Structural and functional characterization of Yersinia enterocolitica type III secretion effectors and chaperones [Elektronische Ressource] / von Renate Carina Büttner
144 Pages
English
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Structural and functional characterization of Yersinia enterocolitica type III secretion effectors and chaperones [Elektronische Ressource] / von Renate Carina Büttner

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Learn all about the services we offer
144 Pages
English

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Structural and functional characterization of Yersinia enterocolitica type III secretion effectors and chaperones Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades einer Doktorin der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Renate Carina Büttner aus Dresden 1. Referent: Honorarprofessor Dr. Dirk Heinz 2. Referentin: Professor Dr. Petra Dersch eingereicht am: 07.01.2008 mündliche Prüfung (Disputation) am: 08.02.2008 Druckjahr 2008 Vorveröffentlichungen der Dissertation Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Publikationen Büttner, C.R., Cornelis, G.R., Heinz, D.W. & Niemann, H.H. (2005). Crystal structure of Yersinia enterocolitica type III secretion chaperone SycT. Protein Sci. 14: 1993-2002 Büttner, C.R., Sorg, I., Cornelis, G.R., Heinz, D.W. & Niemann, H.H. (2008). Structure of the Yersinia enterocolitica type III secretion translocator chaperone SycD. J. Mol. Biol. 375: 997-1012 Tagungsbeiträge Büttner, C.R., Niemann, H.H., Heinz, D.W.

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Published 01 January 2008
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Structural and functional characterization
of Yersinia enterocolitica type III secretion
effectors and chaperones



Von der Fakultät für Lebenswissenschaften

der Technischen Universität Carolo-Wilhelmina

zu Braunschweig

zur Erlangung des Grades einer

Doktorin der Naturwissenschaften

(Dr. rer. nat.)

genehmigte

D i s s e r t a t i o n








von Renate Carina Büttner
aus Dresden


















































1. Referent: Honorarprofessor Dr. Dirk Heinz
2. Referentin: Professor Dr. Petra Dersch
eingereicht am: 07.01.2008
mündliche Prüfung (Disputation) am: 08.02.2008
Druckjahr 2008




Vorveröffentlichungen der Dissertation

Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für
Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab
veröffentlicht:



Publikationen

Büttner, C.R., Cornelis, G.R., Heinz, D.W. & Niemann, H.H. (2005). Crystal structure of
Yersinia enterocolitica type III secretion chaperone SycT. Protein Sci. 14: 1993-2002

Büttner, C.R., Sorg, I., Cornelis, G.R., Heinz, D.W. & Niemann, H.H. (2008). Structure of the
Yersinia enterocolitica type III secretion translocator chaperone SycD. J. Mol. Biol. 375: 997-
1012


Tagungsbeiträge

Büttner, C.R., Niemann, H.H., Heinz, D.W. Crystal structure of the type III secretion
ndchaperone SycT from Yersinia enterocolitica. (Poster) 22 European Crystallographic
Meeting, Eötvös Loránd University, Budapest, Hungary (2004).

Büttner, C.R., Heinz, D.W., Niemann, H.H. Crystal structure of the type III secretion
chaperone SycT from Yersinia enterocolitica. (Poster) Recent Advances in Macromolecular
Crystallization, Le Bischenberg, France (2005).

Büttner, C.R., Heinz, D. W., Niemann, H.H. The crystal structure of the Yersinia
thenterocolitica type III secretion chaperone SycT. (Vortrag) 8 Heart of European
Crystallography. Karlovy Vary, Czech Republic (2005).

Büttner, C.R., Heinz, D.W., Niemann, H.H. Crystal structure of the type III secretion
chaperone SycT from Yersinia enterocolitica. (Poster) Murnau Conference on Structural
Biology of Macromolecular Recognition, Murnau (2005). Posterpreis

Büttner, C.R., Heinz, D.W., Niemann, H.H. Crystal structure of the type III secretion
chaperone SycT from Yersinia enterocolitica. (Poster) Annual Meeting of the German Society
for Crystallography, University of Freiburg (2006). Posterpreis

Büttner, C.R., Heinz, D.W., Niemann, H.H. Crystal structure of the Yersinia enterocolitica
type III secretion chaperone SycT. (Poster) Crystallography School, Como, Italy (2006).

Büttner, C.R., Heinz, D.W., Niemann, H.H. Structure of the Yersinia enterocolitica type III
secretion translocator chaperone SycD. (Poster) Murnau Conference on Structural Biology of
Disease Mechanisms, Murnau (2007).
CONTENTS i


Contents
Abbreviations............................................................................................................................v
Summary ...................................................................................................................................1
1 Introduction ......................................................................................................................2
1.1 The pathogenic bacterium Yersinia........................................................................................................... 2
1.2 Type III secretion - Yersinia’s stratagem .................................................................................................. 3
1.3 T3SS translocators and effectors in Yersinia ............................................................................................ 6
1.4 The Yersinia effector YopT....................................................................................................................... 8
1.5 T3SS chaperones....................................................................................................................................... 9
1.5.1 Classes of T3SS chaperones .......................................................................................................... 10
1.5.2 Postulated functions of T3SS chaperones...................................................................................... 12
1.6 The Yersinia T3SS chaperones SycT and SycD...................................................................................... 15
1.7 Aims of the thesis.................................................................................................................................... 16
2 Results..............................................................................................................................17
2I Characterization of the effector YopT and its cognate chaperone SycT ..................17
2I.1 Strategies to improve the stability of YopT .......................................................................................... 17
2I.1.1 Expression test analysis of new yopT and yopT/sycT constructs .................................................. 17
2I.1.2 In vitro translation of YopT deletion mutants................................................................................ 18
2I.1.3 Refolding of YopT......................................................................................................................... 19
2I.1.4 Follow-up investigations of the chaperone-binding site in YopT.................................................. 20
2I.1.5 Summary of the attempts to improve the YopT stability............................................................... 21
2I.2 Binding studies between the YopT/SycT complex and RhoA.............................................................. 22
2I.3 Modeling of the YopT structure............................................................................................................ 23
2I.4 Biochemical characterization of SycT .................................................................................................. 24
2I.4.1 Generation of sycT expression constructs ..................................................................................... 24
2I.4.2 Gene expression and protein purification of SycT ........................................................................ 25
2I.4.3 SycT forms homodimers in solution.............................................................................................. 26
2I.5 Crystallization of SycT ......................................................................................................................... 26
2I.5.1 Wild-type SycT ............................................................................................................................. 26
2I.5.2 Selenomethionine-labeled SycT .............................................................................................. 27 1-122
2I.6 Structure determination of SycT ........................................................................................................... 28
2I.7 Structural analysis of SycT ................................................................................................................... 31
2I.7.1 Structural overview of SycT.......................................................................................................... 31
2I.7.2 A closed cavity in the SycT dimerization interface ....................................................................... 34
2I.7.3 Hydrophobic surface patches in SycT ........................................................................................... 34
2I.7.4 Interaction of the C-terminal peptide with hydrophobic surface patches ...................................... 35
2II Type III secretion Class II chaperone SycD................................................................37
2II.1 Biochemical characterization of SycD.................................................................................................. 37
2II.1.1 Gene expression, isolation and purification of full-length SycD................................................ 37 ii CONTENTS

2II.1.2 Limited proteolysis of SycD identifies a stable fragment...........................................................38
2II.1.3 New expression construct SycD ..........................................................................................39 21-163
2II.1.4 Surface engineering in SycD ......................................................................................................40
2II.1.5 Oligomerization state of SycD in solution..................................................................................41
2II.2 Complex formation studies of SycD with translocator YopD...............................................................43
2II.2.1 YopD peptides....................................................................................................................43 278-300
2II.2.2 Binding studies and co-crystallization of SycD with YopD ...............................................44 278-300
2II.3 Crystallization of SycD .........................................................................................................................45
2II.3.1 Large, beautiful looking SycD crystals diffract only poorly ...............................................45 21-163
2II.3.2 Engineering of the tetragonal SycD crystals using dehydration..........................................46 21-163
2II.3.3 Reductive methylation of SycD yields well-diffracting crystals .........................................47 21-163
meth
2II.3.4 Engineering of the SycD crystals via heavy atom derivatization ...................................48 21-163
2II.4 Structure determination of SycD ....................................................................................................48 21-163
2II.4.1 How to pack 34 SycD molecules ?......................................................................................48 21-163
meth2II.4.2 Attempts to solve the SycD structure by anomalous phasing........................................49 21-163
meth
2II.4.3 Molecular replacement of SycD and PHASER sensitivity............................................50 21-163
2II.5 Structural overview of SycD ..........................................................................................................54 21-163
2II.6 Ambiguity of the SycD quaternary structure.........................................................................................57
meth2II.6.1 SycD packing analysis reveals two possible dimer assemblies .....................................57 21-163
2II.6.2 Characterization of dimer assembly alternatives 1 and 2............................................................58
2II.6.3 SycD forms head-to-head dimers in solution..............................................................................59
2II.6.4 A third dimer assembly indicates flexible SycD dimerization....................................................64
2II.7 Relevance of the SycD dimerization interface in vivo...........................................................................67
2II.8 Determination of further interaction surfaces in SycD..........................................................................69
3 Discussion........................................................................................................................ 71
3I SycT – a typical and special T3SS effector chaperone ? ............................................ 71
3I.1 SycT lacks the dimerization helix .........................................................................................................71
3I.2 The hydrophobic surface patches in SycT are spatially conserved .......................................................72
3I.3 Does effector binding require conformational rearrangements of SycT?..............................................73
3I.4 The cavity in the SycT dimerization interface is closed and uncharged................................................76
3I.5 YopT structural model and chaperone-binding domain ........................................................................77
3II SycD – a multi-faceted Class II chaperone with multiple faces................................ 78
3II.1 Comparison with the LcrH/SycD homology model ..............................................................................78
3II.2 Alternative dimerization of SycD..........................................................................................................79
3II.3 Affinity of the SycD homodimer...........................................................................................................81
3II.4 Mapping of mutagenesis results onto the SycD structure......................................................................82
3II.5 Implications for further interaction surfaces in SycD............................................................................85
3II.6 The dimerization interface in SycD – a new interaction surface? .........................................................87
3III Concluding remarks ..................................................................................................... 88
4 Outlook............................................................................................................................ 89 CONTENTS iii

4I SycT and its cognate effector YopT..............................................................................89
4II Translocator chaperone SycD ......................................................................................90
5 Materials and Methods ..................................................................................................91
5.1 Chemicals and material ........................................................................................................................... 91
5.2 Molecular weight standards .................................................................................................................... 91
5.3 Enzymes.................................................................................................................................................. 91
5.4 Special chemicals.................................................................................................................................... 92
5.5 Oligonucleotides ..................................................................................................................................... 92
5.5.1 Primers used in polymerase chain reactions .................................................................................. 92
5.5.2 Site-directed mutagenesis .............................................................................................................. 93
5.6 Plasmids .................................................................................................................................................. 94
5.7 Oligopeptides .......................................................................................................................................... 97
5.8 Bacterial strains....................................................................................................................................... 97
5.9 Antibodies ............................................................................................................................................... 98
5.10 Protein biochemistry ............................................................................................................................. 98
5.10.1 Solutions and buffers.................................................................................................................. 98
5.10.2 Media.......................................................................................................................................... 99
5.10.3 Absorption coefficients of proteins used for concentration determination............................... 100
5.11 Protein production............................................................................................................................... 100
5.11.1 YopT/SycT and SycT............................................................................................................... 100
5.11.2 SycD......................................................................................................................................... 101
5.11.3 Production of selenomethionine-labeled substituted SycT....................................................... 102
5.11.4 Gene expression using auto-inducing medium......................................................................... 102
5.11.5 Expression test.......................................................................................................................... 103
5.11.6 In vitro translation .................................................................................................................... 103
5.11.7 Reductive methylation of lysines ............................................................................................. 103
5.12 Protein analytical methods .................................................................................................................. 104
5.12.1 Gel electrophoresis ................................................................................................................... 104
5.12.2 Limited proteolysis................................................................................................................... 104
5.12.3 Western blotting ....................................................................................................................... 104
5.12.4 N-terminal sequencing.............................................................................................................. 104
5.12.5 Dynamic light scattering........................................................................................................... 105
5.12.6 Surface plasmon resonance spectroscopy................................................................................. 105
5.12.7 Circular dichroism.................................................................................................................... 105
5.13 Bioinformatics..................................................................................................................................... 105
5.14 X-ray structural analysis ..................................................................................................................... 106
5.14.1 Crystallization experiments ...................................................................................................... 106
5.14.2 Crystal engineering................................................................................................................... 108
5.14.3 Data collection and processing................................................................................................. 109
5.14.4 Anomalous dispersion in structure solution ............................................................................. 109
5.14.5 Structure determination ............................................................................................................ 110
5.14.6 Model building and structure refinement ................................................................................. 111
5.14.7 Structural analysis and visualization ........................................................................................ 111 iv CONTENTS

5.15 Experiments in vivo .............................................................................................................................111
References ............................................................................................................................. 113
Appendix............................................................................................................................... 126
Danksagung .......................................................................................................................... 129
Lebenslauf............................................................................................................................. 132
ABBREVIATIONS v

Abbreviations
Å Ångstrøm (0.1 nm)
aa Amino acids
AEC Anion exchange chromatography
R
Amp Ampicillin resistance
APS Ammonium persulfate
AraC Transcriptional activator
BESSY Berliner Elektronenspeicherring-Gesellschaft für Syn-
chrotronstrahlung
BLAST Basic Local Alignment Search Tool
c Concentration
C- Carboxy terminus
CAPS N-cyclohexyl-3-aminopropane sulfonic acid
CBD Chaperone-binding domain
CCD Charge-coupled device
Cdc42 Small GTPase of the Rho family
C/H/D Catalytic triad in YopT-like cysteine proteases, cys-
teine/histidine/aspartate
R
Cm Chloramphenicol resistance
12
Da Dalton (equals the mass of 1/12 of the carbon C isotope)
DC Dendritic cell
DESY Deutsches Elektronensynchrotron
DLS Dynamic /quasi-elastic light scattering
E. coli Escherichia coli
ECL Enhanced chemiluminescence
ECM Extracellular matrix
EDTA Ethylenediaminetetraacetic acid
EHEC Enterohemorrhagic E. coli
EPEC Enteropathogenic E. coli
ESI Electro-spray ionization
ESRF European Synchrotron Radiation Facility
FPLC Fast protein liquid chromatography
GAP GTPase activating protein
GDI Guanine nucleotide dissociation inhibitor
GEF Guanine nucleotide exchange factors
GenBank Genetic sequence database maintained by the US National
Center for Biology Information
GFP Green fluorescent protein
GL Glycine linker
GSH Seph Glutathione sepharose
vi ABBREVIATIONS

GST Glutathione S-transferase, used as affinity tag
His Six successive histidine residues, affinity tag 6
HPLC High performance lipid chromatography
IB Inclusion body
IPTG Isopropyl-β-D-thiogalactopyranoside
K Kelvin
RKan Kanamycin resistance
K Affinity in terms of equilibrium dissociation constant D
kDa Kilodalton
LcrH SycD homolog in Y. pestis and Y. pseudotuberculosis
LDAO N,N-Dimethyldodecylamine-N-oxide
MALDI-TOF-MS Matrix-assisted laser desorption ionization-Time of Flight-
Mass spectrometry
MAPK Mitogen activated protein kinase
meth Reductively methylated
MLD Membrane localization domain
MME Monomethylether
MR Molecular replacement
M Molecular mass r
M Theoretical molecular mass derived from amino acid sequence r theo
M Experimental molecular mass derived from SEC r SEC
MWCO Molecular weight cut-off
N- Amino terminus
NCS Non-crystallographic symmetry
NiNTA Nickel nitrilotriacetic acid
OD Optical density at wavelength
oN Over night
PAGE Polyacrylamide gel electrophoresis
PBS Phosphate buffered saline
PCR Polymerase chain reaction
PcrH Pseudomonas aeruginosa translocator chaperone
PDB Protein data bank
PEG Polyethylene glycol
Rac1 Small GTPase of the Rho family
RFZ Rotation function Z-score
rH Relative humidity
r Hydrodynamic radius H
RhoA Small GTPase of the Rho family
r.m.s.d. Root mean square deviation
Standard deviation
SA Surface area

lsl