Crystal Structure Analysis of Septum Site-determining Protein MinD from Pyrococcus horikoshii OT3 Bacterial cell division requires the formation of a s e p t u m a t m i d c e l l , c i r c u m f e r e n t i a l i n v a g i n a t i o n o f t h e c y t o p l a s m i c m e m b r a n e , a n d s y n t h e s i s o f a p e p t i d o g l y c a n l a y e r . T h e k e y s t e p i n s e p t u m f o r m a t i o n i s t h e p o l y m e r i z a t i o n o f e s s e n t i a l c e l l di vi si on pr ot ei n Ft sZ at th e po te nt ia l di vi si on si te . F t s Z r e c r u i t s s e v e r a l o t h e r e s s e n t i a l p r o t e i n s t o f o r m m a t u r e c e l l d i v i s i o n m a c h i n e r y a n d t h e c e l l d i v i s i o n p r o c e s s t h e n p r o g r e s s e s . R o d - s h a p e d b a c t e r i a s u c h a s E s c h e r i c h i a c o l i h a v e t h r e e potential division sites in a cell. One of them is at the midcell position, while the others are adjacent to the cell poles. Thus the precise placement of the FtsZ ring at the cell center is a prerequisite for the accurate cell division of bacteria. In E. coli , the cell d i v i s i o n s i t e i s d e t e r m i n e d b y t h e c o o p e r a t i v e a c t i v i t y o f m i n o p e r o n p r o d u c t s M i n C , M i n D , a n d MinE. MinC is a nonspecific inhibitor of the septum protein FtsZ, and MinE is the suppressor of MinC. M i n D p l a y s a m u l t i f u n c t i o n a l r o l e . I t i s a m e m b r a n e - a s s o c i a t e d A T P a s e a n d i s a s e p t u m s i t e - d e t e r m i n i n g factor through the activation and regulation of MinC and MinE ( Fig. 1 ). MinD is also known to undergo a rapid pole-to-pole oscillation movement in vivo as observed in fluorescent microscopy [1]. We studied recombinant MinD from Pyrococcus horikoshii OT3 ( PH0612 ) expressed in E. coli , and determined the three-dimensional structure at 2.3 Å r e s o l u t i o n b y X - r a y c r y s t a l l o g r a p h y u s i n g t h e S e - M e t M A D m e t h o d a t b e a m l i n e B L 4 1 X U [ 2 ] . T h e c r y s t a l s t r u c t u r e c o n s i s t s o f a β - s h e e t w i t h s e v e n p a r a l l e l a n d o n e a n t i p a r a l l e l s t r a n d s a n d e l e v e n peripheral α -helices ( Fig. 2 ). Although we made no a t t e m p t t o a d d A T P o r A D P m o l e c u l e s i n t h e p u r i f i c a t i o n o r c r y s t a l l i z a t i o n s t e p , t h e e l e c t r o n density clearly shows that MinD from P. horikoshii con tai ns bou nd ADP and a mag nes ium ion at the (b) (a) (c) potential division site potential division site essential division site FtsZ MinE MinC MinD p o c k e t c l o s e t o t h e e d g e o f t h e β - s h e e t o n t h e surface of the MinD molecule ( Fig. 3 ). It has been s h o w n t h a t t h e A D P m o l e c u l e w a s b o u n d d u r i n g the growth of the E. coli cells, and MinD from E. coli ha s AT Pa se ac ti vi ty in th e pr es en ce of Mg 2+ io n. Therefore, the observed coordination of ADP in the pre sen t cry sta l is con sid ere d to be the pro duc t of the molecule’s ATPase activity. F i g . 1 . T h e s c h e m a t i c d i a g r a m o f t h e b a c t e r i a l c e l l d i v i s i o n m a c h i n e r y . ( a ) T h e r o d - s h a p e d bacteria have three potential division sites in the cell. The MinCD complex inhibits division at all potential division sites in the cell. (b) The MinE r i n g i s f o r m e d , a n d t h e a c t i v i t y o f c e l l d i v i s i o n inhibition of MinCD is suppressed by MinE. The M i n C D c o m p l e x u n d e r g o e s a r a p i d p o l e - t o - p o l e osci llat ion move ment . (c) MinE recr uits the cell division proteins as FtsZ at midcell division sites. The cir cum fer ent ial inv agi nat ion wit h squ eez e of FtsZ ring starts. 7 References [1] D.M. Raskin and P.A.J. de Boer, Proc. Natl. Acad. Sci. USA 96 (1999) 4971. [ 2 ] N a o k i S a k a i , M i n Y a o , H i r o s h i I t o u , N o b u h i s a Watanabe, Fumiaki Yumoto, Masaru Tanokura, and Isao Tanaka , Structure 9 (2001) 817. Structure analysis shows that MinD is most s i m i l a r t o n i t r o g e n a s e i r o n p r o t e i n w h i c h i s a mem ber of the fam ily of the P-l oop con tai nin g t h e n u c l e o t i d e t r i p h o s p h a t e h y d r o l a s e supe rfam ily of prot eins . Unli ke nitr ogen ase or other member proteins that normally work as a dimer, MinD was present as a monomer in the c r y s t a l . M i n D i s a l s o k n o w n t o b e h a v e l i k e a m o t o r p r o t e i n i n E . c o l i c e l l s . T h e p r e s e n t a n a l y s i s h a s s h o w n t h a t M i n D h a s a l i m i t e d structural similarity with family of motor proteins. A l t h o u g h t h e t e r t i a r y s t r u c t u r e o f A T P a s e a c t i v i t y s i t e i s s i m i l a r i n t h e s e p r o t e i n s , t h e o v e r a l l t o p o l o g y i s d i f f e r e n t . T h u s , t h e y a r e distantly related if at all. Both the 31 P NMR and Malachite Green method exhibited relatively low levels of ATPase activity. These facts suggest that there are some additional factor(s) for MinD to exhibit ATPase activity in the cell and MinD m a y w o r k a s a m o l e c u l a r s w i t c h i n t h e multiprotein complex in bacterial cell division. Naoki Sakai, Min Yao and Isao Tanaka Hokkaido University E-mail: yao @ castor.sci.hokudai.ac.jp Fig. 2. The ribbon diagram of the crystal structure of MinD from P. horikoshii with transparent molecular surface. Bound ADP is shown as ball and stick and m a g n e s i u m i o n i s s h o w n a s a n o r a n g e b a l l . T h e nucleotide binding pocket is close to the edge of the β -sheet. This pocket is exposed to solvent region. ADP H 2 O H 2 O H 2 O Fig . 3. Ele ctr on den sit y of Mg- ADP reg ion . The sigma-weighted omit map is calculated at 2.3 Å resolution and contoured to 3.0 σ . Mg 2+ 8
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