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The Aryl Hydrocarbon Receptor: Structural Analysis and Activation Mechanisms

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The Aryl Hydrocarbon Receptor: Structural Analysis and Activation Mechanisms The Aryl Hydrocarbon Receptor: Structural Analysis and Activation Mechanisms This thesis is submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in the School of Molecular and Biomedical Sciences (Biochemistry), The University of Adelaide, Australia Fiona Whelan, B.Sc. (Hons) 2009 2 Table of Contents THESIS SUMMARY................................................................................. 6 DECLARATION....................................................................................... 7 PUBLICATIONS ARISING FROM THIS THESIS.................................... 8 ACKNOWLEDGEMENTS...................................................................... 10 ABBREVIATIONS ................................................................................. 12 CHAPTER 1: INTRODUCTION ............................................................. 17 1.1 BHLH.PAS PROTEINS ............................................................................................17 1.1.1 General background..................................................................................17 1.1.2 bHLH.PAS Class I Proteins.........................................................................18 1.2 THE ARYL HYDROCARBON RECEPTOR......................................................................19 1.2.1 Domain Structure and Ligand Activation ..............................................19 1.2.2 AhR Expression and Developmental Activity .......................................21 1.2.3 Mouse AhR Knockout Phenotype ...........................................................23 1.2.4 Xenobiotic Toxicity Through Activation of AhR Target Genes .........25 1.2.5 Endogenous Activation of AhR...............................................................29 1.2.6 Epidermal Effects of TCDD Toxicity ........................................................30 1.3 SUMMARY AND AIMS ..............................................................................................33 CHAPTER 2: MATERIALS AND METHODS........................................ 35 2.1 MATERIALS ..............................................................................................................35 2.1.1 Chemicals, reagents and kits....................................................................35 2.1.2 Plasmids........................................................................................................37 2.1.3 Oligonucleotides and primers..................................................................39 2.1.4 Antibodies ...................................................................................................41 2.1.5 Cell Lines ......................................................................................................42 2.1.6 Bacterial Strains ..........................................................................................42 2.2 METHODS ..............................................................................................................42 2.2.1 Solutions.......................................................................................................42 2.2.2 Bacterial Co-Expression and Purification of bHLH.PAS A Dimers .....44 2.2.3 Purification of Oligonucleotides for Crystallization.............................48 2.2.4 Crystallization of bHLH.PAS A/XRE Complexes....................................49 2.2.5 AFM Analysis...............................................................................................51 2.2.6 SAXS Analysis..............................................................................................52 2.2.7 Cell Culture ..................................................................................................52 2.2.8 Treatment and Denaturing Ni-IMAC of Full-Length HisMyc mAhR..54 CHAPTER 3: PURIFICATION AND CRYSTALLIZATION OF TRANSCRIPTION FACTOR AHR/ARNT COMPLEXED WITH A XENOBIOTIC RESPONSE ELEMENT.................................................. 56 3.1 INTRODUCTION: BHLH TRANSCRIPTION FACTOR FAMILY AND SUBGROUPS BHLH.LZ, BHLH.O AND BHLH.PAS......................................................................................56 3.1.1 bHLH Transcription Factors......................................................................56 3.1.2 Per-ARNT-Sim (PAS) Domains:..................................................................59 3 3.1.3 Dimer Interactions Mediated by PAS Domains .....................................59 3.1.4 Dimeric bHLH.PAS Transcription Factors...............................................61 3.2 RESULTS .................................................................................................................64 3.2.1 Recombinant expression, purification and solubilisation of hAhR/ARNT.................................................................................................64 3.2.2 Crystallization and Partial Proteolysis of bHLH.PAS A Heterodimer hAhR/ARNT.................................................................................................71 3.3 SUMMARY AND DISCUSSION....................................................................................79 CHAPTER 4: ATOMIC FORCE MICROSCOPY AND SMALL ANGLE X- RAY SCATTERING ANALYSIS OF AHR/ARNT BHLH.PAS A HETERODIMER .....................................................................................81 4.1 INTRODUCTION ......................................................................................................81 4.1.1 The Mechanics of DNA Packaging and Transcriptional Regulation.81 4.1.2 Transcription Factors as Effectors of Localized Chromatin Remodelling.................................................................................................83 4.1.3 The AhR/ARNT Heterodimer Affects Chromatin Structures in the Enhancer of Cyp1a1...................................................................................85 4.1.4 Atomic Force Microscopy of Proteins and DNA ...................................88 4.1.5 Small Angle X-Ray Scattering Analysis of Biological Molecules.........89 4.1.6 Summary and Aims ....................................................................................92 4.2 RESULTS .................................................................................................................93 4.2.1 Atomic Force Microscopy of AhR284/H6ARNT362/Cyp1a1 Enhancer Fragment Complexes.................................................................................93 4.2.2 SAXS Analysis of AhR284/H6ARNT362 XRE Complexes..................95 4.2.3 Hypothetical bHLH.PAS A Domain Topology of AhR/ARNT Heterodimer ................................................................................................97 4.3 DISCUSSION............................................................................................................97 CHAPTER 5: RATIONAL MUTAGENESIS OF MAHR LIGAND BINDING DOMAIN TO DISCOVER MUTANTS WITH ALTERED INDUCIBILITY......................................................................................104 5.1 INTRODUCTION ................................................................................................... 104 5.1.1 PAS domains as environmental sensors through cofactor and ligand binding ...................................................................................................... 104 5.1.2 AhR Ligands; Synthetic and Natural Compounds............................. 105 5.1.3 PAS domain flexibility correlates with ligand binding mechanisms ......................................................................................................................110 5.1.4 LBD Homology Models and Mutagenesis Studies............................. 112 5.1.5 Summary and Aims ................................................................................. 115 5.2 RESULTS............................................................................................116 5.2.1 Homology based targeted mutagenesis of the Ligand Binding Domain of Mouse AhR............................................................................ 116 5.2.2 Histidine 285 in the LBD of mAhR is critical for suspension culture activity, but is not required for atypical AhR ligand YH439 inducibility ................................................................................................ 118 5.2.3 YH439, unlike prototypical PAH ligands (TCDD, 3MC and B[a]P),has a novel binding mode which tolerates mutation of Histidine 285 to Tyrosine..................................................................................................... 119 4 5.2.4 Mutation of mAhR Histidine 285 to Tyrosine abolishes endogenous activation paradigms.............................................................................. 120 5.3 CONCLUSIONS ..................................................................................................... 120 CHAPTER 6: IDENTIFICATION OF POST-TRANSLATIONAL MODIFICATION OF THE MOUSE ARYL HYDROCARBON RECEPTOR ............................................................................................................. 123 6.1 INTRODUCTION.................................................................................................... 123 6.1.1 PTMs induce conformational change and form docking sites for Protein:Protein Interactions .................................................................. 123 6.1.2 Post-Translational Modification of Transcription Factor p53.......... 126 6.1.3 AhR Regulation by Post-Translational Modification........................ 129 6.2 RESULTS .............................................................................................................
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