ð àCüyfÆî¿^pN¿Ì½OãÉ¨¯é¡Ì»Ìì»Æ\¢ÉîÃ­½@\\EFV ÛCI^Åf Ia ÉæéAN`ÌCü@\\ßºr¡sYÆåw¶½Èw §603_8555 s{sskæãêÎ{RÃºp¾sYÆåw¶½Èw §603_8555 s{sskæãêÎ{Rv |EFVÛ©çªå³êém ADP {V»ÅfÅé Ia ÍCAN`Ì Arg177ðÁÙIÉ ADP {V»·éBADP {V»Ì½@\ðméÉÍCIa ÆAN`Ì¡Ì\¢Ìð¾ªdvÅ éªC{Ìî¿Åé NAD{ ^¨æÑ Ia_ADP {V»AN`Ì¡Ì\¢Í¾ç©ÅÍÈ©Á½B»êçÌ¡ÌÌ\¢
ð¾é×­»»ðâ X üñÜÀ±ÌðÈÇsöëðJèÔµCNAD{ ªµÄ¢È¢A|^ Ia_AN`¡Ì»ð¾½B±ÌA|^ Ia_AN`¡Ì»ðî¿ntCRÜntÉÙÈÁ½ðÅZÐ·é±ÆÅ NAD{ ^ Ia_AN`Æ Ia_ADP {V»AN`Ì¡Ì»ðìèª¯é±ÆÉ¬÷µCªð\Ì\¢ð¾ç©Éµ½BADP {V»½ÌOãÌ\¢©çCADP {V»Ì½@\ÆµÄgstrain­allevia­tioniÙ£ÆÉajfhðñ¥µ½iPNAS2008, PNAS2013jBIL\JxjECIÌ 2 ÂÌÔÌðoÄAN` Arg177ÖÌ ADP {V»ÌCü½ªN«éÆl¦çêéB±ÌðàÅÍA|^¡Ì©çÌ NAD{ ^¡Ì¨æÑ Ia_ADP {V»AN`¡Ì»Ìì»ÆC»êçÌ\¢ÉîÃ­ ADP {V»Ì½@\ðÐî·éB­XIjðCü·é toxin complex component C3

1. ADP {V»Åf(TccC3jâC¼Ì^Cv III ÆÍÙÈè GTP ^p

ADP  { V  » Æ Í C ADP  { V  î ] Ú y fN¿ðWIÆµÈªçàAXpMÅÍÈ­O^~i ART j É æ Á Ä ø « N ± ³ ê é ½  Å C NAD{ © çðCü·é TccC5 ÈÇVµ¢^CvÌ ART àñ³ê

ADP {[XªªØèæçêWI^pN¿ÉÁ¦çÄ¢é11)BéiFig. 1jBm ADP {VART Í×EÉ´õ·é½ßÉ ADP {V»yféÁèÌ×ÛÅfÌa´«JjYÉÖW·éªÉÁ¦Aªð¿CêÂÌyfÌÉ¼ûÌvfêé|óãCüÌêÂÅ»Í±Æª¾ç©É³êC»êçÌ ART Í»ê¼êÌWI^ðÂàÌàpN¿ÉîÃ«C¢­Â©Ì^CvÉªÞ·é±ÆªÅjbgÆµÄÂàÌàêÎCyfªÆAªðÊXÌTuéiFig. 2AjBART ÍChåÌ« é B Fig. 2A É ¦ · æ ¤ É ^ C v I Æ µ Ä R   ÅGhTCg[VXÌ@\ðpµGh\[ðæè¨f1_3)CSúPÅf4)Æå°ÛÕM«GegLV5)È

ÆµÄ×EàÉüèÞB©gªæÁ½Gh\[ªèC»êçÍwe}[Ì GTP ^pN¿Ì\\[ÉBµªððó¯éOÉATujbVXeCÜ½ÍAMjðWIÆµÄ¢éB^Cv

gÉæèGh\[©ç×E¿ÉÚs·éÆl¦çêII ÉªÞ³êéWteAÅfÆÎ^ÛÅfª elongationé12_16)BÇªfactor 2 ÌWt^~hðCü·é6) B^Cv III ÆµÄCäXÍC^Cv III Æ^Cv IV ÌÅfð¤ÎÛÆµ{ckXÛÌ C3 ÅfÍCGTP ^pN¿ÌAXÄ¢éBOÒÍC×EàÅÌVOi`BnÉdvÈáªpMðWIÆµ7) C^Cv IV ÅÍC{ckXÛÌqÊ G ^pN¿Å

C2 Åf8,9)ÆEFVÛÌCI^Åf10)ªCAN`Ì¿ðyÚµC»Ìe¿ÌêÂÆµÄÔÚIÉ×EinÉAMjð ADP {V»·éBßNC¼Ì^Cv

àe¿µ×EÌ`ÔªÛÅ«È­ÈéiFig. 2BjBãÒIV ÆÍÙÈèAN`ðWIÆ·éªAMjÅÍÈÌ^Cv IV ÅfÍAN`ðÁÙIÉCü·é±ÆÅ¼

é Rho ðÁÙIÉCüµlXÈeúËõ Sept. 2014 Vol.27 No.5  233(C) 2014 The Japanese Society for Synchrotron Radiation ResearchFig. 1

Catalytic mechanism of ADP­ribosylation reaction. Step 1: Cleavage of nicotinamide moiety and production ofoxocarbenium cation. Step 2: deprotonation of Arg177 of actin and making bond between NC1 of N­ribose andguanidyl nitrogen of Arg177.

Fig. 2A) Classication of ARTs based on their target specicity. B) Eect of ADP­ribosylation by Type III and TypeIV ARTs.

Ú×EinÉìpµC×EªÛ­`ÔÏ»ðN±·B×IV ÉªÞ³êAN`Ì Arg177ðÁÙIÉ ADP {VÛªÌàÉüèÝ°ÇàÅBµCÊÆµÄ×Eª»·éBIa ÉÍ ART Éæ­Û¶³ê½ 3 ÂÌÌæª¨«÷OÑÌ£ÈÇ°Ì@\ª¸íêºÈÇÌÇóªèC»ÌêÂÉ ADP_ribosylating Turn Turn (ARTTjø«N±³êéiFig. 2B)17,18)B[vÆÄÎêé ADP {V»Éå«­Ö^µÄ¢éEFVÛÍlXÈÅfðY¶·éªC»ÌêÂÅéCI^ÅfÍyfTujbgÅjbgÅé Ia ÆATué Ib Å\¬³êéBCI^ÅfÍC^Cv234  úËõ Sept. 2014 Vol.27 No.5Ìæªé19) B»ÌÌæÌÉ³çÉ 378Glu_X_Glu380(EXEj`[tª

èC±Ì Glu378Æ Glu380ÌñÂÌO^~_Í ADP {V»«ÉsÂÅéBðà ¡ CüyfÆî¿^pN¿Ì½OãÉ¨¯é¡Ì»Ìì»Æ\¢ÉîÃ­½@\äXÍC2003NÉ Ia PÌ20) C2008NÉÍ NAD{ ÌAiOÅé bTAD ð p ¢ ½ Ia _A N `  Ì ¡  Ì \¢21)ð¾ç©ÉµC\¢ÉîÃ­ ADP {V»Ì½
@\ÉÖ·ém©ð¾½Bµ©µC{Ìî¿Åé

NAD{ ªµ½¡Ìâ ADP {V»³ê½AN`ÈÇ½@\Ìð¾ÉKvÈC¾çêÄ¢È¢\¢îñà½­cÁÄ¢½B2. Cü½OãÉ¨¯é¡Ì»Ìì»±êÜÅÉ¨æ»90,000ð´¦éÌ^pN¿Ì Xü  » \ ¢ ª Protein Data Bank É o ^ ³ ê Ä ¢ éi2014N 9 jB»à»à»ÆÍÏêÈ¨¿ª®RÆÀñ¾àÌÅèC^pN¿à¯lÉ»ÆÈéB±Ì^pN¿Ì»ð¾é½ßÉ¤ÒÍ½­ÌXN[jOðs¤BÁÉÝìp·é^pN¿¯mÌ¡ÌÌ»ÈÇÍ»ÌÝìpÌ­³ÖÌl¶ÈÇC³çÈéHvªKvÅéBCI^Åf Ia É¨¢ÄCNADH Ü½Í NADPH ªµ½ Ia PÌÆ IaCbTADCAN`ÌOÒ¡Ìð¾ç©ÉµÄ«½20,21) B±Ì\¢ÍAMjÁÙI ARTÌÈ©ÅÌ¡Ì\¢ÅÁ½Bµ©µC{Ìî¿Åé NAD{ ÆÌ¡ÌâCADP {V»ðßç¦Ä¨ç¸C»Ì½@\ÌÚ×Ís¾ÅÁ½B±êçÌ¡Ì\¢ðð¾µCADP {V»ðß¨·é½ßÉ IaCAN`CNAD{ Ì»»ðsÁÄ«½Bµ©µCIaCAN`CNAD{ ÌOÒð¬µ»»ðÝÄà»ð¾é±ÆªÅ«È©Á½B±êÍCOÒªµ ADP {VîªAN`ÉtÁ³êéÆ Ia
Æ ADP {V»³ê½AN`ªð£·é½ßÅéÆl¦çê½B»ÌsöëÌÅCäXÍ Ia ÆAN`ÌÝCÂÜè NAD{ ÌµÄ¢È¢A|^Ì Ia_AN`¡ÌÌ»ð¾é±ÆÉ¬÷µ½iFig. 3AjBÜ
½CX üñÜÀ±ÌÛÌX»ª X üÌ½ËÌ×ÆÈé½ßCX»Ì¶¬ðh~·é½ßÌRÜÆµÄICCOZ[ðp¢ÄÅKðÌ¢ðsÁ½ÊCG`OR[É¨¢ÄÅàÇ¢½Ëª¾çê
½BG`OR[Ì ADP {V»ÖÌe¿ð©

Fig. 3Soaking method to create NAD{ binding Ia­actin complexand Ia­ADP­ribosylated actin from common apo­Ia­actin
crystal.

½Æ±ëCã¢ÈªçàG`OR[ª ADP {V»ðjQ·é±ÆªmF³ê½B±êçÌÛð¥ÜQ ÌêCADP {V»ª½ãÌ3CjBêûCìü¦ÄCA|^¡Ì»ÉÎµÈºÌìðsÁ½iFig.

»ð¾é±ÆªÅ«½BiFig. 3CjB3BjBP A|^¡Ì»ð NAD{ ÆG`OR[üðÜÞ»»ntÉZÐ3. ¡Ì\¢©çÌîñQ A|^¡Ì»ð NAD{ ðÜÞ»»ntÉZ
ü±Ìæ¤ÉÙÈéóµÌºÅ NAD{ ntÉA|^ Ia_ÐãCG`OR[ðÜÞ»»ntÉÚAN`Ì¡Ì»ðZ·±ÆÉæÁÄCNAD{_Ia_AµZÐN`Æ Ia_ADP {V»AN`Ìf[^ZbgðP ÅÍCNAD{ ÍµÄ¢½ª ADP»ÌÊCìü»ê¼ê1.75 Å Æ2.2 Å Ìªð\Å¾½B\¢ðÍÌ

{V»ÍN«Ä¢È¢»ð¾é±ÆªÅ«½iFig.

ÊC¡ÌÌSÌ\¢ÍA|^iFig. 4AjCNAD{ ^úËõ Sept. 2014 Vol.27 No.5  235

Fig. 4Overall structures of Ia­actin complex during the reaction. A) Apo­Ia­actin, B) NAD{­Ia­actin, C) Close­upview of NAD{­Ia­actin, D) bTAD­Ia­actin, E) Ia­ADP­ribosylated actin, and F) Close­up view of Ia­ADP­
ribosylated actin. ARTT loops are shown with dotted line in C and F.

iFig. 4BjCIa_ADP {V»AN`iFig. 4EjÆàÙÚ¯¶`ÅÁ½ªCî¿É¾ç©Èá¢ª©çê

ADP {V»³ê½ G_AN`Ì\¢ÍCSpvB Éæ è C ü ³ ê ½ à Ì ª Margarit ç É æ è ñ  ³ ê ½
iPDB: 2GWJ, 2GWK)22) ªCAN`{ÌÌ\¢Í¾ç

½B
NAD{_Ia_AN`É¨¢Äî¿ÊÉÍÍÁ«è©É³êÄ¢½àÌÌ ADP {VîÍC»Ìhç¬ÉÆµ½ NAD{ Ìdq§xª©çê½iFig. 3CjBADP æè©é±ÆÍÅ«Ä¢È©Á½Bµ©µCäXÍC¡ªÍ Ia Ì Asn335, Gln300, Arg295, Arg352Éæè¬çêÌ»ÌÅ ADP {V»½ðsÁ½½ßC½ãéæ¤ÉµÁ©èÆÅè³êCN {[XªÆjR`Ì Ia Æ ADP {V»AN`Ìð£ªW°çêC»A~hªÍ»ê¼ê Glu380Æ Arg296å½ÉæèÅÌÊCADP {Vîª Ia ÉÅè³êé±ÆÅhç®

è³êCjR`A~hªªüÈµ½\¢ðÆéiFig.

±ÆÈ­C±ÌÂ»É¬÷µ½Æl¦Ä¢é23)B

4CjBART É¨¯é NAD{ ÌüÈµ½\¢Í¼Ì ARTÅà©o³êÄ¨è¤ÊµÄ¢éÁÙIÈ\¢ÅéB

Ia_ADP {V»AN`ÅÍCADP {[Xª4. ½@\Í NAD{ ^Æ¯lÉ Ia Ì 4 ÂÌA~m_ÉµÁ©è

ADP {V»AN`CA|^CNAD{ ^CIa_ÆÅè³êÄ¢½Bµ©µCNAD{ ^É©çê½ÉbTAD ^C»ê¼êÌ¡Ì\¢©ç¾çê½æq[ÉÜêÈªÁÄ¢½ N {[XªÆjR`A~hÌ\¢IÁ¥ðîÉäXÍÈºÌæ¤ÉgStrain­Allevia­

ªÅÍCjR`A~hªÌdq§xªÁ¸µCNtion model i Ù £ Æ É a  f  jh ð ñ ¥ µ ½ i Fig.

{[XÌdq§xÍV½É Arg177ßTÉ©o³ê½B5A)21,23)B±ÌÆ«CADP {V»AN`Ì Arg177Í Ia ÌûÖø«o³êCADP {[XÆµÄ¢½iFig. 4FjB236  úËõ Sept. 2014 Vol.27 No.5

NAD{_Ia_AN`\¢©çCj´qÅéAN`Arg177¤½Ì N ´qÆ N {[XÌ NC1 ÆÌÔÌ£ðà ¡ CüyfÆî¿^pN¿Ì½OãÉ¨¯é¡Ì»Ìì»Æ\¢ÉîÃ­½@\Fig. 5

Í8.2 Å Å

Schematic illustrated mechanism of ADP­ribosylation. A) Cartoon representation of ``Strain­Alleviationmodel'', B) Schematic mechanism of ADP­ribosylation. Arrowheads indicate the nitrogen atoms of N­ribose(NC1) and side chain of Arg177 (N).

Á½B±Ì£ÅÍ¼Ú½·é±ÆÍÅ«`Iª\¢Ï»µÄæñÔÌª¶¬³êéBiFig. 5A,¸C·éÉÍ½ç©Ìû@Å±Ì8.2 Å ðkßéKv5B(iii)j±êÉæè N {[XÌ NC1 Æ Arg177ÌOA

ªjWfÌÚßðÂ\Éµ½B³çÉ ARTT [vÌéBµ©µCArg177ªßÃ­¾¯ÅÍê»Ì£ªkÜé±ÆÍÈ­ NAD{ ðÜÞ Ia ¤Å½©µçÌANA~m_ª­«©¯é±ÆÅCââ Arg177ªø«o³VªN±éKvªéÆl¦½Bê C Å I I É N  { [ X Æ Arg177 Ì  £ Í 8.2 Å © çNAD{ ª   · é Û É ADP  ª Í Ia Ì Asn335,1.49 Å ÉÜÅkßçê ADP {Vîª Arg177ÆGln300, Arg295, Arg352ÉæèÅè³êéBÁÉ Asn335

Å«éæ¤ÉÈéiFig. 5A, 5B(iv)jB

ÍAfjCGln300Í A {[XðC»µÄ Arg295Æ
Arg352ÍC2 ÂÌ_ÆÝìpµÄCNAD{ ðÉxÉÜèÈ°¢Ù£óÔisÀèÈóÔjÉµÄ¨èC±Ìæ¤ÈÁÙIÈ NAD{ Ì\¢ªCNAD{hADP {[5. ADP {V»³ê½AN`ÌüÛ»ÖÌe¿AN`Í×EàÉåÊÉ¶ÝµCæ­Û¶³ê½^XiIL\JxjEJ`Ij{jR`A~hÌ½
tðà½ç·Æª·éiFig. 5A, 5B(i)jB»±ÉAN`pN¿Åª·é±ÆÅ IaCÁÉ ARTT [vª\¢Ï»µ`ÆÉ«Ì§äÈÇdvÈ×E@\ÉÖ^µÄ¢éBÜ

iFig. 4C, 4FjCjR`A~hªØf³êæêÔÌÅ

½C×EiÌåvÈ\¬vfÆµÄCG_AN`iPèC×E^®«Cûk«C×EàAC×EÌé F_AN`i½ÊÌjÌ

éIL\JxjEJ`Iª¶¬³êéBjR`AÊÌjÆtBgóÅ~hØfãCIL\JxjEJ`IÍ Tyr251ÆJñóÔÌÔÅÏ»µÄ¢éBüÛ»µ½AN`Í 2 {`I_p ÝìpÉæèÀè»³êÄ¢éªCjR`ªQçêéæ¤ÉµÄAN`tBgð`¬µÄ¢

A~hªØê½¾¯ÅÍ8.2 Å Ì£ÍkÜçÈ¢iFig.

éiFig. 6AjBÊíÍ G_AN`Æ F_AN`ÌñóÔ5A, 5B(ii)jB

ÌÔÅ½tðÛ¿Èªç×EÌ\¢â`ÔðÛµÄ¢é

ADP {V»ãÌ\¢É¨¢Ä ADP ªÍËRµªC×EÌ^®«â×EªôÈÇKvÉ¶AN`tBÁ©èÆÅè³êÄ¢½±Æ©çCNAD{ÍjR`A~gÌLkªN±èClXÈêÅdvÈððÊ½hªØê½±ÆÅ Arg296ÉæéjR`A~hÌÅèªµÄ¢é24,25)Bð¯CN {[XªÙ£óÔ©çE·éB»ÌãC

ÅÍC±Ì ADP {V»³ê½AN`ÍÇÌæ¤ARTT [vÌA~m_Ì­«Éæè N {[X¤ÉüÛ»ðjQ·éÌ¾ë¤©BF_AN`ÍCPÊÌ

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6. ÜÆßÈãÌæ¤ÉA| Ia_AN`¡ÌÌ»ð ADP {V»Ì½êÆµÄpµC³çÉ NAD{ ÆRÜÌZÐðÆµÄ NAD{ ÆG`OR[Ì¯\[LOàµ­ÍiK\[LOðp¢C±êçÌ»©ç NAD{ _Ia_AN`Æ Ia_ADP {V»AN`Ìªð\Ì\¢ð¾ç©Éµ½B±ÌAN`ÌAMj  É t ^ ³ ê ½ ADP  { V  î Ì Â  » C ³ ç ÉNAD{ Æ Ia_AN`¡ÌCADP {V»AN`Æ Ia Ì¡ÌÍßÄÌñÅèC±êçÌ\¢©çAN`ÌAMjÌ ADP {V»Ì½@\ÆµÄgStrain­Alleviation model_Ù£ÆÉafhðñ¦µ½B·×ÄÌCüyf_î¿^pN¿ÉÂ¢Ä¡ñÌæ¤
ÉA|^¡Ì»ª¾çêéÆÍÀçÈ¢ªC±Ì¡Ì»ª¾é±ÆªÅ«êÎî¿ÌZÐÌdûæÅ Ia_AN`Æ¯lÉ½OãÌ»ð¾é±ÆªÅ«C½
@\ð¾ç©É·éêÆµÄg¤±ÆªÅ«éÆl¦Ä¢
Fig. 6Fitting an ADP­ribosylated actin into the lamentous actin
structure. A) Actin laments undergo treadmilling. Actinmonomers add at the ({) barbed end and dissociate fromthe (|) pointed end. B) The structure of intact actin molec­
ules are tted into the cryo­microscopy electron density map(EMD_5168). C) Superposed structure of ADP­ribosylated
actin on (B).

éB{¤ÍVwpÌæ¤u×EVOiO¡ÌÉæéVOimE`BEÌ\¢Iîbvi¤ÛèÔ
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F_AN`Ì G_AN`ÌzüðÏ@µC»êð³ÉtBgAN`Ìf\¢ðñÄµ½27) B³çÉÅßÅÍCNCIdq°÷¾ðp¢½è@Éæè6.6Å27) ¨æÑ6.0 Å28,29) ªð\Ìdq§xª¾çê½iFig.
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AN`ªqÆ§ÌáQðN±µÄ¨èCÅ«È¢±
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å·É³öðoÄ2010Næè»EBúËõ Sept. 2014 Vol.27 No.5  239

Modication reaction mechanism based on thecomplex structure of enzyme­substrate protein atpre­ and post­reaction states\The reaction mechanism of iota toxin fromClostridium perfringens \Toshiharu TSURUMURA

Kyoto Sangyo University, Kamigamo Motoyama Kita­ku, Kyoto, 603_8555 Japan

Hideaki TSUGE

Kyoto Sangyo University, Kamigamo Motoyama Kita­ku, Kyoto, 603_8555 Japan

Abstract

Ia is mono­ADP­ribosylating toxin secreted by Clostridium perfringens , which ADP­ribosylates toarginine 177 of actin specically. To understand the mechanism of ADP­ribosylation, it is neces­sary to reveal the complex structure of Ia­actin, which is enzyme­substrate protein. However, thecomplex structure of NAD{­Ia­actin and Ia­ADP­ribosylated actin are open question. To obtainthese structures, we rened the conditions of crystallization and X­ray diraction experiment.
Through trial and error, we succeeded to obtain the Ia­actin complex crystal without NAD{.
Using two dierent conditions of crystal soaking, we obtained the Ia­actin complex crystal hold­ing NAD{ and Ia­ADP­ribosylated actin complex crystal, and then we revealed high­resolutionstructures of them. These structures are correspond to pre­ and post­states of ADP­ribosylation,respectively. According to these results, we suggested ``strain­alleviation model'' as an ADP­
ribosylation mechanism in two articles (PNAS2008, PNAS2013). In this model, Ia ADP­ribosy­
lates actin arginine 177 via two oxocarbenium ion intermediates.
We introduce ADP­ribosylation mechanism based on these structures using complex crystals ofmodication enzyme and substrate protein; NAD{­Ia­actin and Ia­ADP­ribosylated actin.

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