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

Cbu

Sujets

• Biologie

Informations

• Publié par: technische_universitat_carolo-wilhelmina_zu_braunschweig
• Publié le: 01 janvier 2008
• Nombre de lectures: 38
• Langue: English
• Poids de l'ouvrage: 10 Mo

Extrait

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.........................................................