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Solution Structure of Apocalmodulin bound to a Binding DomainPeptide from the IQ motifs of Myosin V

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I nt r oduct i on

Myosi ns ar e a l ar ge f ami l y of mol ecul ar mot or s t hat move al ong act i n f i l ament s. Myosi n I I ( convent i onal myosi n) gener at es cont r act i on i n muscl e, whi l e myosi n V ( unconvent i onal myosi n) t r anspor t s car go as a monomer i n non- muscl e cel l s. Al l myosi ns have a gl obul ar N- t er mi nal mot or domai n t hat cont ai ns t he act i n and ATP- bi ndi ng si t es, f ol l owed by an el ongat ed α - hel i cal l ever ar m ( l ever ) . The l ever i s composed of speci al sequences cal l ed I Q mot i f s ( I QxxxRGxxxR, wher e consensus r esi dues ar e under l i ned and x i s any ami no aci d) , each of whi ch st abi l i zed by t he bi ndi ng of cal modul i n ( CaM) . Despi t e t he pr eval ence of

t hi s mot i f i n myosi n, t her e was pr evi ousl y no at omi c- r esol ut i on st r uct ur e of a CaM- myosi n heavy chai n compl ex unt i l r ecent l y.

  Recent l y r epor t ed 2. 5 Å r esol ut i on st r uct ur e of cal ci um f r ee CaM ( apoCaM) bound t o t he f i r st t wo I Q mot i f s of t he mur i ne myosi n V heavy chai n has r eveal ed an unusual CaM conf or mat i on

1)

. The C- t er mi nal l obe of each CaM adopt s a semi - open conf or mat i on t hat gr i ps t he f i r st par t of t he I Q mot i f ( I QxxxR) , wher eas t he N- t er mi nal l obe adopt s a cl osed conf or mat i on t hat i nt er act s mor e weakl y wi t h t he second par t of t he mot i f ( GxxxR) . I t i s i mpor t ant t o know whet her t he cr yst al st r uct ur e i s a st abl e st r uct ur e even i n sol ut i on, because a sol ut i on st r uct ur e i s di r ect l y Sol ut i on St r uc t ur e of Apoc al modul i n bound t o a Bi ndi ng Domai n

Pe pt i de f r om t he I Q mot i f s of Myos i n V

Yos hi nobu I z umi , Hi r oki Ebi s a wa , Yuj i J i nbo

Gr aduat e Pr ogr am of Human Se ns i ng and Func t i onal Se ns or Engi ne e r i ng, Gr aduat e Sc hool of Sc i e nc e and Engi ne e r i ng, Y amagat a Uni v e r s i t y ,

4- 3- 16 J o- Nan, Y one z awa, Y amagat a, 992- 8510, J apan

(平成 21 年 10 月8日受理)

Abs t r ac t

The s ol ut i on s t r uc t ur e s of c ompl e xe s be t we e n a poc a l modul i n

(a

poCa M

a nd a bi ndi ng doma i n of t he I Q

mot i f s of myos i n V ha ve be e n de t e r mi ne d by s ma l l - a ngl e X- r a y s c a t t e r i ng

(SAXS)

wi t h us e of s ync hr ot r on

r a di a t i on a s a n i nt e ns e a nd s t a bl e X- r a y s our c e . We us e d t hr e e s ynt he t i c pe pt i de s of r e s i due s 772- 786

(I

Q1

,

795- 810

(I

Q2

, a nd 772- 810

(I

Q

(1+2))

of t he myos i n V t o c ompa r e t he s ol ut i on s t r uc t ur e s wi t h t he

c or r e s pondi ng c r ys t a l s t r uc t ur e

(PDB:

2i x7

. The r a di us of gyr a t i on of a poCa M bound t o t he I Q1 or I Q2 a t

a mol a r r a t i o of 1: 1 i nc r e a s e d by 4. 8±0. 3Å or 3. 8±0. 3Å, r e s pe c t i ve l y , a s c o mpa r e d wi t h t he c or r e s pondi ng

c r ys t a l s t r uc t ur e . The e xpe r i me nt a l Kr a t ky pl ot s i ndi c a t e d t ha t a poCa M bound t o t he I Q1 or I Q2 a dopt s a

dumbbe l l - s ha pe d s t r uc t ur e . I n c ont r a s t t o t he s e c ompl e xe s , t he s ol ut i on of a poCa M/ I Q

(1+2)

a t a mol a r r a t i o

of 2: 1 be c a me t ur bi d, i ndi c a t i ng t ha t t he s ol ut i on c ont a i ns s e ve r a l t ype s of a ggr e ga t e s . The t ur bi d s ol ut i on

wa s c e nt r i f uge d a nd t he s upe r na t a nt wa s us e d f or t he SAXS me a s ur e me nt s . The SAXS r e s ul t s s ugge s t e d t ha t

t he s upe r na t a nt i s c ompos e d of a mi xt ur e of a poCa M/ I Q

(1+2)

a nd a poCa M. The r a di us of gyr a t i on of

a poCa M/ I Q

(1+2)

a t a mol a r r a t i o of 1: 2 i nc r e a s e d by 0. 8±0. 6Å, a s c ompa r e d wi t h t he c or r e s pondi ng c r ys t a l

s t r uc t ur e . The e xpe r i me nt a l Kr a t ky pl ot wa s c ompa r e d wi t h c a l c ul a t e d c ur ve s of bot h s ol ut i on s t r uc t ur e s

ba s e d on t he dumbbe l l - s ha pe d s t r uc t ur e a nd t he c r ys t a l s t r uc t ur e .

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r el at ed t o t he f unct i on of myosi n V.

  I n t hi s r epor t , we have i nvest i gat ed t he sol ut i on st r uct ur e of t he compl exes by smal l - angl e X- r ay scat t er i ng ( SAXS) wi t h use of synchr ot r on r adi at i on as an i nt ense and st abl e X- r ay sour ce, whi ch i s a usef ul met hod t o det ect di r ect i nt er act i on bet ween CaM and t ar get pept i des t hr ough measur abl e st r uct ur al changes of CaM. We used t hr ee synt het i c pept i des of r esi dues 772- 786 ( I Q1) , 795- 810 ( I Q2) , and 772- 810 ( I Q( 1+2) ) of t he myosi n V t o compar e t he sol ut i on st r uct ur e wi t h t he cr yst al st r uct ur e. The mai n SAXS r esul t s i ndi cat e t hat t he sol ut i on st r uct ur e i s di f f er ent f r om t he cor r espondi ng cr yst al st r uct ur e and t hat apoCaM bound t o t he I Q mot i f adopt s a dumbbel l - shaped st r uct ur e.

Exper i ment al

 Mat e r i al s . The r ecombi nant CaM based on t he sequence of r at CaM was expr essed as descr i bed i n r ef . 2. CaM f r act i on was obt ai ned as descr i bed i n r ef . 3. The pept i des wer e synt hesi zed and pur i f i ed as descr i bed i n r ef . 4.

Tabl e 1 summar i zes t he pr i mar y sequences of t hr ee synt het i c pept i des, I Q1, I Q2, and I Q( 1+2) .  SR- SAXS Measur ement s. The basi c medi um used f or t he SAXS measur ement s was 50 mM Tr i s- HCl , pH 7. 6, and 120 mM NaCl . A compl ex of apoCaM wi t h I Q1 or I Q2 was pr epar ed by mi xi ng 1. 0 mol of t he pr ot ei n wi t h 1. 1 mol of t he pept i de and t hese sol ut i ons wer e cl ear . On t he ot her hand, a compl ex of apoCaM wi t h I Q( 1+2) was pr epar ed by mi xi ng 1. 0 mol of t he

pr ot ei n wi t h 0. 5 mol of t he pept i de and t he sol ut i on was t ur bi d i n t he r ange of concent r at i on f r om 3 t o 10 mg/ mL. The t ur bi d sol ut i on was cent r i f uged and t he super nat ant was used f or t he measur ement s. Her e t he mol ar r at i os of t he pept i de t o t he pr ot ei n wer e det er mi ned accor di ng t o r ef . 1. The pr ot ei n concent r at i ons f or bot h apoCaM/ I Q1 and apoCaM/ I Q2 wer e 7. 5, 10. 0, 12. 5, 15. 0, and 17. 5 mg/ mL, whi l e t hose f or apoCaM/ I Q( 1+2) wer e 2. 26, 2. 99, 5. 6, and 8. 7 mg/ mL. The concent r at i ons f or bot h apoCaM/

I Q1 and apoCaM/ I Q2 wer e det er mi ned by t he met hod descr i bed i n r ef . 5, whi l e t hose f or t he super nat ant s of apoCaM/ I Q( 1+2) wer e det er mi ned spect r oscopi cal l y.

  The SAXS pr of i l es f or al l sampl es wer e acqui r ed usi ng t he i nst r ument f or SAXS i nst al l ed at BL- 10C of Phot on Fact or y, KEK, Tsukuba. The det ai l s of t he opt i cs and i nst r ument s ar e gi ven el sewher e

6)

. An X- r ay wavel engt h of 1. 488 Å was used. The sampl es wer e cont ai ned i n a mi ca cel l wi t h a vol ume of 70 μ L, and t he t emper at ur e was kept at 25. 0±0. 1 ℃ by ci r cul at i ng wat er t hr ough t he cel l hol der . The r eci pr ocal par amet er , s , equal s t o ( 2 si n θ ) / λ , was cal i br at ed by t he obser vat i on of a chi cken col l agen, wher e 2 θ i s t he scat t er i ng angl e and λ i s t he X- r ay wavel engt h. Scat t er i ng dat a wer e col l ect ed f or 300 s at var i ous concent r at i ons.

I r r adi at i on of al l sampl es f or per i ods up t o 1800 s pr oduced no change i n t he scat t er i ng pr of i l es.

 Sc at t e r i ng Dat a Anal y s i s . Two met hods of dat a anal ysi s wer e used. The f i r st met hod was t hat of Gui ni er and Four net

7)

. I n t he case of a gr oup of i dent i cal par t i cl es, t he scat t er i ng i nt ensi t y of I ( s , c ) measur ed as a f unct i on of s at a f i ni t e concent r at i on, c, i s gi ven by

   I ( s , c ) = I ( 0, c ) exp{ - ( 4 π

2

/ 3) R

g

( c )

2

s

2

} ( a) Her e I ( 0, c ) i s t he scat t er i ng i nt ensi t y at s = 0 and R

g

( c ) i s t he r adi us of gyr at i on at a concent r at i on c . I n t he di l ut e l i mi t , I ( 0, c ) and R

g

( c ) ar e gi ven by

   K c / I ( 0, c ) = 1/ M + 2 A

2

c + … ( b) Tabl e 1: Pr i mar y Sequences of Thr ee Pept i des of

I Q1, I Q2, and I Q( 1+2) Synt hesi zed i n Thi s Wor k

peptide name primary sequence AA

a

IQ1 RIQKTIRGWLLRKRY 15

IQ2 TVQRYVRGYQARCYAK 16

IQ(1+2) RIQKTIRGWLLRKRYLCMQRAAITVQRYVRGYQARCYAK 39

a

AA represents the number of amino acid residues; Consensus residues are underlined;

Abbreviations for the amino acid residues are: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe;

G, Gly; H, His; I, Ile; K, Lys; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V,

Val; W, Trp; and Y, Tyr.

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  R

g

( c )

2

= R

02

- B

if

c + … , ( c) r espect i vel y. Her e K i s an opt i cal const ant , M i s t he mol ecul ar wei ght of t he pr ot ei n, A

2

i s t he second vi r i al coef f i ci ent , R

0

i s t he r adi us of gyr at i on at i nf i ni t e di l ut i on, and B

if

i s t he par amet er of t he i nt er par t i cl e i nt er f er ence

8)

. The i nt er cept and t he i ni t i al sl ope of t he Gui ni er pl ot ( l n I ( s , c ) ver sus s

2

) det er mi ne I ( 0, c ) and R

g

( c ) , r espect i vel y. Usi ng eqs. b and c, t he f our par amet er s M , A

2

, R

0

, and B

if

ar e eval uat ed.

  I n t he case of a gr oup of sever al t ypes of par t i cl es, an ext ended met hod of Gui ni er and Four net was used

7)

. I n t he di l ut e l i mi t , I ( s , c ) i s gi ven by

  I ( s , c ) = ∑p

k

I

k

( s , c ) ( d) Her e t he summat i on encompasses al l t ypes of par t i cl es i n t he assembl age, p

k

i s t he pr obabi l i t y t hat one of t he N par t i cl es i s of t he t ype k and I

k

( s , c ) i s t he scat t er i ng i nt ensi t y of t he par t i cl e of t ype k . I

k

( s , c ) i s gi ven by t he same f or m as eq. a   I

k

( s , c ) = I

k

( 0, c ) exp{ - ( 4 π

2

/ 3) R

gk

( c )

2

s

2

} ,   ( a ’ ) By devel opi ng eq. a ’ under t he condi t i on of ( 4 π

2

/ 3) R

gk

( c )

2

s

2

<<1 ( t he condi t i on of Gui ni er ) , I ( s , c ) i s compi l ed i nt o t he same f or m as eq. a:

  I ( s , c ) = I ( 0, c ) exp{ - ( 4 π

2

/ 3) R

gz

( c )

2

s

2

} ( e) But t he i nt er cept and t he i ni t i al sl ope of t he Gui ni er pl ot gi ve t he wei ght - aver age mol ecul ar wei ght ( M

w

) and t he z- aver age r adi us of gyr at i on ( R

gz

) , r espect i vel y. M

w

i s obt ai ned by   I ( 0, c ) = ∑p

k

I

k

( 0, c ) = K c M

w

, ( f ) wher e K i s a opt i cal const ant and t he t ot al concent r at i on c i s gi ven by:

  c = ∑c

k

( g) R

gz

i s gi ven by

  R

gz

( c )

2

= ∑p

k

I

k

( 0, c ) R

gk

( c )

2

/ ∑p

k

I

k

( 0, c )   ( h)   The second met hod was t hat of Kr at ky, whi ch i s def i ned by t he pl ot of s

2

I ( s ) ver sus s ( t he Kr at ky pl ot ) i n t he case of a gr oup of i dent i cal par t i cl es

9)

. The Kr at ky pl ot s pr ovi de t he st r uct ur al char act er i st i cs ( e. g. , mol ecul ar shape) of a chai n pol ymer and a bi opol ymer . The Kr at ky pl ot f r om a gr oup of sever al t ypes of par t i cl es was cal cul at ed by

  s

2

I ( s , c ) / I ( 0, c ) = s

2

∑p

k

I

k

( s , c ) / ∑p

k

I

k

( 0, c ) ( i )  Cal c ul at i on of SAXS Pr of i l e of Known T e r t i ar y S t r uc t ur e s . PDB: 2i x7 was used as t he coor di nat es f or t he cr yst al st r uct ur e. Coor di nat es f or sol ut i on st r uct ur es wer e newl y cr eat ed by separ at i ng t he N- t er mi nal l obe of apoCaM f r om t he or i gi nal posi t i on on t he cr yst al st r uct ur e. I n t he cal cul at i on of t he scat t er i ng pr of i l es, we f i xed t he r adi us of gyr at i on of new sol ut i on st r uct ur e t o t he val ue of 25. 4 Å, whose val ue cor r esponds t o t hat of apoCaM/ I Q( 1+2) at a mol ar r at i o of 2: 1. The scat t er i ng pr of i l es wer e cal cul at ed by usi ng t he ext ended Debye ’ s f or mul a and nor mal i zed, as descr i bed pr evi ousl y

10)

.

Res ul t s

  Gui ni e r Re gi on of t he Sc at t e r i ng Pr of i l e . An exampl e of Gui ni er pl ot s f or a compl ex of apoCaM/ I Q1, apoCaM/ I Q2, and apoCaM/ I Q( 1+2) over t he concent r at i on ser i es i s shown i n Fi gur e 1A- C.

I n al l of t he sampl es st udi ed her e, t her e i s no evi dence of any upwar d cur vat ur e at l ow s

2

val ues i n t he Gui ni er pl ot s, whi ch i ndi cat es t hat t he dat a ar e f r ee f r om t he aggr egat i on of sampl es. The val ues of K c / I ( 0, c ) eval uat ed f r om t he i nt er cept s of t he Gui ni er pl ot s f or al l sampl es ar e shown i n Fi gur e 2A as a f unct i on of pr ot ei n concent r at i on. The pl ot s ar e l i near over t he ent i r e concent r at i on r ange, and t he val ue of [ K c / I ( 0, c ) ]

c=0

ext r apol at ed t o i nf i ni t e di l ut i on f or each compl ex has t he i nver se mol ecul ar wei ght appr opr i at e f or t he sol ubl e par t i cl e. The sl opes of t hese l i nes ar e posi t i ve f or al l compl exes, i ndi cat i ng t hat t he val ues of A

2

ar e posi t i ve, t hat i s, t he i nt er act i on bet ween compl exes i s r epul si ve

8,10,11)

.

Al l val ues of mol ecul ar wei ght ( M ) i n Tabl e 2 agr ee wel l wi t h t he cal cul at ed val ues denot ed i n par ent heses wi t hi n t he exper i ment al er r or of about 7 %. Ther ef or e, i t i s r easonabl e t o concl ude t hat apoCaM bi nds each pept i de wi t h t he mol ar r at i o descr i bed i n Tabl e 2.

  Radi i of gyr at i on at i nf i ni t e di l ut i on ( R

0

) wer e

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cal cul at ed f r om t he sl opes of t he Gui ni er pl ot s and ar e shown i n Fi gur e 2B. The l i near i ncr ease wi t h decr easi ng pr ot ei n concent r at i on was obser ved i n al l compl exes. The sl opes of t hese l i nes, whi ch ar i se f r om i nt er par t i cl e i nt er f er ence ef f ect s, r epr esent a vi r i al coef f i ci ent

8,10,11)

. Tabl e 2 al so compi l es t he val ues of R

0

. The R

0

val ue f or apoCaM/ I Q1 and t hat f or apoCaM/ I Q2 ar e

21. 8±0. 3 Å and 21. 3±0. 3 Å , r espect i vel y, a val ue t ypi cal of t he dumbbel l - shaped st r uct ur e

4, 11-13)

. I n cont r ast , t he R

crystal

val ue f or t he cr yst al st r uct ur e of apoCaM/ I Q1 i s 17. 0 Å and t hat f or apoCaM/ I Q2 i s 17. 5 Å . The R

0

val ue of apoCaM bound t o t he I Q1 or I Q2 at a mol ar r at i o of 1: 1 i ncr eased by 4. 8±0. 3Å or 3. 8±0. 3Å, r espect i vel y, as compar ed wi t h t he cor r espondi ng st r uct ur e, i ndi cat i ng t hat t he sol ut i on st r uct ur e i s di f f er ent f r om t he cr yst al st r uct ur e. I n t he cr yst al st r uct ur e, t he C- t er mi nal l obe of each CaM adopt s a semi - open conf or mat i on t hat gr i ps t he f i r st par t of t he I Q mot i f ( I QxxxR) , whi l e t he N- t er mi nal l obe adopt s a cl osed conf or mat i on t hat i nt er act s mor e weakl y wi t h t he second par t of t he mot i f ( GxxxR) .   The l ar ger val ues of R

0

f or apoCaM/ I Q1 and apoCaM/ I Q2 i ndi cat e t hat t he N- t er mi nal Tabl e 2: Mol ecul ar Wei ght ( M ) , Radi us of Gyr at i on

at I nf i ni t e Di l ut i on ( R

0

) f or a Compl ex of apoCaM/ I Q1, apoCaM/ I Q2, and apoCaM/ I Q( 1+2) .

______________________________________________________________________

System 10

-3M

(g/mol) (calcd. value)

a R0

()

Rcrystal

()

b

______________________________________________________________________

ApoCaM/IQ1 (1:1) 19.41.4 (18.7) 21.80.3 17.0

ApoCaM/IQ2 (1:1) 17.51.2 (18.7) 21.30.3 17.5

ApoCaM/IQ(1+2)(2:1) 35.92.5 (38.2) 25.20.6 24.6 ______________________________________________________________________

aM

for a complex of apoCaM/IQ1 and apoCaM/IQ2 at a molar ratio of 1:1, and apoCaM/IQ(1+2) at a molar ratio of 2:1.

b

Radius of gyration for the corresponding crystal structure.

-4 -3.5 -3 -2.5 -2

0 0.5 1 1.5

ln I ( s ) (a.u.)

10

4

s

2

-2

) A

5 4 3 2 1

Fi gur e 1: Gui ni er pl ot s f or a compl ex of apoCaM/ I Q1 at a mol ar r at i o of 1: 1 ( A) , apoCaM/ I Q2 at a mol ar r at i o of 1: 1 ( B) , and apoCaM/ I Q( 1+2) at a mol ar r at i o of 2: 1 ( C) at var i ous concent r at i ons. The st r ai ght l i nes wer e obt ai ned wi t h t he dat a poi nt s sat i sf yi ng t he Gui ni er condi t i on by t he l east - squar ed met hod. ( A) and ( B) : ( 1) 7. 5 mg/ mL; ( 2) 10. 0 mg/ mL; ( 3) 12. 5 mg/ mL; ( 4) 15. 0 mg/ mL; ( 5) 17. 5 mg/ mL. ( C) : ( 1) 2. 26 mg/ mL; ( 2) 2. 99 mg/ mL; ( 3) 5. 6 mg/ mL; ( 4) 8. 7 mg/ mL.

-4 -3.5 -3 -2.5 -2

0 0.5 1 1.5

ln I ( s ) (a.u.)

10

4

s

2

-2

) B

5 4 3 2 1

-5 -4.5 -4 -3.5 -3 -2.5 -2

0 0.5 1 1.5

ln I ( s ) (a.u.)

10

4

s

2

-2

) C

3

2 4

1

0 0.005 0.01 0.015 0.02

10

5

K c / I (0, c ) (mol g

-1

)

c (g cm

-3

)

A 8

6

4

2

Fi gur e 2: Zi mm pl ot s ( A) and t he squar e of t he r adi us of gyr at i on, R

g2

, ( B) f or a compl ex of apoCaM/ I Q1, apoCaM/ I Q2, and apoCaM/ I Q( 1+2) . (

) apoCaM/ I Q1 at a mol ar r at i o of 1: 1; (

) apoCaM/ I Q2 at a mol ar r at i o of 1: 1; (

) apoCaM/ I Q( 1+2) at a mol ar r at i o of 2: 1.

400 500 600

0 0.005 0.01 0.015 0.02

R

g

2

2

)

c (g cm

-3

)

B

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l obe i s separ at ed f r om t he second par t of t he mot i f i n t he sol ut i on. On t he ot her hand, t he R

0

val ue f or apoCaM/ I Q( 1+2) i s 25. 2±0. 6 Å , whose val ue i s sl i ght l y l ar ger t han t he cor r espondi ng val ue of t he cr yst al st r uct ur e ( 24. 6 Å ) .

Kr at k y Re gi on of t he Sc at t e r i ng Pr of i l e . Fi gur es 3A and 3B show t he Kr at ky pl ot s f or apoCaM/ I Q1 and apoCaM/ I Q2, r espect i vel y, whi ch ar e char act er i zed by t he pr esence of a br oad peak near s = 0. 015 Å

-1

, i ndi cat i ng t hat each compl ex adopt s a dumbbel l - shaped st r uct ur e whi ch i s cl ose t o a dumbbel l - shaped st r uct ur e r epor t ed pr evi ousl y

4,10)

. Al l scat t er i ng pr of i l es conver ge t o t hat at t he hi ghest concent r at i on. On t he ot her hand, t he scat t er i ng pr of i l e cal cul at ed f r om t he cor r espondi ng cr yst al st r uct ur e i s shown by a bl ack sol i d l i ne i n Fi gur es 3A or 3B, whi ch i s char act er i zed by t he pr esence of a shar p peak near s = 0. 016 Å

-1

. Fr om a compar i son bet ween t hese pr of i l es, i t i s r easonabl e t o concl ude t hat t he sol ut i on st r uct ur e i s obvi ousl y di f f er ent f r om t he cor r espondi ng cr yst al st r uct ur e, suggest i ng t hat t he N- t er mi nal l obe of CaM i s separ at ed f r om t he second par t of I Q mot i f i n t he sol ut i on.

  Fi gur e 3C shows t he Kr at ky pl ot f or apoCaM/

I Q( 1+2) , whi ch i s char act er i zed by t he pr esences of a br oad and asymmet r i c peak near s = 0. 013 Å

-1

and a shoul der ar ound s = 0. 025 Å

-1

. I n cont r ast ,

t he cal cul at ed pr of i l e of t he cor r espondi ng cr yst al st r uct ur e ( a bl ack sol i d l i ne) , i s char act er i zed by t he pr esences of a shar p peak near s = 0. 011 Å

-1

and a shoul der ar ound s = 0. 025 Å

-1

. The r esul t s show t hat t he pr of i l e cal cul at ed f r om onl y t he cr yst al st r uct ur e i s not suf f i ci ent t o r epr oduce t he dat a poi nt s of t he sol ut i on st r uct ur e, suggest i ng t hat ot her component s ar e cont ai ned.

Di s cus s i on

  The measur ement s of t he mol ecul ar wei ght s, t he r adi i of gyr at i on and t he Kr at ky pl ot s f or apoCaM/ I Q1 and apoCaM/ I Q2 pr esent ed her e show t hat t he sol ut i on st r uct ur e adopt s a dumbbel l - shaped st r uct ur e, whose st r uct ur e i s t ot al l y di f f er ent f r om t he cr yst al st r uct ur e

1)

. Combi ned wi t h t he r esul t s of t he cr yst al st r uct ur e, t he most st r ai ght f or war d i nt er pr et at i on of t he t hese SAXS r esul t s i s t hat t he N- t er mi nal l obe of apoCaM i s separ at ed f r om t he second par t of t he I Q mot i f i n sol ut i on, whi l e t he C- t er mi nal l obe st i l l gr i ps t he f i r st par t of t he mot i f .

  I n cont r ast , t he scat t er i ng pr of i l es cal cul at ed f r om t he cr yst al st r uct ur e f or apoCaM/ I Q( 1+2) i s not suf f i ci ent t o r epr oduce t he exper i ment al pr of i l e, t hough t he exper i ment al val ues of M and R

0

ar e i n good accor dance wi t h t he cal cul at ed val ues f or t he cr yst al st r uct ur e. I n t hi s poi nt , we r ecal l t hat an upwar d cur vat ur e

0 2 4 6 8 10

0 0.01 0.02 0.03 0.04 0.05 0.06 7.5 10.0 12.5 15.0 17.5 cal/1

10

5

s

2

I ( s )/ I (0) (Å

-2

)

s (Å

-1

) A

Fi gur e 3: Kr at ky pl ot s f or a compl ex of of apoCaM/ I Q1 at a mol ar r at i o of 1: 1 ( A) , apoCaM/ I Q2 at a mol ar r at i o of 1: 1 ( B) , and apoCaM/ I Q( 1+2) at a mol ar r at i o of 2: 1( C) at var i ous concent r at i ons. The number s i n t he f r ame of A- C denot e concent r at i ons of mg/ mL. The bl ack l i nes ( cal / 1) i n A- C wer e cal cul at ed by usi ng t he cor r espondi ng at omi c coor di nat es f r om t he PDB: 2i X7.

0 2 4 6 8 10

0 0.01 0.02 0.03 0.04 0.05 0.06 7.5 10.0 12.5 15.0 17.5 cal/1

10

5

s

2

I ( s )/ I (0) (Å

-2

)

s

-1

) B

0 1 2 3 4 5 6

0 0.01 0.02 0.03 0.04 0.05 0.06 5.6 8.7 cal/1

10

5

s

2

I ( s )/ I (0) (Å

-2

)

s (Å

-1

)

C

(6)

at l ow s

2

val ues i n t he Gui ni er pl ot was obser ved i n a si mpl e sol ut i on of I Q( 1+2) ( dat a i s not shown) , i ndi cat i ng t hat t he sol ut i on cont ai ns var i ous associ at es. A t ur bi d sol ut i on was obt ai ned by mi xi ng t he sol ut i on of I Q( 1+2) wi t h t hat of apoCaM. Af t er t he cent r i f ugat i on, t he super nat ant was separ at ed f r om t he pr eci pi t at es and used f or t he SAXS measur ement s.

Fr om t he exper i ment al val ues of M and R

0

, i t i s cl ear t hat t he mai n component of t he super nat ant i s composed of t he compl ex of apoCaM/ I Q( 1+2) at a mol ar r at i o of 2: 1. Not i ng t hat t he val ue of M i s smal l er t han t he cal cul at ed val ue, ot her component s wi t h a l ower mol ecul ar wei ght mi ght be cont ai ned.

Her e, we assume t hat apoCaM i s cont ai ned i n t he super nat ant as a mi nor component . Then, t he r adi us of gyr at i on and mol ecul ar wei ght f or apoCaM/ I Q( 1+2) ar e r epl aced by M

w

i n eq. f and R

gz

i n eq. h, r espect i vel y. Usi ng M

w

def i ned by

p

k

M

k2

/ ∑ p

k

M

k

, t he exper i ment al val ue of M

w

( 35. 9x10

3

) was r epr oduced by sel ect i ng p

1

= 0. 8 f or apoCaM/ I Q( 1+2) and p

2

= 0. 2 f or apoCaM, r espect i vel y. Usi ng t hese val ues of p

1

and p

2

, t he val ue f or t he r adi us of gyr at i on of apoCaM/

I Q( 1+2) at a mol ar r at i o of 2: 1 was eval uat ed as 25. 4 Å. The r esul t s i ndi cat e t hat t he component

of apoCaM shoul d be subt r act ed i n advance f r om t he exper i ment al dat a i n Fi gur e 3C.

  Fur t her mor e, t he r esul t s of apoCaM/ I Q1 and apoCaM/ I Q2 showed t hat t he sol ut i on st r uct ur e i s evi dent l y di f f er ent f r om t he cor r espondi ng cr yst al st r uct ur e. Fi gur e 4A shows a schemat i c sol ut i on st r uct ur e f or apoCaM/ I Q1 or apoCaM/

I Q2, i n whi ch t he N- t er mi nal l obe of apoCaM i s separ at ed f r om t he second par t of t he I Q mot i f . Two t ypes of sol ut i on st r uct ur es ar e shown.

Fi gur e 4B shows t hr ee t ypes of sol ut i on st r uct ur es f or apoCaM/ I Q( 1+2) .

  Fi gur e 5 shows t he cal cul at ed r esul t s of t he scat t er i ng pr of i l es f or t hr ee t ypes of sol ut i on st r uct ur es as wel l as t he cr yst al st r uct ur e i n Fi gur e 4B. The dat a poi nt s f or apoCaM/ I Q( 1+2) at a mol ar r at i o of 1: 2 ar e r epr oduced by t hese cal cul at ed pr of i l es. By usi ng t he sol ut i on st r uct ur es, a di f f er ence obser ved near s = 0. 01 Å

-1

i s f ai r l y i mpr oved. However , a di f f er ence i n t he r ange of 0. 01< s ( Å

-1

) <0. 03 i s not subst ant i al l y i mpr oved by t he use of t he sol ut i on st r uct ur e.

A f ur t her sear ch of coor di nat es f or an appr opr i at e sol ut i on st r uct ur e wi l l be desi r ed t o i mpr ove t hi s poi nt . Resear ch i s i n pr ogr ess al ong t hi s l i ne.

C

N IQ1

Solution Structure

N C

IQ2

ApoCaM/IQ1

ApoCaM/IQ2

C N IQ1

N C IQ2

+ Crystal Structure

C N

N

IQ1 IQ2

C

Fi gur e 4: Cr yst al and sol ut i on st r uct ur es f or a compl ex of apoCaM/ I Q1, apoCaM/ I Q2, and apoCaM/ I Q( 1+2) . I n t he cr yst al st r uct ur e ( PDB: 2i x7) , t he N- t er mi nal l obe of apoCaM i nt er act s wi t h t he second par t ( GxxxR) of I Q mot i f s, whi l e t he C- t er mi nal l obe i nt er act s wi t h t he f i r st par t ( I QxxxR) . I n Fi gur e 4A, t he cr yst al st r uct ur e was separ at ed i nt o t wo par t s: apoCaM/ I Q1 and apoCaM/ I Q2. I n Fi gur e 4B, onl y t he N- t er mi nal l obe of apoCaM i s separ at ed f r om t he second par t of t he I Q1 ( t he sol ut i on st r uct ur e 1) . I n t he sol ut i on st r uct ur e 2, onl y t he N- t er mi nal l obe of apoCaM i s separ at ed f r om t he second par t of t he I Q2. I n t he sol ut i on st r uct ur e 3, each N- t er mi nal l obe of apoCaM i s separ at ed f r om t he second par t of t he cor r espondi ng I Q mot i f .

Solution Structure 1

Solution Structure 2

Solution Structure 3 Crystal

Structure

C C N

N

IQ1 IQ2

C C N

N

IQ1 IQ2

IQ1 N N C C

IQ2

N N C C

IQ1 IQ2

(7)

Acknowl edgment s

We t hank Dr . Kat sumi Kobayashi f or hi s hel p i n t he SAXS measur ement s at KEK and Dr . Nobuhi r o Hayashi of Tokyo I nst i t ut e of Technol ogy f or pr ovi di ng pl asmi ds f or t he expr essi on of CaM i n Es c he r i c hi c ol i and f or t he spect r oscopi c det er mi nat i on of pr ot ei n concent r at i ons. The SAXS measur ement s wer e per f or med wi t h appr oval of t he Phot on Fact or y Commi t t ee, KEK, Tsukuba, Japan ( Pr oposal Nos. 05G299, 07G545 and 09G068) .

Ref er ences

1) A. Houdusse, J. - F. Gaucher , E. Kr ement sova, S. Mui , K. M. Tr ybus, and C. Cohen: Cr yst al st r uct ur e of apo- cal modul i n bound t o t he f i r st t wo I Q mot i f s of myosi n V r eveal s essent i al r ecogni t i on f eat ur es, PNAS 103- 51, pp. 19326- 19331( 2006) .

2) N. Hayashi , M. Mat subar a, A. Takasaki , K.

Chi t ani , and H. Tani guchi : An expr essi on syst em of r at cal modul i n usi ng T7 phage

pr omot er i n Escher i chi a col i , Pr ot ei n Expr essi on Pur i f . 12- 1, pp. 25- 28 ( 1998) . 3) M. Yazawa, M. Sakuma, and K. Yagi :

Cal modul i ns f r om muscl es of mar i ne i nver t ebr at es, scal l op and sea anemone, J.

Bi ochem. ( Tokyo) 87- 5, pp. 1313- 1320 ( 1980) . 4) Y. I zumi , S. Kuwamot o, Y. Ji nbo, and H.

Yoshi no: I ncr ease i n t he mol ecul ar wei ght and r adi us of gyr at i on of apocal modul i n i nduced by bi ndi ng of t ar get pept i de:

Evi dence f or compl ex f or mat i on, FEBS Let t . 495- 1&2, pp. 126- 130 ( 2001) .

5) O. H. Lower y, N . J. Rosenbr ough, A. L. Far r , and R. J. Randal l : Pr ot ei n measur ement wi t h t he Fol i n phenol r eagent , J. Bi ol . Chem. 193- 1, pp. 265- 275 ( 1951) .

6) T. Ueki , Y. Hi r agi , M. Kat aoka, Y. I noko, Y.

Amemi ya, Y. I zumi , H. Tagawa, and Y.

Mur oga: Aggr egat i on of bovi ne ser um al bumi n upon cl eavage of i t s di sul f i de bonds, st udi ed by t he t i me- r esol ved smal l - angl e X- r ay scat t er i ng t echni que wi t h synchr ot r on r adi at i on, Bi ophys. Chem. 23- 1&2, pp. 115- 124 ( 1985) .

7) A. Gui ni er and G. Four net , Smal l - Angl e Scat t er i ng of X- Rays, John Wi l ey & Sons, New Yor k ( 1955) pp. 65- 67 & pp. 126- 129.

8) Y. I zumi , M. Waki t a, H. Yoshi no, and N.

Mat sushi ma: St r uct ur e of t he pr ot eol yt i c f r agment F34 of cal modul i n i n t he absence and pr esence of mast opar an as r eveal ed by sol ut i on scat t er i ng, Bi ochemi st r y 31- 48, pp. 12266- 12271 ( 1992) .

9) O. Kr at ky, i n Smal l - Angl e X- r ay Scat t er i ng ( Edi t ed by H. Br umber ger ) , Gor don &

Br each, Sci ence Publ i sher s, New Yor k ( 1967) pp. 63- 129.

10 ) Y. I zumi , H. Wat anabe, N. Wat anabe, A.

Aoyama, Y. Ji nbo, and N. Hayashi : Sol ut i on X- r ay scat t er i ng r eveal s a novel st r uct ur e of cal modul i n compl exed wi t h a bi ndi ng domai n pept i de f r om t he HI V- 1 mat r i x pr ot ei n p17, Bi ochemi st r y 47- 27, pp. 7158- 0 0.01 0.02 0.03 0.04 0.05 0.06

10 5 s 2 I ( s )/ I (0) (Å -2 )

s (Å -1 ) 0

1 5 4 3 2

Fi gur e 5: Compar i son among t he scat t er i ng cur ves cor r espondi ng t o t hr ee t ypes of st r uct ur es shown i n Fi gur e 4B. The dat a poi nt s ( open bl ue ci r cl es) wer e obt ai ned by subt r act i ng t he dat a poi nt s f or apoCaM f r om t hose f or apoCaM/

I Q( 1+2) , t aki ng i nt o account of t he per cent age.

The bl ack sol i d l i ne denot es t he cr yst al

st r uct ur e; t he bl ue sol i d l i ne denot es t he

sol ut i on st r uct ur e 1; t he gr een sol i d l i ne

denot es t he sol ut i on st r uct ur e 2; t he r ed sol i d

l i ne denot es t he sol ut i on st r uct ur e 3.

(8)

7166 ( 2008) .

11 ) T. Yokouchi , H. Nogami , Y. I zumi , H.

Yoshi no, K. Nakashi ma, and M. Yazawa:

Sol ut i on X- r ay scat t er i ng dat a show st r uct ur al di f f er ences among chi mer as of yeast and chi cken cal modul i n: I mpl i cat i ons f or st r uct ur e/ f unct i on, Bi ochemi st r y 42- 7, pp. 2195- 2201( 2003) .

12 ) N. Mat sushi ma, Y. I zumi , T. Mat suo, H.

Yoshi no, T. Ueki , and Y. Mi yake: Bi ndi ng

of cal ci um and mast opar an t o cal modul i n i nduces a l ar ge change i n t he t er t i ar y st r uct ur e, J. Bi ochem. ( Tokyo) 105- 6, pp.

883- 887 ( 1989) .

13) M. Noguchi , Y. I zumi , and H. Yoshi no:

Tar get r ecogni t i on by cal modul i n: t he r ol e

of aci d r egi on cont i guous t o t he cal modul i n-

bi ndi ng domai n of cal ci neur i n A, FEBS

Let t . 573- 1~3, pp.121-126 (2004).

Tabl e 1  summar i zes t he pr i mar y sequences of t hr ee  synt het i c  pept i des, I Q1, I Q2, and  I Q( 1+2)

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