Crystal structure, nucleic-acid binding properties, and dimerization model of Pur-α [Pur-alpha] [Elektronische Ressource] / Almut Graebsch

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München


Crystal structure,
nucleic-acid binding properties,
and dimerization model
of Pur-α



Almut Graebsch

aus Berlin
2010 Erklärung
Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung
vom 29. Januar 1998 von Herrn Dr. Dierk Niessing betreut und von Herrn Prof. Karl-
Peter Hopfner vor der Fakulät für Chemie und Pharmazie vertreten.



Ehrenwörtliche Versicherung
Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe erarbeitet.




München, am 31. März 2010
Almut Graebsch





Dissertation eingereicht am: 31. März 2010
1. Gutachter: Karl-Peter Hopfner
2. Gutachter: Klaus Förstemann
Mündliche Prüfung am: 17. Mai 2010
ii Chapter 1 Introduction......................................................................................................1
1.1 Abstract.................................................................................................................................1
1.2 Scientific background..........2
1.2.1 Learning and memory.....2
1.2.2 Active transport...............................................................................................................2
1.2.3 Dendritic localization of mRNAs...................3
Composition of the mRNA transport granule.............. 5
Localization elements 7
Translocation ................................................................................................................................ 7
1.2.5 Activity-dependent translation........................8
1.3 Pur-α .....................................................................9
1.3.1 Cellular functions of Pur-α...........................................................11
Transcription............................................................................................... 11
Replication.................................. 12
Cell cycle control........................................................ 13
Tumor suppression ..................................................................................... 13
RNA binding.............................. 13
1.3.2 Fragile X Tremor/Ataxia syndrome..............................................14
1.3.3 Pur-α knockout mouse..................................................................15
1.4 Objectives............................................................17
Chapter 2 Results............................................................................ 18
2.1 Structural studies ...............................................18
2.1.1 Crystal structure of Pur-α.............................18
2.1.1.1 Human Pur-α....................... 18
Crystallization trials of human Pur-α......................................................................................... 18
Identification of PUR repeats in Pur-α 22
2.1.1.2 Borrelia burgdorferi Pur-α................................. 23
Crystallization of B. burgdorferi Pur-α...................... 25
Structure determination and refinement ..................................................................................... 28
Crystal structure of B. burgdorferi Pur-α................... 30
Surface assessment ................................ 32
Systematic structural comparison............................................................................................... 33
2.1.1.3 Drosophila melanogaster Pur-α......................... 35
Crystallization of D. melanogaster Pur-α repeats I-II 35
Structure determination and refinement ..................................................................................... 38
Crystal structure of D. melanogaster Pur-α repeats I-II............................ 41
Surface assessment ................................ 42
Comparison with the crystal structure of B. burgdorferi Pur-α................. 44
Systematic structural comparison............................................................................................... 45
Crystallization trials of D. melanogaster Pur-α repeat III ......................................................... 47
Co-crystallization trials of D. melanogaster Pur-α repeats I-II with nucleic acids ................... 48
2.1.2 Solution structure of Pur-α ...........................................................51
2.1.2.1 Analytical size-exclusion chromatography ......................................................................... 51
2.1.2.2 Small angle X-ray scattering............................... 52
SAXS of D. melanogaster Pur-α repeats I-II............. 55
SAXS of D. melanogaster Pur-α repeats I-III........... 57
iii
2.2 Functional studies...............................................................................................................60
2.2.1 Nucleic-acid binding of Pur-α......................60
2.2.1.1 Human Pur-α....................... 60
Filter binding assays................... 60
2.2.1.2 B. burgdorferi Pur-α ........................................................................................................... 62
Filter binding assays................... 62
Filter binding assays with mutant Pur-α.................... 64
2.2.1.3 D. melanogaster Pur-α........ 66
Filter binding assays with Pur-α repeats I-III ............................................................................ 67
Electrophoretic mobiliy shift assays with full length Pur-α....................... 68
EMSAs with Pur-α repeats I-II .................................. 69
EMSAs with mutant Pur-α repeats I-II...................................................... 70
Circular dichroism spectra of mutant Pur-α repeats I-II............................ 74
EMSAs with Pur-α repeat III..................................................................... 75
EMSA with quadruplex RNA and full length Pur-α.. 76
2.2.2 Protein binding of Pur-α...............................................................78
2.2.2.1 Yeast-two-hybrid screen..................................................................... 78
Interactions with known proteins 79
Arrrestin1................................................................ 79
Btb VII... 80
CG5758.................................................................. 80
Cka......................................................................... 80
Eye......... 81
Lamin C. 81
Pur-α...................................................................... 82
Interactions with non-existing proteins ...................................................................................... 82
Chapter 3 Discussion ...................................... 84
3.1 Structure of Pur-α.............84
3.1.1 PUR repeats are highly conserved structural units .......................................................84
3.1.2 B. burgdorferi Pur-α is a functional PUR protein........................84
3.1.3 Two PUR repeats form a PUR domain.........................................................................85
3.1.4 The PUR domain is similar to bacteriophage coat and Whirly proteins ......................86
3.1.5 Dimerization of Pur-α is mediated by PUR repeat III..................88
3.1.6 PUR repeat III is predicted to form a PUR domain................................90
3.1.7 Interaction of the PUR repeats is likely specific...........................91
3.1.8 The PUR domain is likely conserved in different orthologs ........................................92
3.2 Interaction with nucleic acids ................................93
3.2.1 The PUR domain binds nucleic acids...........................................93
3.2.2 PUR repeat III only weakly contributes to nucleic-acid binding..................................94
3.2.3 Pur-α binds DNA as well as RNA................95
3.2.4 The Pur-α dimer bears two nucleic-acid binding domains...........97
3.2.5 The nucleic-acid binding surface of a PUR domain is reminiscent of an RRM
domain ...................................................................................................................................99
iv
3.3 Interaction with proteins .................................................................................................101
3.3.1 Protein interactions are mediated by PUR repeats......................101
3.3.2 Yeast-two hybrid screen.............................102
3.3.2.1 Potential artifacts.....103
3.3.2.2 Arr1, LamC, Eye and Cka are putative binding partners of Pur-α..........................104
Arrestin1................................................................................................................................... 104
LaminC ..................................... 105
Eye............ 106
Cka................................................................................................ 106
Pur-α......... 106
Chapter 4 Conclusion ................................... 107
Chapter 5 Materials and Methods............................................................................... 108
5.1 Consumables and chemicals............................108
5.2 Oligonucleotides...............1