about the structure of the molecular array. In the case of muscle, the molecules of myosin and actin a r e a r r a n g e d p e r i o d i c a l l y i n h e l i c e s . T h i s arrangement gives rise to a number of line-shaped reflections (layer lines) across the long axis of the m u s c l e f i b e r s , a s i n t h e p a t t e r n s r e c o r d e d a t beamline BL45XU [2] (Fig. 2 ). Figure 2a shows the diff ract ion patt ern reco rded from stre tche d musc le fibers, in which the myosin and actin filaments do not overlap and therefore the myosin heads cannot in te ra ct wi th ac ti n. A fe w, we ak la ye r li ne re fl ec ti on s are seen, and they are based on actin repeat. The F i g . 1 . S t r u c t u r e o f t h e c o n t r a c t i l e machinery of muscle. ( a ) Structure of a sarcomere consisting of two sets of filaments (myosin and actin). ( b ) M e c h a n i s m o f c o n t r a c t i o n , w h i c h i s c a u s e d b y t h e s l i d i n g o f t h e f i l a m e n t s relative to each other. ( c ) Structure of a single myosin molecule. ( d ) Structure of a myosin head, consisting of motor and lever arm domains. ( e ) C o n v e n t i o n a l e x p l a n a t i o n o f t h e m e c h a n i s m o f c o n t r a c t i l e f o r c e p r o d u c t i o n , c a u s e d b y t h e s w i n g o f t h e l e v e r a r m d o m a i n o n t h e m o t o r d o m a i n b o u n d t o a n a c t i n f i l a m e n t i n a stereospecific manner. 25 H i r o y u k i I w a m o t o a , K a z u h i r o O i w a b a n d Tetsuro Fujisawa c (a) SPring-8 / JASRI (b) Communications Research Lab. (c) SPring-8 / RIKEN E-mail: iwamoto @ spring8.or.jp p a t t e r n i n F i g . 2 b w a s t a k e n a f t e r m y o s i n h e a d s ( p r e p a r e d b y s e v e r i n g t h e w h o l e m o l e c u l e w i t h pr ot ea se ) we re di ff us ed in to th e st re tc he d mu sc le fibers as in Fig. 2a in the absence of ATP. This is th e co nd it io n eq ui va le nt to ri go r mo rt em , in wh ic h m i t o c h o n d r i a n o l o n g e r p r o d u c e A T P , a n d s t r o n g stereospecific interactions are known to be formed between actin and myosin (this makes the muscle very stiff). In the diffraction pattern, the actin-based layer lines, notably the one at 5.9 nm (arrow), are strongly enhanced. The enhancement is caused by t h e m y o s i n h e a d s , w h i c h a r e b o u n d t o t h e a c t i n filament and now follow the actin repeat. It is also noticed that the peak of the reflection at 5.9 nm has shifted inwards. Addition of ATP to the muscle fibers creates a condition equivalent to contraction. The heads had b e e n c r o s s - l i n k e d t o a c t i n s o t h a t t h e y w o u l d n o t d i s s o c i a t e . I n s u c h m u s c l e f i b e r s t h e h e a d s hydrolyze ATP at a very high rate, because the two contractile proteins are held in close proximity. It is expected from the conventional theory that in such h i g h l y a c t i v a t e d m u s c l e f i b e r s , s t e r e o s p e c i f i c interactions are formed substantially and therefore t h e d i f f r a c t i o n p a t t e r n w o u l d b e m o r e o r l e s s l i k e that in Fig. 2b . However, the recorded pattern ( Fig. 2 c ) w a s v e r y s i m i l a r t o t h a t i n F i g . 2 a , i . e . , t h e pattern from naked actin filaments [3]. The results are exp lai ned onl y if the myo sin hea d is swi ngi ng a s a w h o l e i n t h i s h i g h l y a c t i v a t e d a c t i n - m y o s i n c o m p l e x , a n d l i t t l e s t e r e o s p e c i f i c i n t e r a c t i o n s a r e f o r m e d . I t i s p r o b a b l e t h a t t h e m o t o r d o m a i n o f m y o s i n p l a y s a m o r e d y n a m i c r o l e t h a n s i m p l y providing a scaffold for the lever arm swing. Fi g. 2. Bi rd ’ s- ey e vi ew s of th e di ff ra ct io n pa tt er ns re co rd ed fr om an ar ra y of ov er st re tc he d si ng le ra bb it skeletal muscle fibers. ( a ) Pattern recorded in the absence of exogenously introduced myosin head. Layer lines typical of bare actin filaments are observed. ( b ) Pattern recorded after myosin heads had been exogenously i n t r o d u c e d i n t h e a b s e n c e o f A T P . T h e a c t i n - b a s e d l a y e r l i n e s a r e s t r o n g l y e n h a n c e d b e c a u s e o f t h e stereospecific labeling of the actin filaments by the myosin heads. ( c ) Pattern recorded in the presence of ATP after exogenously introduced myosin heads had been cross-linked. The actin-based layer lines are as weak as those of bare actin filaments and there is little sign of stereospecific binding. The arrow indicates the layer line indexed to the 5.9 nm repeat of actin monomers. References [1] I. Rayment et al. , Science 261 (1993) 50. [2] T. Fujisawa et al. , J. Appl. Cryst. 33 (2000) 797. [3] H. Iwamoto, K. Oiwa, T. Suzuki and T. Fujisawa, J. Mol. Biol. 305 (2001) 863. 26