Crystallographic Analyses of the Runx-1/AML1 (Runt domain) – DNA Interaction and Its Allosteric Control by CBF

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(a) (b) (c) thei r DNA bind ing ( e.g. , the “sci ssor s grip ” by the Fo s- Ju n he te ro di me r ). On th e ot he r ha nd , so me t r a n s c r i p t i o n f a c t o r s c o n s i s t o f o n e s u b u n i t t h a t b i n d s t o D N A a n d o n e t h a t d o e s n o t . I n m o s t ca se s of th is ty pe of pr ot ei n, it re ma in s un kn ow n how a non DNA-binding subunit contributes to DNA b i n d i n g a n d w h e t h e r t h e m e c h a n i s m b y w h i c h a no n- DN A- bi nd in g su bu ni t st im ul at es DN A bi nd in g is shared with different transcription factors. In this type, very little structural information is available to d a t e , e . g . t h e G A B P α - G A B P β - D N A c o m p l e x ( G A B P : G A - b i n d i n g p r o t e i n ) . O n e w e l l - s t u d i e d e x a m p l e o f th i s ty p e o f tr a n s c r i p ti o n fa c to r i s th e c o r e b i n d i n g f a c t o r ( C B F ) , w h i c h f u n c t i o n s a s a heterodimer consisting of a DNA-binding α subunit and a non-binding β subunit. T h e C B F h e t e r o d i m e r i c t r a n s c r i p t i o n f a c t o r s c o m p r i s e d o f A M L / C B F A / P E B P 2 α / R u n x a n d C B F β / P E B P 2 β s u b u n i t s a r e e s s e n t i a l f o r d i f f e r e n t i a t i o n o f h e m a t o p o i e t i c a n d b o n e c e l l s , a n d t h e i r m u t a t i o n i s i n t i m a t e l y r e l a t e d t o t h e de ve l op me nt of ac ut e l eu ke mi a an d cl ei do cr an i al d y s p l a s i a ; ( A M L : a c u t e m y e l o g e n o u s l e u k e m i a , C B F A : c o r e - b i n d i n g f a c t o r A a n d P E B P : p o l y o m a v i r u s e n h a n c e r - b i n d i n g p r o t e i n ) . H e r e w e p r e s e n t t h e crys tal stru ctur es of the AML1 / Runx -1 / CBF α (Run t d o m a i n ) - C B F β ( c o r e d o m a i n ) - C / E B P β ( b Z i p ) - D N A , A M L 1 / R u n x - 1 / C B F α ( R u n t d o m a i n ) - C / E B P β ( b Z i p ) - DNA, and AML1 / Runx-1 / CBF α (Runt domain) -DNA c o m p l e x e s , w h i c h a r e a b b r e v i a t e d t o C B F α - β - C / E B P β - D N A , C B F α - C / E B P β - D N A a n d C B F α - D N A r e s p e c t i v e l y , o b t a i n e d f r o m t h e d i f f r a c t i o n e x p e r i m e n t s a t b e a m l i n e s B L 4 1 X U a n d B L 4 5 X U [1]. Our focus is on the recognition of specific DNA s e q u e n c e s b y C B F α a n d o n t h e m e c h a n i s m b y which CBF β facilitates DNA binding. T h e s t r u c t u r e o f a c o m p l e x c o m p r i s i n g t h e C B F α R u n t d o m a i n a n d t h e C B F β c o r e d o m a i n bound to a 26-bp DNA fragment from the CSF-1R prom oter , toge ther with the C/ EBP β basi c leuc ine zipp er regi on homo dime r, is show n in Fig. 1(a- c) . Not e tha t the str uct ure s of DNA -bo und CBF α are Crystallographic Analyses of the Runx-1 / AML1 (Runt domain) - DNA Interaction and Its Allosteric Control by CBF β β T h e p r o m o t e r r e g i o n s o f m o s t g e n e s c o n t a i n binding sites for multiple transcription factors whose interactions are essential for the correct regulation o f t r a n s c r i p t i o n . S u c h i n t e r a c t i o n s i n c l u d e t h o s e b e t w e e n d i f f e r e n t t r a n s c r i p t i o n f a c t o r s a s w e l l a s those between subunits of the same factor. In the cas e of som e het ero dim eri c fac tor s ( e.g . , Jun / Fos an d My c /M ax ) bo th su bu ni ts bi nd to sp ec if ic DN A sequences, and recent X-ray studies of this type of protein have clarified the molecular mechanisms of Fig. 1. Overviews of the CBF α - β -C / EBP β -DNA quaternary complex from three perspectives [1]. Vie ws ( a ) fro m the fro nt, ( b ) fro m the sid e and ( c ) fr om th e to p. Wi th in CB F α , β st ra nd s an d loops are depicted as red arrows and pink tubes, respe ctivel y; withi n CBF β , α helic es, β stran ds a n d l o o p s a r e d e p i c t e d a s g r e e n r i b b o n s , b l u e a r r o w s a n d c y a n t u b e s , r e s p e c t i v e l y . T h e C - t e r m i n a l r e g i o n o f t h e C / E B P β h o m o d i m e r cont aini ng the bZip doma in is show n as yell ow ribbons. Areas I and II are marked with circles. C/EBP β CBF α CBF β C/EBP β CBF β CBF β CBF α C/EBP β CBF α Area l Areal II 18 v e r y s i m i l a r , w i t h o r w i t h o u t C B F β : t h e r . m . s . d e v i a t i o n of the α carbon atoms was only about 0.28 Å. Both the CBF α Runt domain and the CBF β core domain f o l d p r i n c i p a l l y a s β b a r r e l a r c h i t e c t u r e s , w i t h t h e structure of the former being classified as an s-type immunoglobulin fold. The Runt domain recognizes sp ec if ic ba se s ( TG TG GT T ) in bo th th e ma jo r an d minor grooves of the DNA, mainly using loops. The C B F β c o r e d o m a i n i n t e r a c t s w i t h t h e C B F α R u n t d o m a i n a t a s i t e d i s t a n t f r o m t h e p r o t e i n - DNA interface. The CBF α -CBF β interface extends linearly from the DNA and c a n b e di vi de d in to tw o pr ed om in an tl y hy dr op hi li c i n t e r a c t i o n a r e a s − o n e p r o x i m a l t o D N A (Area I) and one distal (Area II ) − with an int erv eni ng hyd rop hob ic are a ( Fig s. 2 and 1b ). Contrary to earlier protein-protein and protein-DNA binding assays [2,3], analyses o f c r y s t a l s o f t h e C B F α - β - C / E B P β - D N A a n d C B F α - C / E B P β - D N A c o m p l e x e s revealed no direct interaction between the C / E B P β b a s i c l e u c i n e z i p p e r d o m a i n a n d th e CB F α Ru nt do ma in or th e CB F β co re domain. Fig. 3. Schematic representation of the stabilization network between CBF β (cyan), CBF α (pink) and DNA (gray) [1]. CBF α -DNA binding is enhanced several folds in the presence of CBF β . To examine the regulation of CBF α -DNA binding by CBF β , we compared the structure of the CBF α - β -C / EBP β -DNA complex with t h o s e o f t h e C B F α - C / E B P β - D N A a n d C B F α - D N A c o m p l e x e s , w h i c h l a c k t h e C B F β s u b u n i t . S o m e w h a t s u r p r i s i n g l y , C B F β b i n d i n g c a u s e s n o d r a m a t i c s t r u c t u r a l c h a n g e s i n t h e C B F α R u n t domain, which implies that CBF β does not alter the F i g . 2 . C B F α - C B F β h e t e r o d i m e r s t r u c t u r e i n c o m p l e x w i t h t h e D N A [ 1 ] . T w o p r e d o m i n a n t l y hydrophilic interaction areas between CBF α and CBF β (Areas I and II ) are marked with dotted circles. 19 o v e r a l l c o n f o r m a t i o n o f t h e C B F α R u n t d o m a i n , b u t o n l y s t a b i l i z e s i t i n a c o n f o r m a t i o n s u i t a b l e f o r D N A b i n d i n g . A s des cri bed abo ve, CBF β int era cts wit h CBF α v i a t w o p r e d o m i n a n t l y h y d r o p h i l i c r e g i o n s − Are as I and II, and the ir sta bil iza tio n aff ect s t h e c o n f o r m a t i o n s o f t h e f l e x i b l e l o o p s i n c o n t a c t w i t h t h e D N A t o g r i p t h e D N A m o l e c u l e a t b o t h t h e m a j o r a n d m i n o r grooves ( Fig. 3 ). A c u t e l e u k e m i a a n d r e l a t e d d i s e a s e s a s s o c i a t e d w i t h b i a l l e l i c a n d h e t e r o z y g o u s po in t mu ta ti on s of AM L1 / Ru nx -1 ha ve be en re po rt ed . In th e ca se of bi al le li c mu ta ti on s, no functional AML1 / Runx-1 allele remains; in the cas e of het ero zyg ote s, the DNA bin din g a c t i v i t y o f A M L 1 / R u n x - 1 f r o m o n e a l l e l e i s i m p a i r e d , w i t h A M L 1 / R u n x - 1 - C B F β h e t e r o d i m e r i z a t i o n a c t i v i t y r e t a i n e d . B a s e d o n t h e t h r e e - d i m e n s i o n a l s t r u c t u r e o f t h e CBF α -CBF β heterodimer bound to the DNA, p o i n t m u t a t i o n s i t e s i n t h e h e t e r o z y g o u s ca se s ar e ma pp ed ex cl us iv el y to re gi on s of the CBF α -DNA interface ( Fig. 4 ). It is readily a p p a r e n t t h a t t h e s e m u t a t i o n s i m p a i r t h e c a p a c i t y o f C B F α t o b i n d t o D N A w i t h o u t a f f e c t i n g t h e o v e r a l l f o l d i n g a r c h i t e c t u r e o f C B F α o r C B F α - C B F β h e t e r o d i m e r i z a t i o n , wh ic h co ul d ex pl ai n th ei r do mi na nt ne ga ti ve behavior. References [1] T.H. Tahirov, T. Inoue, M. Sasaki, K. Kimura, H. M o r i i , A . F u j i k a w a , M . S h i i n a , K . S a t o , T . Kumasaka, M. Yamamoto, S. Ishii, K. Ogata, Cell 104 (2001) 755. [ 2 ] D . E . Z h a n g e t a l . , M o l . C e l l . B i o l . 1 6 ( 1 9 9 6 ) 1231. [3] M.S. Petrovick et al. , Mol. Cell. Biol. 18 (1998) 3915. Fig. 4. Structural maps of CBF α point mutations o c c u r r i n g i n a c u t e m y e l o g e n o u s l e u k e m i a a n d r e l a t e d d i s e a s e s [ 1 ] . T h e m u t a t e d r e s i d u e s a r e shown in green ball and stick presentations. The p e p t i d e b a c k b o n e s o f C B F α a n d C B F β a r e depicted as pink and cyan tubes, respectively. Tahir H. Tahirov a,b , Taiko Inoue-Bungo a and Kazuhiro Ogata a,b,c (a) Kanagawa Academy of Science and Technology ( KAST ) (b) SPring-8 / RIKEN (c) Yokohama City University School of Medicine E-mail: ogata @ med.yokohama-cu.ac.jp Acute Myelogenous Leukemia and related diseases 20