165 Pages
English
Gain access to the library to view online
Learn more

Structural and mutational characterisation of Shigella pathogenicity factors [Elektronische Ressource] / vorgelegt von Naomi Tidten

-

Gain access to the library to view online
Learn more
165 Pages
English

Description

Structuralandmutationalcccchhhhaaaarrrraaaacccctttteeeerrrriiiissssaaaattttiiiioooonnnn ofShigellaPathogenicityFactors DissertationzurErlangungdesDoktorgradesderNaturwissenschaften(Dr.rer.nat.)demFachbereichPharmaziederPhilipps!UniversitätMarburgvorgelegtvonNaomi&idtenausBonnMarburg/Lahn2007 DieUntersuchungenzurvorliegendenArbeitwurdenaufAnregungvonHerrnPD Dr. Klaus Reuter und Herrn Prof. Dr. Gerhard Klebe am Institut fürPharmazeutische Chemie des Fachbereichs Pharmazie der Philipps!Universität Marburg in der Zeit von Mai 2004 bis Dezember 2007durchgeführt.VomFachbereichPharmaziederPhilipps!UniversitätMarburgalsDissertationam15.02.2008angenommen.Erstgutachter:Prof.Dr.GerhardKlebeZweitgutachter:PDDr.KlausReuterTagdermündlichenPrüfung:15.02.2008InErinnerunganmeinenGroßvater EsgibtmehrLeute,diekapitulieren,alssolche,diescheitern.HenryFord(1863!1947)Tableofcontents ITableofontentsTableofcontents.......................................................................................... I FiguresandTables ................................................................................... V 1 Introduction............................................................................................. 1 1.1 Bacillarydysentery–Shigellosis.........................................................

Subjects

Informations

Published by
Published 01 January 2008
Reads 39
Language English
Document size 8 MB

Exrait

Structuralandmutational
cccchhhhaaaarrrraaaacccctttteeeerrrriiiissssaaaattttiiiioooonnnn
ofShigellaPathogenicityFactors

Dissertation
zurErlangungdesDoktorgrades
derNaturwissenschaften
(Dr.rer.nat.)
demFachbereichPharmazie
derPhilipps!UniversitätMarburg
vorgelegtvon
Naomi&idten
ausBonn
Marburg/Lahn2007
DieUntersuchungenzurvorliegendenArbeitwurdenaufAnregungvonHerrn
PD Dr. Klaus Reuter und Herrn Prof. Dr. Gerhard Klebe am Institut für
Pharmazeutische Chemie des Fachbereichs Pharmazie der Philipps!
Universität Marburg in der Zeit von Mai 2004 bis Dezember 2007
durchgeführt.
VomFachbereichPharmaziederPhilipps!UniversitätMarburgalsDissertation
am15.02.2008angenommen.
Erstgutachter:Prof.Dr.GerhardKlebe
Zweitgutachter:PDDr.KlausReuter
TagdermündlichenPrüfung:15.02.2008InErinnerunganmeinenGroßvater
EsgibtmehrLeute,diekapitulieren,alssolche,diescheitern.
HenryFord(1863!1947)
Tableofcontents I
Tableofontents
Tableofcontents.......................................................................................... I
FiguresandTables ................................................................................... V
1 Introduction............................................................................................. 1
1.1 Bacillarydysentery–Shigellosis......................................................... 1
Shigellaeclassification........................................................................... 1
1.2 PathogenesisbyShigellae.................................................................. 2
1.3 MolecularbasisoftheShigellainvasionmechanism........................... 6
1.3.1 PathogenicityfactorIpaA,itschaperoneSpa15andthehostcell
proteinVinculin .......................................................................... 7
1.3.2 PathogenicityfactorIpgDanditschaperoneIpgE ........................ 9
1.3.3 PathogenicityfactorOspD1....................................................... 10
1.3.4 PathogenicityfactorIpgB2......................................................... 11
1.3.5 PathogenicityfactorVirF ........................................................... 11
1.4 tRNAguaninetransglycosylase,thegeneproductofvacCisatarget
forstructure!baseddrugdesign...................................................... 12
1.5 Aimsoftheproject ......................................................................... 14
1.5.1 PathogenicityfactorsIpaA,IpgE,IpgB2andOspD1.................... 14
1.5.2 SubstrateselectivityandspecificitystudyoftRNAguanine
transglycosylasesofthethreedomainsoflife,basedona
ZymomonasmobilisTGTmodelsystem..................................... 14
1.6 References ...................................................................................... 16
2 SelectedShigellaPathogenicityFactors................................................... 21
2.1 CloningandExpressionofipaA,spa15,vinc_hd,ipgB2,ospD1,
ipgE ................................................................................................ 22
2.2 PurificationofPathogenicityFactorsIpgB2,OspD1andIpgE ............ 26
2.2.1 IpgB2........................................................................................ 26
2.2.2 OspD1 ...................................................................................... 27
2.2.3 IpgE.......................................................................................... 27
2.3 CrystallisationofPathogenicityFactorsOspD1andIpgE .................. 28
2.4 HomologyModellingofPathogenicityFactorsIpaAandIpgE ............ 29
2.5 SummaryandOutlook..................................................................... 31
2.6 MaterialsandMethods .................................................................... 33
2.6.1 PrimersusedforPCRamplificationofpathogenicityfactorgenes
fromthevirulenceplasmidpCP301........................................... 33
II Tableofcontents
2.6.2 Buffers,Enzymes ...................................................................... 34
2.6.3 CleavageoftheGST!tag ........................................................... 34
2.6.4 Homolgymodelling................................................................... 35
2.7 References ...................................................................................... 35
2353 Glu iscriticalforsubstrateselectivityinTGT ...................................... 39
3.1 Results............................................................................................ 46
3.1.1 CrystalstructureofwildtypeZ.mobilisTGTincomplexwith
guanine .................................................................................... 46
3.1.2 EnzymaticcharacterisationofwildtypeZ.mobilisTGT.............. 47
3.1.3 ConstructionandenzymaticcharacterisationofZ.mobilis
235TGT(Glu Gln) .......................................................................... 48
2353.1.4 CrystalstructureanalysesofTGT(Glu Gln).............................. 49
3.2 Discussion ...................................................................................... 53
3.3 Summary ........................................................................................ 57
3.4 MaterialsandMethods .................................................................... 58
3.4.1 CloningandTGTpreparation .................................................... 58
Tyr3.4.2 PreparationoftRNA ............................................................... 58
3.4.3 Kineticparameters .................................................................... 58
3.4.4 Crystalstructureanalyses ......................................................... 60
3.4.5 ProteinDataBankAccessionCodes ........................................... 62
3.4.6 CrystallographicTables............................................................. 62
3.4.7 Acknowledgements................................................................... 65
3.5 References ...................................................................................... 65
4 CharacterisationofamodelsystemforthehumanTGTbindingpocket .. 69
4.1 Introduction.................................................................................... 69
4.2 Results............................................................................................ 73
4.2.1 Sequencecomparisonsandhomologymodelling....................... 73
4.2.2 ConstructionandenzymaticcharacterisationofZ.mobilis TGT
variants .................................................................................... 75
4.2.3 Crystalstructures...................................................................... 79
4.2.4 Moleculardynamicssimulation.................................................. 84
4.2.5 Co!crystallisationoftheTGTvariantswithpreQ derivatives ..... 87 1
4.3 Discussion ...................................................................................... 90
4.3.1 TheturnovernumbersoftheTGTvariantsdecrease
dramatically.............................................................................. 90
4.3.2 TheturnovernumbersofTGTvariantsincreaseforthesubstrate
basepreQ ................................................................................ 90 1Tableofcontents III
4.3.3 IncreasedK forpreQ .............................................................. 91 M 1
4.3.4 Z.mobiliswild!typeTGTiseventuallyabletobindqueuine....... 93
4.3.5 Outlook .................................................................................... 95
4.4 Summary ........................................................................................ 96
4.5 Supplement..................................................................................... 97
4.5.1 MALDI!TOF/MSanalysisoftheRNAproduct.............................. 97
4.5.2 MALDI!TOF/MSassaydrawbacks ............................................ 100
4.6 MaterialsandMethods .................................................................. 102
4.6.1 Homolgymodelling................................................................. 102
4.6.2 ConstructionoftheTGTmutantsC158V,V233G,C158V!V233G,
A232S!V233G,C158V!A232S!V233G..................................... 102
4.6.3 Expression,Purification........................................................... 103
4.6.4 Determinationofkineticparameters ....................................... 103
4.6.5 MALDI!TOF/MSanalysis.......................................................... 104
4.6.6 Crystallisation,datacollection,andprocessing........................ 105
4.6.7 MolecularDynamicsSimulation ............................................... 110
4.6.8 AlignmentandFigures ............................................................ 111
4.6.9 ProteinDataBankAccessionCodes ......................................... 111
4.7 Acknowledgements ....................................................................... 111
4.8 References .................................................................................... 111
5 Appendix............................................................................................. 119
5.1 X!rayrefinementToolkit!Manual ................................................ 119
5.1.1 Arrangementoffoldersandsubfolders ................................... 119
5.1.2 default.txt............................................................................... 120
5.1.3 Ligandrestraints(shelxldfxfile) ............................................. 121
5.1.4 Proteinsequencefile............................................................... 121
5.1.5 start.pdb................................................................................. 121
5.1.6 Firstcnsrun............................................................................ 121
5.1.7 Secondandfollowingcnsruns ................................................ 123
5.1.8 Firstshelxlrun........................................................................ 124
5.1.9 Secondandfollowingshelxlruns ............................................ 126
5.1.10 Utilisingthetoolkitafteramanualrefinement......................... 127
5.1.11 Additionalscripts.................................................................... 128
5.1.12 Templatefilesprovidedbythetoolkit ..................................... 129
5.2 MultipleSequencealignments ....................................................... 130
5.2.1 MultiplesequencealignmentofhumanTGTwithselectedbacterial
TGTstructures........................................................................ 130
IV Tableofcontents
5.2.2 MultiplesequencealignmentofeukaryoticandbacterialTGTs. 132
5.2.3 MultiplesequencealignmentofIpaAandIpgEhomologues ..... 133
5.3 Kineticmeasurements ................................................................... 134
35.3.1 [8! H]!guanineTGTassay...................................................... 134
3 Tyr5.3.2 [8! H]!guanine!tRNA TGT'washoutassay'.......................... 138
Asp5.4 MALDI!TOF/MSanalysisofRNAoligo reactionproducts ............ 139
5.5 References .................................................................................... 143
5.6 Abbreviations................................................................................ 144
Zusammenfassung ............................................................................... 147
Danksagungen ..................................................................................... 150
Erklärung ............................................................................................. 152
CurriculumVitae................................................................................... 153
Tableofcontents V
FFFFiiiigggguuuurrrreeeessssnnnndddd&&&&aaaabbbblllleeeessss
Figure1:Intra!andintercellularspread....................................................... 3
Figure2:PathogenesisofS.flexneri[7]. ...................................................... 4
Figure3:EntrancestructureduringShigellainvasion.................................... 6
Figure4:EffectofIpaAonthecytoskeletalrearrangementsoftheenty
focus[9]......................................................................................... 8
Figure5:IpaAVinculininteraction. .............................................................. 9
Figure6:MALDI!TOFmassspectrumofIpgE[2]. ....................................... 28
Figure7:homologymodelsofIpaAandIpgE.............................................. 30
Figure8:Queuinemodificationpathway. ................................................... 41
Figure9:BaseexchangemechanisminbacterialTGT................................. 42
Figure10:CrystalstructuresofbacterialandarchaealTGT. ....................... 44
235Figure11:CrystalstructuresofTGT(Glu Gln). ......................................... 50
Figure12:Homologymodeling.................................................................. 74
3 TyrFigure13:Excorporationof[8! H]!guaninefromlabelledtRNA inthe
presenceofqueuine. .................................................................... 79
233 158 232 233Figure14:Uncomplexed(apo)TGT(V G)andTGT(C V/A S/V G)crystal
structures..................................................................................... 80
233 158 232 233Figure15:preQ boundTGT(V G)andTGT(C V/A S/V G)crystal1
structures..................................................................................... 81
158 232 233Figure16:CrystalstructureofTGT(C V/A S/V G)co!crystallisedwith
queuine........................................................................................ 82
158 232 233Figure17:MDsimulationofTGT(C V/A S/V G)incomplexwith
queuine........................................................................................ 85
Figure18:MDsimulationofapowild!typeTGT. ........................................ 86
Figure19:preQ derivatives....................................................................... 87 1
158Figure20:CrystalstructureofBoc!preQ boundtotheTGT(C V/1
232 233A S/V G). ................................................................................ 88
Figure21:QueuinedockedintothehTGThomologymodelafter
minimisationwiththeMABforcefieldofMOLOC[42].................... 92
Figure22:Okadaetal.[21],Figure2. ........................................................ 93
Figure23:MALDI!TOF/MSanalysisofnativeandpreQ !modifiedRNA1
Aspoligo ......................................................................................... 97
AspFigure24:MALDI!TOF/MSanalysisofRNAoligo reactionproductsupon
incubationwithqueuine................................................................ 99
VI Tableofcontents

Table1:InvivoandinvitrosubstratesofTGTsofthethreedomains[42]. . 15
Table2:Constructscontainingthediffenentgenesorcombinationsofgenes,
expressionhostsandlevels[1,2]. ................................................. 23
Table3:IpgEcrystallisationconditionsidentifiedbyhighthroughput
screening[2]. ............................................................................... 29
Table4:PrimersusedforamplificationofgenesviaPCRattachingrestriction
sitestothe3'!and5'!ends. ......................................................... 33
Table5:StructuresofnaturalsubstratesofbacterialTGTandtheinhibitor
2!butyl!1H!imidazole!4,5!dicarboxylicacidhydrazide(BIH). ....... 40
235Table6:KineticparametersforwildtypeTGTandTGT(Glu Gln). ............. 47
Table7:Crystallographicdatacollectionandrefinementstatistics. ............ 62
Table8:Threedomainsoflife:FinaltRNAmodificationsandsubstratebases
ofTGTs. ....................................................................................... 70
Table9:KineticcharcteriationoftheTGTvariants...................................... 77
Table10:BindingpocketvolumescalculatedwithCASTp[43].................... 86
Table11:RelativemassdifferencesofnativeandmodifiedRNAsubstrates.97
AspTable12:MALDI!TOF/MSanalysisofRNAoligo reactionproducts.......... 98
Table13:Oligonucleotidesusedinmutagenesis. ..................................... 103
Table14:Crystallographictable............................................................... 107