129 Pages

Functional and structural studies of the FGFR1 oncogene partner protein and biochemical investigations of the retinoblastoma protein and its binding partners [Elektronische Ressource] / Aleksandra Mikolajka


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Published 01 January 2007
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Language English
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Max Planck Institut für Biochemie
Abteilung Strukturforschung
Biologische NMR Arbeitsgruppe

Functional and Structural Studies of the FGFR1
Oncogene Partner Protein
Biochemical Investigations of the
Retinoblastoma Protein and its Binding Partners

Aleksandra Miko łajka

Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität
München zur Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigte Dissertation.

Vorsitzender: Univ.-Prof. Dr. St. J. Glaser

Prüfer der Dissertation: 1. Priv.-Doz. Dr. N. Budisa

2. Univ.-Prof. Dr. J. Buchner

Die Dissertation wurde am 22.11.2006 bei der Technischen Universität München
eingereicht und durch die Fakultät für Chemie am 05.02.2007 angenommen. PUBLICATIONS

Smialowski P., Martin-Galiano A., Mikolajka A., Girschick T., Holak T.A., Frishman D.
(2006). Protein solubility: sequence based prediction and experimental verification
Bioinformatics, submitted.

Mikolajka A., Yan X, Popowicz G., Smialowski P., Nigg E.A., Holak T.A. (2006).
Structure of the N-terminal domain of the FOP (FGFR1OP) protein and the
mechanism of its dimerization and centrosomal localization.
J Mol Biol 359: 863-75.

Singh M., Krajewski M., Mikolajka A., Holak T.A. (2005). Molecular determinants for
the complex formation between the retinoblastoma protein and LXCXE sequences.
J Biol Chem 280: 37868-76.

Smialowski P., Singh M., Mikolajka A., Majumdar S., Joy J.K., Nalabothula N.,
Krajewski M., Degenkolbe R., Bernard H.U., Holak T.A. (2005). NMR and mass
spectrometry studies of putative interactions of cell cycle proteins pRb and CDK6
with cell differentiation proteins MyoD and ID-2.
Biochim Biophys Acta 1750: 48-60.

1D one-dimensional
2D two-dimensional
Å Ångstrom
AP alkaline phosphatase
APS ammoniumperoxodisulfate
bHLH basic helix-loop-helix
BSA bovine serum albumin
CAP350 centrosome associated protein 350 kDa
CDK cyclin-dependent kinase
-1Da Dalton (g mol )
DMSO dimethylsulfoxide
DNA deoxyribonucleic acid
DTT dithiothreitol
EB1 end-binding protein 1
EDTA ethylenediaminetetraacetic acid
E. coli Esherichia coli
FGFR1 fibroblast growth factor receptor 1
HAT histone acetyltransferase
HDAC histone deacetylase
HSQC heteronuclear single quantum coherence
IPTG isopropyl- β-thiogalactopyranoside
ITC isothermal titration calorimetry
K association constant A
K dissociation D
kDa kilodalton
LB Luria-Broth medium
LisH LIS1 homology motif
MAD multiwavelength anomalous diffraction
MAPK the mitogen-activated protein kinase
MIR multiple isomorphous replacementMR molecular replacemen
MTs microtubule
MW molecular weight
NC nitrocelulose
NiNTA nickel-nitrilotriacetic acid
NMR nuclear magnetic resonance
OD optical density
PAGE polyacrylamide gel electrophoresis
PAI plasminogen activator inhibitor
PBS phosphate buffered saline
PCM pericentriolar material
PEG polyethylene glycol
PMSF phenylmethylsulfonylflouoride
ppm parts per million
RMSD root mean square deviation
SDS sodium dodecyl sulphate
SIR single isomorphous replacement
STAT signal transducer and activator of transcription
TEMED N,N,N’,N’- tetramethylethylendiamine
Tris tris(hydroxymethyl)aminomethane

Amino acids and nucleotides are abbreviated according to either one or three letter IUPAC
code. Table of contents
1.1 The cytoskeleton 1
1.1.1 The centrosome 1 Fibroblast growth factor receptor 1 oncogene partner (FOP) 3
1.2 Cell differentiation and cell cycle 6
1.2.1 The retinoblastoma protein, pRb 7
1.2.2 Viral oncoproteins 9
1.2.3 Histone deacetylase HDAC1 9
1.2.4 Plasminogen activator inhibitor-2 10
1.2.5 Helix-loop-helix (HLH) protein family 11 Id-2 12 MyoD 13
1.3 X-ray protein crystallography 14
1.3.1 The unit cell, space group and symmetry 14
1.3.2 The Bragg´s law 15
1.3.3 Data collection and scaling 16
1.3.4 The phase problem 16
2.1 Materials 21
2.1.1 Chemicals 21
2.1.2 Enzymes 21
2.1.3 Kits and reagents 21
2.1.4 Oligonucleotides 21 Primers for FOP constructs cloning 21 Primers for FOP mutagenesis 22 Primers for FOP-FGFR1 cloning 24 Primers for FOP-FGFR1 mutagenesis 24 Primers for CAP350 cloning 24 Primers for pRB cloning 25 Primers for pRb mutagenesis 25 Primers for E7 mu25
I Table of contents Primers for PAI2 cloning 25
2.1.5 Strains and plasmids 26 Cloning strains 26 Protein expression strains 26
2.1.6 Vectors 26
2.1.7 Antibiotics 26
2.1.8 Antibodies 26
2.1.9 Protein and nucleic acids markers 27
2.1.10 Peptides 27
2.1.11 Isotopically enriched chemicals 27
2.1.12 Cell growth media and stocks solutions 27 Stock solution of glucose 27 Stock solution of ampicillin 27 Stock solution of kanamycin 27 Stock solution of chloramfenicol 28 Stock solution of IPTG 28 Luria Bertani (LB) 28 Minimal medium (MM) for uniform enrichment with N 28 Defined medium for selective N labeling of proteins
(also for Sel-Met labeling) 29 Solution for making chemically competent E. coli cells 30
2.1.13 Buffer for DNA agarose gel electrophoresis 30
2.1.14 Protein purification buffers 30 Ion exchange and gel filtration chromatography buffers 30
2.1.15 Buffers for Ni-NTA chromatography under native conditions 31
2.1.16 atography under denaturing conditions 32
2.1.17 Protease buffers 33
2.1.18 Refolding Buffers 34
2.1.19 Buffers for crystallization, NMR and ITC 34
2.1.20 Reagents and buffers for SDS-PAGE and Western blots 35 Buffers for the SDS-PAGE 35 Protein visualization 36 Reagents for the Western blots 36
2.1.21 Apparatus 36
II Table of contents ÄKTA explorer 10 purification system 36 Chromatography equipment, columns and media 37 NMR spectrometers 37 Other apparatus 37
2.2 Methods 38
2.2.1 DNA techniques 38 Isolation of the plasmids 38 Screening of positive colonies 38 DNA sequencing 39 Cloning to LIC vectors 39 Ligation 40 Gene synthesis 40 Site directed mutagenesis 40
2.2.2 Transformation of E. coli 42 Transformation by heat shock 42 ation by electroporation 42
2.2.3 Preparation of chemically competent cells 42
2.2.4 Protocol for electrocompetent cells 42
2.2.5 Protein expression 43 E. coli expression in LB medium 43 E. coli expression in the minimal medium (MM) 44
2.2.6 Protein purification and refolding 44 Protein purification under native conditions 44 Protein purification under denaturating conditions 45
2.2.7 Protease digestion 45
2.2.8 Handling and storing of the proteins 46
2.2.9 SDS polyacrylamide gel electrophoresis (SDS-PAGE) 46
2.2.10 Western immunoblotting 47
2.2.11 Determination of protein concentration 47
2.2.12 Pull down assays 48
2.2.13 Mass spectrometry 48
2.2.14 Binding tests on gel filtration column 48
2.2.15 PAI-2 activity tests 48
2.2.16 Crystallization trials 50
III Table of contents
2.2.17 Isothermal titration calorimetry (ITC) 50
2.2.18 NMR spectroscopy 51
2.2.19 Immunofluorescence microscopy 51
2.2.20 Yeast two hybrid system 52 Yeast transformation protocol 52
3.1 Centrosomal proteins - structural and functional studies 54
3.1.1 Results 54 Plasmid constructions and protein expression 54 Protein purification strategies 56 In vitro binding tests between CAP320 and FOP 58 Optimizing the constructs of FOP and CAP350 for crystallization 58 Crystallization and data collection 60 The molecular structure of the N-terminal domain of FOP 64 The LisH motif is necessary but not sufficient for FOP
homodimerization 67 The N-terminal FOP domain is sufficient for centrosome localization 70
3.1.2 Discussion 71 LisH motif as a part of dimerization domain 71 Structural neighbors of FOP 72 Role of the LisH domain in protein-protein interactions 73
3.2 Cell differentiation and cell cycle proteins – interaction studies 75
3.2.1 Results 75 Cloning and plasmids 75 Protein expression 75 Purification and protease digestion 76 Activity tests of pRb, MyoD, Id-2 and PAI2 ΔCD 77 Activity tests of PAI-2 78 Peptide synthesis 79 Interactions between pRb and the HLH domain of MyoD or Id-2 79 pRb and L/IXCXE peptides from HPV E7, SV40 large T antigen,
HDAC1 and PAI-2 85
3.2.2 Discussion 90
IV Table of contents The HLH proteins MyoD and Id-2 does not interact directly with pRb 90 Role of the complex formation between pRb and LXCXE proteins
and peptides 92

7.1 List of programs and web-pages used to analyse sequences and, structures 117
7.1.1 Nucleic acid 117
7.1.2 Proteins
7.1.3 Expression system 118
7.1.4 Protocols
7.1.5 NMR data analysis 118
7.1.6 X-ray data analysis 118
VChapter 1 Introduction
1.1 The cytoskeleton
Dynamic remodeling of the cytoskeleton is responsible for many cell behaviors such as cell
migration, its shape and mechanical strength, as well as the organelles and chromosomes
segregation. All these processes have to be precisely regulated in response to external and
internal signals (Dormann and Weijer 2006). The cytoskeleton of higher-eukaryotic cells is
made up of three kinds of protein filaments: actin filaments (also called microfilaments),
which average 8 nm in diameter, intermediate filaments - 10 nm in diameter, and
microtubules - 25 nm, as well as a growing number of associated proteins. Within the
digestive epithelia, members of these three protein families are actins, keratins and
tubulins, respectively (Ku et al. 1999). The relative motion between filaments are induced
by motor proteins (Kruse and Julicher 2006). One of the most important cytoskeletal
activities is the mitotic spindle formation, which is regulated by a microtubule network
(Nigg 2006). MTs dynamics are controlled by coordinated interactions with actin
cytoskeleton and proteins that form adhesion sites, as well as by kinases and phosphatases
via signaling cascades. There are two major groups of microtubule motors: kinesins (most
of these move toward the plus end of the microtubules) and dyneins (which move toward
the minus end). In animal cells, the microtubules originate at the centrosome.
1.1.1 The centrosome
The centrosome is the major microtubule-organizing center of animal cells. It influences
not only the formation of the bipolar mitotic spindle but also determines the cell shape and
polarity in interphase (Loffler et al. 2006). In addition, the centrosomes are also required
for cytokinesis, segregation of signaling molecules, and developing some of the neurons
(Andersen et al. 2003). Each mammalian somatic cell typically contains one centrosome,
which is duplicated in coordination with DNA replication; thus the proper centrosomal
cycle is tightly connected with cell cycle regulation. Both processes require cyclin-
dependent kinase 2 (CDK2), in association with cyclins E and A, polo-like kinase 4 Plk4,
as well as phosphorylated retinoblastoma protein pRb (Meraldi et al. 1999; Bettencourt-
Dias et al. 2005; Habedanck et al. 2005). Numerical and structural centrosome aberrations
have been implicated in cancer. Abnormalities in the centrosomal number were present in