Structure and function of virulence associated proteins from Pseudomonas aeruginosa and Yersinia enterocolitica [Elektronische Ressource] / von Gregor Hagelüken
154 Pages
English
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Structure and function of virulence associated proteins from Pseudomonas aeruginosa and Yersinia enterocolitica [Elektronische Ressource] / von Gregor Hagelüken

Downloading requires you to have access to the YouScribe library
Learn all about the services we offer
154 Pages
English

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Structure and function of virulence associated proteins from Pseudomonas aeruginosa and Yersinia enterocolitica Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Gregor Hagelüken aus Paderborn Honorarprofessor Dr. Dirk Heinz 1. Referent: Professor Dr. Petra Dersch 2. Referentin: 01.10.2007 eingereicht am: 03.12.2007 mündliche Prüfung (Disputation) am: 2007 Druckjahr: Vorveröffentlichungen der Dissertation Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für Lebenswissen-schaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Publikationen Hagelueken, G., Adams, T. M., Wiehlmann, L., Widow, U., Kolmar, H., Tümmler, B., Heinz, D. W. & Schubert, W. D. (2006). The crystal structure of SdsA1, an alkylsulfatase from Pseudomonas aeruginosa, defines a third class of sulfatases. Proc. Natl. Acad. Sci U. S. A 103, 7631-7636 Hagelueken, G., Wiehlmann, L., Adams, T. M., Kolmar, H., Heinz, D. W., Tümmler, B. & Schubert, W. D. (2007). Crystal structure of the electron transfer complex rubredoxin-rubredoxin reductase of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci U. S.

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Published 01 January 2007
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Language English
Document size 459 MB

Exrait


Structure and function of virulence associated
proteins from Pseudomonas aeruginosa and
Yersinia enterocolitica



Von der Fakultät für Lebenswissenschaften

der Technischen Universität Carolo-Wilhelmina

zu Braunschweig

zur Erlangung des Grades eines

Doktors der Naturwissenschaften

(Dr. rer. nat.)

genehmigte

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


















von Gregor Hagelüken
aus Paderborn





































Honorarprofessor Dr. Dirk Heinz 1. Referent:
Professor Dr. Petra Dersch 2. Referentin:
01.10.2007 eingereicht am:
03.12.2007 mündliche Prüfung (Disputation) am:
2007 Druckjahr:

Vorveröffentlichungen der Dissertation

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




Publikationen

Hagelueken, G., Adams, T. M., Wiehlmann, L., Widow, U., Kolmar, H., Tümmler, B., Heinz,
D. W. & Schubert, W. D. (2006). The crystal structure of SdsA1, an alkylsulfatase from
Pseudomonas aeruginosa, defines a third class of sulfatases. Proc. Natl. Acad. Sci U. S. A
103, 7631-7636

Hagelueken, G., Wiehlmann, L., Adams, T. M., Kolmar, H., Heinz, D. W., Tümmler, B. &
Schubert, W. D. (2007). Crystal structure of the electron transfer complex rubredoxin-
rubredoxin reductase of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci U. S. A 104, 12276-
12281

Tagungsbeiträge

Hagelüken, G., Adams, T.M., Wiehlmann, L., Kolmar, H., Tümmler, B., Heinz, D.W., Schu-
bert, W.-D.: Crystal structure of PA0740, a zinc hydrolase probably involved in quorum sens-
thing of Pseudomonas aeruginosa. (Poster) 20 Congress of the International Union of
Crystallography, Florenz, Italien, 2005

Hagelüken, G., Adams, T.M., Wiehlmann, L., Kolmar, H., Tümmler, B., Heinz, D.W., Schu-
bert, W.-D.: Crystal structure of PA0740, a novel zinc-dependent sulfatase from the human
pathogen Pseudomonas aeruginosa. (Vortrag) 8. Heart of European Bio-Crystallography
Meeting, Karlsbad, Tschechien, (2005).

Hagelüken, G., Adams, T.M., Wiehlmann, L., Kolmar, H., Tümmler, B., Heinz, D.W., Schu-
bert, W.-D.: The crystal structure of SdsA1, an alkylsulfatase from Pseudomonas aeruginosa,

defines an independent, third mechanistic class of sulfatases. (Vortrag) Jahrestagung der
Deutschen Gesellschaft für Kristallographie, Freiburg (2006).

Hagelüken, G., Adams, T.M., Wiehlmann, L., Kolmar, H., Tümmler, B., Heinz, D.W., Schu-
bert, W.-D.: SdsA1 from P. aeruginosa, defines a new mechanistic class of sulfatases.
rd(Poster) 23 European Crystallographic Meeting, Leuven, Belgien, 2006. Bestes Poster.
Contents I
Contents
SUMMARY 1
I INTRODUCTION 3
IA Structure and function of PA0740 and PA5349 3
IA.1 Pseudomonas aeruginosa 3
IA.2 Quorum sensing is crucial for P. aeruginosa virulence 6
IA.3 Identification of virulence factors by transposon mutagenesis 8
IA.4 PA0740 – an “enzyme of unknown function” 10
IA.5 PA5349 encodes a rubredoxin reductase 11
IB Structure of the type-III secretion effector YopO 12
IB.1 Yersinia spp. 12
IB.2 Genetic differences between pathogenic Yersiniae 14
IB.3 The Yersinia type-III secretion system 15
IB.4 Yops – Yersinia outer proteins 17
IB.5 YopO – a multifaceted T3SS effector 19
IC Aims of this work 22
IC.1 Structure and function of PA0740 and PA5349 22
IC.2 Structure of the type-III secretion effector YopO 22
II RESULTS 24
IIA Structure and function of PA0740 (SdsA1) 24
IIA.1 Protein expression and purification 24
IIA.2 Unravelling the function of PA0740 24
IIA.3 P. aeruginosa utilizes SdsA1 to grow on SDS 26
IIA.4 SdsA1 cleaves SDS and other primary alkylsulfates 27
IIA.5 Activity of SdsA1 is metal dependent 29
IIA.6 Determination of the crystal structure of SdsA1 31
IIA.7 Crystal structure of SdsA1 40
IIA.8 Binding of substrate, product and inhibitor to the active site 46
IIA.9 Mutational analysis to corroborate the inferred mechanism 50
IIB Crystal structure of the RdxR/Rdx complex 54 II Contents
IIB.1 Protein expression, purification 54
IIB.2 RdxR is indispensable for n-alkane oxidation by P. aeruginosa 54
IIB.3 Determination of the RdxR structure 55
IIB.4 Structure of RdxR 57
IIB.5 Determining the crystal structure of the RdxR/Rdx complex 62
IIB.6 Structure of the RdxR/Rdx complex 66
IIB.7 The RdxR and RdxR/Rdx structures reveal a possible RdxR dimer 70
IIC Crystallization of the YopO/Actin complex 74
IIC.1 Protein expression and purification 74
IIC.2 Production and purification of YopO 74 89-729
IIC.3 Surface engineering 78
IIC.4 Purification of the YopO /actin complex 79 89-729
IIC.5 YopO autophosphorylates upon complexation with actin 81 89-729
IIC.6 Crystallization 82
III DISCUSSION 84
IIIA SdsA1 defines a new group of sulfatases 84
IIIA.1 Structural characterization of group-III sulfatases 85
IIIA.2 Mechanism of sulfate ester cleavage by group-III sulfatases 91
IIIA.3 SdsA1 - From structure to function 96
IIIB The electron transfer complex RdxR/Rdx 100
IIIB.1 Relationship of RdxR to other FAD-dependent reductases 100
IIIB.2 RdxR provides a preformed redox scaffold for Rdx 101
IIIB.3 RdxR discriminates between two types of Rdx 102
IIIB.4 Electron Transfer from RdxR to Rdx 104
IIIB.5 Role of the RdxR/Rdx couple is probably not confined to alkane oxidation 109
IIIB.6 Concluding remarks for RdxR/Rdx and SdsA1 109
IIIC YopO – a challenging target for structural analysis 112
IIIC.1 Modification of loop regions by proteolysis assisted surface engineering 112
IIIC.2 Alternative strategies to crystallize YopO 114
IV OUTLOOK 116
IVA.1 Structure and function of SdsA1 and RdxR/Rdx 116
IVA.2 Structure of the type-III secretion effector YopO 118 Contents III
V METHODS 119
VA SdsA1 119
VA.1 Cloning, protein expression and purification 119
VA.2 Site-directed mutagenesis 119
VA.3 Bacterial growth 120
VA.4 Organic synthesis 120
VA.5 Biochemical characterization of sulfatase activity 120
VA.6 Metal exchange 121
VA.7 Crystallization 121
VA.8 Structure determination 121
VB RdxR/Rdx 122
VB.1 Cloning, Protein Expression and Purification 122
VB.2 Alkane oxidation 123
VB.3 Equilibrium binding studies 123
VB.4 Crystallization 123
VB.5 Structure Determination 123
VC YopO 124
VC.1 Cloning, expression and purification 124
VC.2 Purification of the YopO /actin complex 124 89-729
VC.3 Site directed mutagenesis 125
VC.4 CD spectroscopy 125
VC.5 Autophosphorylation of YopO 125
VC.6 Limited proteolysis 125
VC.7 Crystallization 126
REFERENCES 127
DANKSAGUNG 141
LEBENSLAUF 144
IV Abbreviations
Abbreviations
1DA 1-decane sulfonic acid
1DO 1-dodecanol
Å Ångström (0.1 nm)
AA Amino acid
ADP Adenosine diphosphate
AHL Acyl homoserine lactone
AMP Adenosine monophosphate
AMPPNP 5'-adenylyl-β,γ-imidodiphosphate
ATP Adenosine triphosphate
BLAST Basic local alignment search tool
CD-spectroscopy Circular dichroism spectroscopy
CF Cystic fibrosis
CFTR Cystic fibrosis transmembrane conductance regulator
cmc Critical micelle concentration
DALI server Distance alignment server
DLS Dynamic light scattering
ET Electron transfer
EM Electron microscopic
FAD Flavin adenine dinucleotide (oxidized)
FADH reduced FAD
G-actin Globular actin (monomeric)
GAP GTPase activating protein
GC-MS Gas chromatography – mass spectrometry
GDI Guanosine nucleotide dissociation inhibitor
GDP Guanosine diphosphate
GEF Guanosine nucleotide exchange factor
GR Glutathione reductase
GSH reduced glutathione
GST Glutathione-S-transferase
GTP Guanosine triphosphate
His His-His-His-His-His-His 6 Abbreviations V
ICP-MS Inductively coupled plasma – mass spectrometry
k Turnover number cat
k /K Catalytic efficiency cat M
K Dissociation constant D
K Michaelis-Menten constant M
K Substrate inhibition constant S
M cells Microfold cells
2+ M Divalent metal ion
MAD Multiple anomalous dispersion
MBL Metallo-β-lactamase
MW Molecular weight
NAD Nicotinamide-adenine-dinucleotide (oxidized)
NAD(P)H Nicotinamide-adenine-dinucleotide (reduced)
PA0740 Gene product of locus 0740 of P. aeruginosa
PA0740 PA0740 knock-out mutant of P. aeruginosa
PA5349 Gene product of locus 5349 of P. aeruginosa
PA5349 PA5349 knock-out mutant of P. aeruginosa
PA5350 Gene product of locus 5350 of P. aeruginosa
PA5351 Gene product of locus 5351 of P. aeruginosa
Pdx Putidaredoxin
PdxR Putidaredoxin reductase
PEG Polyethyleneglycol
PSK Prostate-derived STE20 (sterile 20) -like kinase
r.m.s.d. Root mean square difference
Rdx Rubredoxin
RdxR Rubredoxin reductase
S6S Sodium hexylsulfate
SAXS Small-angle X-ray scattering
SDS Sodium dodecylsulfate
SdsA1 Synonym for PA0740
SDS-PAGE SDS-polyacrylamide gel electrophoresis
SeMet Selenomethionine
SER Surface-entropy-reduction
SG Structural genomics
DDVI Abbreviations
T3SS Type-3 secretion system
Trx Thioredoxin
TrxR Thioredoxin reductase
V Elution volume in ml e
V Maximum catalytic velocity max
W Water molecule 1 1
W Water molecule 2 2
YopO Yersinia outer protein O (equivalent to YpkA)
YpkA Yersinia protein kinase A (equivalent to YopO)