Structural studies on ferric uptake regulator proteins from Mycobacterium tuberculosis [Elektronische Ressource] / presented by Debora Lucarelli

Dissertation






submitted to the
Combined faculty for the Natural Sciences and for Matematics
of the Rupuerto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences










Presented by


Debora Lucarelli
born in Roma, Italia



Oral examination: ………………………………………..



Structural Studies on
Ferric Uptake Regulator Proteins
from Mycobacterium tuberculosis





















Referees:

Prof. Dr. Irmgard Sinning, University of Heidelberg, Heidelberg

Dr. Elena Conti, EMBL Heidelberg, Heidelberg





This thesis is dedicated to my mum
CONTENTS



Contents...........................................................................................................................i
List of Figures................................................................................................................iv
List of Tables .................................................................................................................vi
List of Abbreviations.....................................................................................................vii
Preface.........................................................................................................................viii
Zusammenfassung..........................................................................................................ix
Summary........................................................................................................................xi

1. INTRODUCTION..................................................................................................... 1
1.1 Metal ion homeostasis ........................................................................................... 1
1.2 Introduction to the biology and pathogenesis of Mycobacterium tuberculosis ........ 2
1.3.1 Iron in Mycobacterium tuberculosis ................................................................ 3
1.3.2 Fur (ferric uptake regulator) family................................................................. 5
1.3.3 Mtb FurA and Mtb FurB............................................................................... 10
1.4 Zinc regulation .................................................................................................... 12
1.5 Objectives ........................................................................................................... 14
2. MATERIALS AND METHODS ............................................................................ 15
2.1 Cloning. expression and purification.................................................................... 15
2.1.1 FurA............................................................................................................. 15
2.1.2 Cloning, expression and purification of FurB................................................ 17
2.2 Site directed mutagenesis..................................................................................... 17
2.3 Bioinformatics analysis........................................................................................ 18
2.4 DNA-binding assays............................................................................................ 19
2.5 FurB-DNA complex purification ......................................................................... 19
i 2.6 MicroPIXE.......................................................................................................... 20
2.6.1 Brief introduction to microPIXE ................................................................... 20
2.6.2 MicroPIXE sample preparation and data collection....................................... 21
2.7 Isothermal Calorimetry........................................................................................ 21
2.7.1 Brief introduction to the Isothermal Calorimeter ........................................... 21
2.7.2 ITC sample preparation and data collection................................................... 22
2.8 EXAFS................................................................................................................ 23
2.8.1 Brief introduction to EXAFS ........................................................................ 23
2.8.2 X-ray absorption spectroscopy sample preparation and data collection.......... 25
2.9 Homology modelling........................................................................................... 27
2.10 Crystallization and crystal structure determination............................................. 27
3. Mtb FurA RESULTS .............................................................................................. 29
3.1 Purification.......................................................................................................... 29
3.2 Metal analysis...................................................................................................... 29
3.3 Isothermal titration .............................................................................................. 31
3.4 EXAFS analysis .................................................................................................. 33
3.4.1 Zinc site........................................................................................................ 33
3.4.2 Iron site ........................................................................................................ 33
3.5 DNA affinity ....................................................................................................... 37
3.6 Homology model................................................................................................. 38
3.7 Proteolysis........................................................................................................... 39
4. Mtb FurB RESULTS .............................................................................................. 40
4.1 Purification.......................................................................................................... 40
4.2 Metal analysis...................................................................................................... 40
4.3 Isothermal titration .............................................................................................. 41
4.4 DNA affinity ....................................................................................................... 44
4.5 FurB-DNA complex ............................................................................................ 45
4.6 EXAFS analysis .................................................................................................. 46
4.6.1 Zinc site........................................................................................................ 47
4.6.2 Cobalt site..................................................................................................... 50
ii 4.7 Proteolysis........................................................................................................... 53
4.8 Mutants ............................................................................................................... 54
4.9.1 Crystallization............................................................................................... 55
4.9.2 Crystal structure............................................................................................ 58
4.9.3 Metal binding sites in the crystal structure .................................................... 61
5. Mtb FurA DISCUSSIONS ...................................................................................... 64
5.1 FurA possesses two metal sites ............................................................................ 64
5.2 The structural site ................................................................................................ 64
5.3 The regulatory site............................................................................................... 68
5.4 DNA affinity ....................................................................................................... 70
5.5 Summary and conclusion..................................................................................... 70
6. Mtb FurB DISCUSSIONS ...................................................................................... 72
6.1 Structure overview............................................................................................... 72
6.2 Metal sites ........................................................................................................... 74
6.3 Remarkable points............................................................................................... 76
6.3.1 EXAFS similarity ......................................................................................... 76
6.3.2 DNA-binding................................................................................................ 78
6.3.3 Alignment considerations.............................................................................. 79
6.4 Conclusion .......................................................................................