m„c“”‘n’’‚8 i9~ m - all journals
TRANSCRIPT
!
11" !
2#
2013$
6%
& ' ( ) *
ChineseJournalofBioinformaticsVol.11No.2Jun.,2013
!"#$
:2012-08-22;%&#$
:2012-09-07.
'()*
:²�-D�51267
(Q20081411)8²�K"M*m³D¶X1267
(20062016)\=>
。
-.��
:́µ¶
,Email:Liuzg77@yahoo.com.cn
doi:103969/j.issn.1672-5565.2013.02.10
m�c���n'��8
I9~�_m
´µ¶
,·
¸
,F
£
,¹
º
(²�K"M*&'KQ*R
,²� �»
430068)
F
G
:�;12zy&�{�y�
pACY1Z���z�
,���t�K�TSz���E[���z|c����3�
�
。12zy� �
50I¡¢z�
,d£
PROCHECK�lT
,¤¥Gz�
A、B���t�K�
,�¦§z�
A1、B1。E[
���z|�¨^_z�
B1©ª«��5¬�
。PROCHECK、ProSai
WHATIF®l�¨¯°�z�
B1±²��3�
�
。¦§&³q-´�,
I(pACY1)&Z���
,L��`��µ¶�·¸¹º-�
。
HIJ
:q-´�,
I;12zy
;�t�K�
;E[���z|
KLDMN
:Q518.2 OPQR?
:A OS>N
:1672-5565(2013)-02-130-06
ConstructionandevaluationofathreedimensionalstructureofporcineaminoacylaseⅠLIUZhigang,DENGBei,YANGBo,HUZheng
(CollegeofBiotechnology,HubeiUniversityofTechnology,Wuhan430068,China)
Abstract:Athree-dimensionalstructureofporcineAminoacylaseI(pACY1)wasconstructedbyhomologymodelling.Themodelsafterenergyminizationwereundertheproceduresofmoleculardynamics(MD)simulationandrationalevaluation.50modelswerebuildedbyhomologymodelling.Amongthemthebesttwomodels(modelA,B)werechoosedbyPROCHECKandwerefutherrefinedbyenergyminization.TherefinedmodelsweresignedasmodelA1andmodelB1frommodelAandmodelBrespectively.ModelB1hasthebetterstablitiythanmodelA1duringMDsimulation.TheevaluationsillustratedmodelB1wasareasonablestructurebyPROCHECK,ProSaandWHATIF.Wegetareasonable3DstrucureofpACY1,whichcouldbefurtherappliedtostudytherelationbetweenstructureandfunctionofpACY1.Keywords:Aminoacylase1;HomologyModeling;EnergyMinization;MolecularDynamicsSimulation
á1¼åVⅠ(AminoacylaseI,EC3.5.1.14;
ACY1)ëì�½åè[
N-¼1
-L-á1â
,&¯
L-á1â
。u&ä�ÕcabW¾¿ãÀôá1
âÅÄ��Áz�&�\¥¦
[1-4]。�ºXÂÃ
Ä�Å|W�¶
ACY11���Ò{V��ZÆ{
ÇÑ�G\
[5-7]。ACY1-?*V
M2012
,WX
»r
Zn2+\SÐ�Èeßà
。Éá1¼åVⅠ
(PorcineaminoacylaseI,pACY1)c¦?á1â\`
�ÊÖ
,c\Ë»Ì1�
406»á1â8Ë»
Zn2+
ã¯
。Zn2+W
pACY1è[��OVW\
,ǹ©ª
«abÍ'Îië©?
Zn2+Øïðè[¥¦
。s
W
,X©ª«abÏÐ�Ñ�Ò<ÓëìÔú�
\¥¦
[8-9]。vÕd¤
pACY1\ëì()XÕÝ
�ÏÐ�\�ÕÄ
ACY1\½åÖ�
。
1997$
,ZhaonÍÎ�w½Éá1¼åVⅠ\
¡e
[10],sØÃk[ó\ëì
。2003$
,LindnerÍ
ν¶á1¼åVⅠ(hACY1)T347G��eq�
Â\¡eëì
。�Èeëì+�û¯¡e²³×Ñ
½
,¹Ñ�wÏë©ëì+\ëì
[11]。�ëì¢
Zn2+ë©X»ØáâÖ�
。/¹ú±��\ë�Ù
¾
,�Èeëì+T\XÂá1â61ÜV\��
ÄÍ'ë©4�£¤
[11]。Úµ
,��Q½É8¶á
1¼åVⅠ\��Pìïð\ú±��©ª÷ÝÍ
'që©?
Zn2+T
[12]。
w7³b^
,�?×Û�Â\ëì()
,¶EÜ
ACY1\Í'ë©ï�ĽåÖäyq½[
。b½
[�v»Z[
,ÜEÜ
pACY1\ëìïð½��8
³å
。��©ä�Ýò
,Þß�w½©ä\
pACY1�Èeßàé<ëì
。vb©ª
pACY1\ëìP
�ÕliàW½1ý
。
1 íY
1.1 BCV_m��
��
BLASTpßá½P
pACY1S12ÄSÐ
�Æm\�»óëì\ßàb�d
,cEW
hACY1\
T347G��e8
Neisseriameningitidismc58succinyldiaminopimelatedesuccinylase,
uëì
�
PDB¢\ÝôÖÔb
1Q7L8
1VGY。â¦
CLUSTALWÜ
pACY1\á1â��Pv�»ßà
\á1â��ïðQÜ
[13]。S�
,�¦
PHYREÜ
pACY1\�êëìïðñòØP�dïðëìQ
Ü
[14]。�YQÜë���Æ/êã8äRïð¶
bHI
。
1.2 ����
5¥SÐQÜ\ë�
,�¦
MODELLER9v7(http://www.salilab.org/modeller/)
Q�
,ì�
pACY1
Ö�\é<åêëì�Ì
。Ü�êëìqæç
8`�d�âw¦ål�êëì{�
,Ü
Zn2+ïð
èÐ{�
,ÜËeïðÜé{�
。Z�w\
50»�
u�ÌP�êëìñòÅĹ£��QÜ\ë�ï
ðÜt
,ßp©ä\�Ì��
PROCHECK3.5Öó
,
�wÆ©ä\Gu�Ì
[15]。
1.3 _m��
ZGu�Ì7ýê8èÖ�
,/ô¦
AMBER7�V¢\
SANDER�0
,ïðÕCÆ/å\³åÓ
ë
。w¦
PARM99ìí8Ï��îîeç�ìí
,
ï,�¤b
1.0,12?\
cutoff,�âð
1000�\Æ
ð�ÛWñY\��
,òióW
2500�\ôõ�
ÛWñY\��
[16-17]。���\�Q¢w¦
40kJ/mol
ö
?2\ìíÜOk\��ïðä÷
。Z³
åô\�Ìïð©ä�Öó
。
1.4 D������
¦
AMBER7�V¢\
GROMACS�0
,ïðÖ
�®ì*��
(moleculardynamicssimulation),øò
³åô�Ì\Ôú�
。��M�ù\UVW
,âð
4ä÷�Ö�®ì*��
。w¦��
2.0fs,��
MÛ
300K,��
200ps,8.0?\
nonbondedcutoff\s¤Òú
。��ÖóËe{�Èe�ÌÖ�®ì
*��³ô£��ì=\
RMSD(rootmeansquaredeviation)̈
Ýû�Ì\Ôú�
。
1.5 ��{��
Tî³åô\�Ì��ïð
PROCHECKÖó
,
ßáßàUü.8ý.ì=J�©ä�+\�Ì
。
Úµ
,â¦
ProSa2003Ü�Ìïð�ÕÖó
,ßá6
1ÕCJÖ4?þÿ\�Ì
[18]。Æô
,�¦
WHATIF6.0
Öó�Ì\61¾<ë�
[19]。
2 ë�P!"
2.1 BC<_m��
â¦
NCBI\
BLASTpK?Öó
pACY1ýá1
�\S�
,Öó\ë�÷Ý
,pACY18*V
M2012¢\u!è[V?kÇm\SÐ�
。#ß
óëìÄSÐ�½m\ßà¥b$ß�d
。¦
PHYREQ�ñò
pACY1ý��
406»á1â��
\�êëì
,ñò\ë�÷Ý
pACY1�
10»ü¤
\α-898
15» β-:;ã¯
。v8*V
M2012¢M¹¤V\üeëìåæ©
。
Z$ß�d\�êëìP
PHYREñò
pACY1\�êëìïðÜQ
,ßá½
hACY1\
T347G�
�eÏë©ëì+
(PDBID:1Q7L)8
Neisseriameningitidismc58succinyldiaminopimelatedesuccinylase(nmDAPE)(PDBID:1VGY)
¥b�d
。
hACY1\
T347G��e\�Èeëì+�û¯¡
e²³Z[½
,O[ó\ëìbÏë©ëì+
。�
SwissPdbViewer¢
,1Q7LÕÇü%£;�
nmDAPE½åëì+T
,ŽP�Èeëì+Fi\/ïÖ
á1â61µ
,u!�êëìDV1o£;�XG
。
Óë
C�\
RMSDÿ
,É�ó�
263»ïð£
;\á1â61¢k
201»61\
C�
RMSDÿb
1.43?。�¦
ClustalWQ��d��P
pACY1\á
1â��ïðQÜ
。«��QÜ�S�d¤st
PHYRE�êëì£;\ë�
,&Cq'êãúº�
898:;¢
,ØÆM{Û(_êã\¤C
。®�
`®HIô\Æô��QÜë�¼¢
1O¾
。�
1Q7L¢\P�Èeëì+Fi\ïÖá1â61
,
��Èeëì+Z[ô�&½ì=\�å
,��v
ïÖ61qÕ¥b�d
,Ø�QÜ¢)ÅØÅ*e
+÷¾
。
131&
2(
CDE
,+
:FGHIJKLMNO5
I6PQRF
L
1 CLUSTALW�
pACY1,T347GmutantofhACY1(1Q7L),mnDAPE(1VGY)��9��BC C
Fig1 SequencealignmentofpACY1,T347GmutantofhACY1(1Q7L),mnDAPE(1VGY)producedbyCLUSTALW
1Q7LrepresentsthechainsA,BandC,D.1VGYrepresentsthechainsA,B.Thedeletedsequencesin1Q7Lareindicatedinitalics
2.2 ����c��
pACY1\ËÈeÖ�¢rkX»ÏÐ�
[8,20],
¹�ó\
hACY1\ëì¢ö»Ì1Jrk�»
ÏÐ�
[11],vÜÝ
pACY1\ÏÐ�ÉÕkqS?
�dßà\ç�û�
。��
,ÜEØÃkst�d
¢ÏÐ�\,Lïð��
。5¥�-�Öó
,Biagini
8
Puigserver�ú
pACY1¢\ÏÐ�ç�61b
ASP112,His79,8
Glu147[21]。ÜE¦
MODELLER\èÐ{�\íY��ÏÐ�,L
。
hACY1��e
T347G[ó\
hACY1é<ëì
WÏë©ëì+
,�?�Èeëì+�롳Õ×
Ñ
,���Èe\Ïë©ëì+,L.å/0
。�
Å
1Q7Lb�dÜ
pACY1�Èeëìïð���
,
�?�d,Lqh9
MODELLEROd¤\åê{
�
,qÕ��»½åëì+¢êäRX»�Èeë
ì
。ÜE,
1Q7L\
AB.8
CD.Ö%¥b�»
�d
,Æ1¯�%��½
pACY1�Èeßàé<ë
ì
。
�¦
MODELLERQ�
,&¯
50»
pACY1\�
u�Ì
。¦
PROCHECKQ�S�øò½v
50»�
Ì\®eå*ë�
。¼÷
1O¾
,ßúu¢Æü\
�»�Ì
A8
BØïðÕCÆ/å
。³åô\�
ÌÜËb
A18
B1。
p
1 PROCHECKDE��9¡¢��£{
Table1 PROCHECKanalyzesthequalitiesofthemodels
ModelRamachandranplotquality(%) Goodnessfactor
Core AllowedGeneralDisall
owed
Dihe
dralsCovalentOverall
A 90.2 7.0 2.4 0.4 -0.07 -0.41 -0.19
B 91.7 7.6 0.4 0.2 -0.06 -0.47 -0.21
A1a 89.2 9.2 1.5 0.1 -0.18 0.04 -0.10
B1a 90.5 1.3 0.2 0 -0.15 0.04 -0.08
Notes:ab
ModelA1andB1minimizedfrommodelAandBrespectively.
2.3 D������
Ü�Ì
A18
B1\ËeÄu�Èeëìïð
½`ä÷Ö�®ì*��
。Öó��³ôëìì=
²ê\
RMSDÅÝû�Ì\Ôú�
。ë�¼¢
2O
¾
,�Ì
A18
B1\Ëeì=���³ôµ�_7
\
RMSD,¹!E\�Èeì=���³ôÕ�ý
£©
,Üݽ�Èeëì½Ëeh7Ôú
。Úµ
,�
Ì
B1\�Èeì=PËeì=²ê\
RMSD\Ý
Ù/?�Ì
A1,vÜÝ�Ì
B1½�Ì
A1?kh
231!
"
#
$
%
&
11'
ü\Ôú�
。
L
2 pACY1|¤¢_m9D������
Fig2 EvolutionofthedimericstructureofpACY1duringMDsimulations
Notes:RMSDsfromthestartingconformationsfortheModelsA1andB1areshownontheplots(a)and(b),respectively.Values
forthemonomersandthewholedimerareindicatedbygrayandblacklines,respectively
2.4 _m��{��
�¦
PROCHECKQ�Ý2½�Ì\®eå*
ë�
。¼÷
1O¾
,B1\®eå*ë�ÝÙ³?u
c�Ì
,ØÄ
B1\é6s¤aLJæ©SÐ��
ë�\¤3
。Ramachandranplotë�Æ÷Ý
(¢
3),�Ì
B190.5%\61��#47�f
,9.3%
L
3 ��
B19
RamachandranplotL
Fig3 RamachandranplottheΨ/ΦdistributionofModelB1
Notes:producedbyPROCHECK.90.5% residuesareinmostfavoredregions;
8.3% residuesareinadditionalallowedregions;
1.2% residuesareingeneralregions
\61�ÆM47�f
,̀61Ö4?q47\�
+f
。vÜÝ�Ì
B1¢\öX»61Jn?©ä
\®eå*ì=¢
,橽®eå*φ,ψ�îfÖ
4\¤3
。�¦
ProSaÝ2�Ì
A18
B1\61\
ÕCÖ4
,¼¢
4O¾
,�Ì
A1k_ïÖ61\ç
ÜÕ
(pairenergy)M?
0,¹�Ì
B1\Ok61\
ÕCn?þÿ
。vÂ÷Ý�Ì
A1ÉÕµ�5Âë
ì67�+¹�Ì
B1¢\61\ì=Jn?ÕC
©ä�+
。WHATIFïX�ø8½�Ì
B161\
¾<UC
,@á1â61²ê\åb�
。¼¢
5O
¾
,�Ì
B1\é»61\ÕCJ�
WHATIF�ú
\©ä��9f
(Zcore≥:
5.0)。
L
4 ProSa¥r��
A1c
B19u¦D§
Fig4 ProSaenergyprofilescalculatedforModelA1,B1
Notes:Thevalueofenergyshouldbeunder0forareliablemodel.Boldline:
PairenergygraphsofModelA1;Dottedline:
PairenergygraphsofModelB1
L
5 WHATIF¥r��
B19¨©£{
Fig5 WHATIFqualitycontrolvaluescalculatedforModelB1
Notes:Thescoresshouldbeaboveª
5forareliablemodel
331&
2(
CDE
,+
:FGHIJKLMNO5
I6PQRF
L
6 pACY1|¤¢_m��
(��
B1)
Fig6 RibbonrepresentationofthefinalpACY1model
Notes:ThebackboneofModelB1iscoloredbysecondarystructure,Thezincionisrepresentedastheredsphere.
3 ë"
��ÜSÐ��\�Ìïð³å8¹6Ý2
,
ÜEÞßú�Ì
B1¥bÜ
pACY1é<ëìSÐ�
�\Æ1ëì
。¼¢
6O¾
,pACY1Ëe�;Ë\
ÏÐ�ë©ëì+8�Èeëì+ã¯
,ö»Ëe
�
10» α898
15» β:;ã¯\
。vPÜE
ñò
pACY1\�êëìåæ
。u¢
,ÏÐ�ë©ë
ì+\
1~1888
311~406á1â61û¯½α8
9¾<βá³\ûË
;�Èeëì+\
189~310á
1â61=û¯�» α898X» βá³
。SÐ
���w\
pACY1�Â�ÈeëìbïX�©ª
pACY1\ëìP�ÕliàW½1ý
。Úµ
,�?
hACY18
pACY1�á1âè@SÐ�øw
89%,v»ëì\�úk�?>ó?¯|@\
hacy11�
��
。
«¬
:!"#$%&'(
Prof.JamesW.Caldwell
)*+
AMBER7。��OP
(References)[1] Mitta,M.,Kato,I.,Tsunasawa,S.Themucleotidesequenceof
humanaminoacylase1[J].BiochimicaetBiophysicaActa,
1993,1174:201203.
[2] Tsunasawa,S.Doesuncontrolledsurvivalofacetylatedpeptides
leadtocellproliferation?DeletionofNterminaldeacetylatingsys
temforprotein/peptideinsmalllungcarcinomacells[J].J.
Lab.Clin.Med.,1992,120:505506.
[3] Scaloni,A.,Jones,W.M.,Pospischil,M.,Sassa,S.,
Schneewind,O.,Popowicz,AM.,Bossa,F.,Graziano,SL.,
Manning,JM.Deficiencyofacylpeptidehydrolaseinsmallcell
lungcarcinomacelllines[J].J.Lab.Clin.Med.,1992,
120:546552.
[4] Maceyka,M.,Nava,V.E.,Milstien,S.,Spiegel,S.Amino
acylase1isasphingosinekinase1interactingprotein[J].FEBS
Letters,2004,568:3034.
[5] Sass,J.O.,Olbrich,H.,Mohr,V.O.,Hart,C.,Woldseth,
B.,Krywawych,S.,Bjurulf,B.,Lakhani,PK.,Buchdahl,
RM.,Omran.H.Neurologicalfindingsinaminoacylase1defi
ciency[J].Neurology,2007,68:21512153.
[6] TylkiSzymanska,A.,Gradowska,W.,Sommer,A.,Heer,
A.,Walter,M.,Reinhard,C.,Omran,H.,Sass,JO.,Ju
recka,A.Aminoacylase1deficiencyassociatedwithautisticbe
havior[J].JInheritMetabDis.,2010,33:14
[7] Sommer,A.,Christensen,E.,Schwenger,S.,Seul,R.,
Haas,D.,Olbrich,H.,Omran,H.,Sass,JO.Themolecular
basisofaminoacylase1deficiency[J].BiochimicaetBiophysica
Acta,2011,1812:685690.
[8] Heese,D.,Berger,S.,RhmK.H.Nuclearmagneticrelaxation
studiesoftheroleofthemetalioninM2+n subtitutedaminoacylase
1[J].Eur.J.Biochem.,1990,188:175180.
[9] Tang,Z.Y.,Yu,J.Y.,Zhou,Q.,He,B.,Wang,ZF.,
Zhou,HM.Secondarystructureofholoandapoaminoacylase
frompredication,circulardichroism,andftramanspectroscopy
[J].J.Biochem.,1995,118:706709.
[10] Zhao,HY.,Meng,WY.,Lin,ZJ.,ZhouHM.Crystallization
andpreliminaryXraystudiesofporcinekidneyaminoacylase1
[J].ChineseScienceBulletin,1997,42:17501752.
[11] Lindner,H.,Lunin,V.V.,Alary,A.,Hecker,R.,Cygler,
M.,Ménard,R.Essentialrolesofzincligationandenzymedim
erizationforcatalysisintheaminoacylase1/M20family[J].J.
Biol.Chem.,2003,278:4449644504.
[12] Lindner,H.,Alary,A.,Wilke,M.,Sulea,T.Probingthe
AcylBindingPocketofAminoacylase1[J].Biochemistry,
2008,47:42664275.
[13] Larkin,MA.,Blackshields,G.,Brown,NP.,Chenna,R.,
McGettigan,PA.,McWilliam,H.,Valentin,F.,Wallace,
IM.,Wilm,A.,Lopez,R.,Thompson,JD.,Gibson,TJ.,
Higgins,DG.ClustalWandClustalXversion2.0[J].Bioin
formatics,2007,23(21):29472948.
[14] Kelley,LA.,Sternberg,MJE.Proteinstructurepredictionon
theweb:acasestudyusingthePhyreserver[J].NatureProto
cols,2009,4:363371.
[15] Laskowski,RA.,MacArthur,MW.,Moss,DS.,Thornton,
431!
"
#
$
%
&
11'
JM.PROCHECK:aprogramtocheckthestereochemicalquality
ofproteinstructures[J].J.App.Cryst.,1993,26:283291.
[16] Wang,J.,Cieplak,P.,Kollman,PA.Howwelldoesare
strainedelectrostaticpotential(RESP)modelperformincalcu
latingconformationalenergiesoforganicandbiologicalmole
cules?[J].J.Comput.Chem.,2000,21:10491074.
[17] Pang,Y.P.,Xu,K.,Yazal,JE.,Prendergas,FG.Successful
moleculardynamicssimulationofthezincboundfarnesyltrans
feraseusingthecationicdummyatomapproach[J].ProteinSci
ence,2000,9:18571865.
[18] Wiederstein,M.,Sippl,MJ.ProSAweb:interactivewebserv
icefortherecognitionoferrorsinthreedimensionalstructuresof
proteins[J].NucleicAcidsResearch,2007,35:W407W410.
[19] Vriend,G.Amolecularmodelinganddrugdesignprogram[J].
J.Mol.Graph.,1990,8:5256.
[20] Pittelkow,S.,Lindner,H.,Rhm,KH.Humanandporcine
aminoacylase1overproducedinabaculovirusexpressionvector
system:evidenceforstructuralandfunctionalidentitywithen
zymesisolatedfromkidney[J].ProteinExpr.Purif.,1998,
12:269276.
[21] Biagini,A.,Puigserver,A.Sequenceanalysisoftheaminoacy
lase1family.Anewproposedsignatureformetalloexopeptidases
[J].Comp.Biochem.Physiol.PartB,2001,128:469481.
531&
2(
CDE
,+
:FGHIJKLMNO5
I6PQRF