Biochemical characterization of the Sec14-PH module from the neurofibromatosis type I protein [Elektronische Ressource] / presented by Stefan Welti

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Dissertationsubmitted to theCombined Faculties for the Natural Sciences and for Mathematicsof the Ruperto-Carola University of Heidelberg, Germanyfor the degree ofDoctor of Natural Sciencespresented byStefan Welti, Diplom-Biologeborn in: HeidelbergOral exam:Biochemical Characterizationof the Sec14-PH Modulefrom the Neurofibromatosis Type I ProteinReferees: PD Dr. Klaus ScheffzekProf.Dr. Michael BrunnerCONTENTSContents1 Abstracts 81.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . 81.2 Zusammenfassung . . . . . . . . . . . . . . . . . . . . . . 92 Abbreviations 103 Introduction 133.1 Neurofibromatosis Type I . . . . . . . . . . . . . . . . . 133.2 The NF1 gene . . . . . . . . . . . . . . . . . . . . . . . . 143.3 Animal models of NFI . . . . . . . . . . . . . . . . . . . 153.3.1 Mouse . . . . . . . . . . . . . . . . . . . . . . . . 153.3.2 Drosophila melanogaster . . . . . . . . . . . . . . 163.4 The NF1 gene product Neurofibromin . . . . . . . . . . . 173.4.1 The GAP related domain and Ras. . . . . . . . . 203.4.2 The tubulin binding region . . . . . . . . . . . . . 233.4.3 The syndecan binding region . . . . . . . . . . . . 253.4.4 The CSRD domain . . . . . . . . . . . . . . . . . 263.4.5 The Sec14 homology - PH like module . . . . . . 273.5 Current situation and goals . . . . . . . . . . . . . . . . 294 Results and discussion 314.1 Improved overlay assays and PIP binding . . . . . . . . . 314.1.

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Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences
presented by
Stefan Welti, Diplom-Biologe
born in: Heidelberg
Oral exam:Biochemical Characterization
of the Sec14-PH Module
from the Neurofibromatosis Type I Protein
Referees: PD Dr. Klaus Scheffzek
Prof.Dr. Michael BrunnerCONTENTS
Contents
1 Abstracts 8
1.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.2 Zusammenfassung . . . . . . . . . . . . . . . . . . . . . . 9
2 Abbreviations 10
3 Introduction 13
3.1 Neurofibromatosis Type I . . . . . . . . . . . . . . . . . 13
3.2 The NF1 gene . . . . . . . . . . . . . . . . . . . . . . . . 14
3.3 Animal models of NFI . . . . . . . . . . . . . . . . . . . 15
3.3.1 Mouse . . . . . . . . . . . . . . . . . . . . . . . . 15
3.3.2 Drosophila melanogaster . . . . . . . . . . . . . . 16
3.4 The NF1 gene product Neurofibromin . . . . . . . . . . . 17
3.4.1 The GAP related domain and Ras. . . . . . . . . 20
3.4.2 The tubulin binding region . . . . . . . . . . . . . 23
3.4.3 The syndecan binding region . . . . . . . . . . . . 25
3.4.4 The CSRD domain . . . . . . . . . . . . . . . . . 26
3.4.5 The Sec14 homology - PH like module . . . . . . 27
3.5 Current situation and goals . . . . . . . . . . . . . . . . 29
4 Results and discussion 31
4.1 Improved overlay assays and PIP binding . . . . . . . . . 31
4.1.1 New tools: the αNF1-SecPH antibody . . . . . . 31
4.1.2 Overlay assays and the PIP binding Site . . . . . 32
4.2 Assessment of typical PH- and Sec14-domain activities . 39
4.2.1 NF1-SecPH does not bind phosphotyrosine . . . . 39
4.2.2 Localization studies in life cells . . . . . . . . . . 41
4.3 Structural investigation of lipid bound NF1-SecPH . . . 45
4.3.1 Improved purification procedure for NF1-SecPH . 45
4.3.2 Crystallization of detergent free NF1-SecPH . . . 47
4.3.3 Structure of glycerophospholipid bound NF1-SecPH 49
4.4 Properties of the lipid binding cage . . . . . . . . . . . . 52
3CONTENTS
4.4.1 Identification of the bound ligands as PtdEtn and
PtdGro . . . . . . . . . . . . . . . . . . . . . . . 52
4.4.2 NF1-SecPH has lipid exchange activity . . . . . . 56
4.5 Patient derived mutations of NF1-SecPH . . . . . . . . . 58
4.5.1 PurificationandcharacterizationofNF1-SecPHmu-
tants . . . . . . . . . . . . . . . . . . . . . . . . . 58
4.5.2 Structure of the ΔK1750 mutant . . . . . . . . . 62
4.5.3 of the TD mutant . . . . . . . . . . . . 66
4.6 Access to the lipid binding cage can be inhibited with PIPs 69
4.7 Conclusions and outlook . . . . . . . . . . . . . . . . . . 71
5 Materials and methods 77
5.1 Common Methods . . . . . . . . . . . . . . . . . . . . . 77
5.2 Expression and purification of NF1-SecPH . . . . . . . . 78
5.3 Protein - lipid overlay assays . . . . . . . . . . . . . . . . 79
5.3.1 Generation of αNF1-SecPH antibodies . . . . . . 79
5.3.2 Protein lipid overlay assays . . . . . . . . . . . . 80
5.3.3 Site directed mutagenesis . . . . . . . . . . . . . . 81
5.4 Crystallographic techniques . . . . . . . . . . . . . . . . 82
5.4.1 Crystallization . . . . . . . . . . . . . . . . . . . 82
5.4.2 X-ray data collection . . . . . . . . . . . . . . . . 83
5.4.3 Structure determination by molecular replacement 85
5.5 AnalysisoftheNF1-SecPH-glycerophospholipidinteraction 86
5.5.1 Lipid extraction . . . . . . . . . . . . . . . . . . . 86
5.5.2 Preparation of liposomes . . . . . . . . . . . . . . 87
5.5.3 Lipid exchange reactions and inhibition . . . . . . 88
5.5.4 Mass spectrometry analysis . . . . . . . . . . . . 88
5.6 Protein characterization . . . . . . . . . . . . . . . . . . 90
5.6.1 Analytical size exclusion chromatography . . . . . 90
5.6.2 Circular dicroism spectroscopy . . . . . . . . . . . 90
5.6.3 Isothermal titration calorimetry . . . . . . . . . . 91
5.7 Mammalian cell culture and microscopy. . . . . . . . . . 92
4CONTENTS
6 Appendix 94
6.1 Publication list . . . . . . . . . . . . . . . . . . . . . . . 94
6.2 Abbreviations used in Fig. 3-4 (p.24) . . . . . . . . . . . 95
6.3 Key to the lipid arrays . . . . . . . . . . . . . . . . . . . 97
6.4 Units, amino acids and prefixes . . . . . . . . . . . . . . 98
References 99
5Acknowledgement
Foremost, I want to thank my supervisor Klaus Scheffzek for arous-
ing my fascination for structural biology, introducing me to X-ray
crystallography and for offering me the opportunity to elaborate this
thesis. ForfurthervaluableinputanddiscussionsIwouldliketothank
the members of my Thesis Advisory Committee Philippe Bastiaens,
Michael Brunner, Luis Serrano and Jochen Wittbrodt. In addition, I
would like to thank Thomas Rausch for participating in the disputa-
tion.
Thisthesiswouldnothavebeenpossiblewithoutthehelpofmanypeo-
ple - I want to thank especially Igor D’Angelo for uncounted help and
advice,SvenFratermanandMatthiasWilmforagreatandfruitfulcol-
laboration as well as Massimiliano Mazza, Ivan Yudushkin, Wolfram
Antonin and Fabien Bonneau for patience and showing me new tech-
niques. For years of friendship, help and a good working atmosphere
I am further grateful to Annabel Parret, Esther Lenherr, Jeanette
Seiler, Vladimir Pena, Vadim Sidorovitch, Kanchan Anand, Ulrike
Laabs, Michael Hothorn, Stefan Reinelt, Hus¨ seyin Besir, Ann-Marie
Lawrence, Ines Racke and Uli Steuerwald. A big thank also to Sonja
Kuhn¨ who gave me the chance to guide her to her Diploma and who
managed to advance two projects at once, as well as to Uli Karst, a
summer student I had the privilege to work with. For additional valu-
able help I also want to thank Oriol Galego, Stefanie Kandels-Lewis,
Simone Prinz, Stephanie Kronenberg, Christopher Roome and Frank
Thommen.
Furhtermore, I am grateful to my parents, Friederike and my brother
Wilfried for their support and I want to address a very special thank
to my wife Henriette for her continuous help, encouragement and un-
derstanding; without you, this thesis would not have been possible.
6fur¨ Henriette und
meine Eltern1 - Abstracts
1 Abstracts
1.1 Abstract
Neurofibromatosis type I (NF1) is an inherited neurocutaneous disor-
der with a high incidence of 1 in 3500 newborns. Clinical manifesta-
tions include pigment anomalies, Lisch nodules and the formation of
differenttumorslikeneurofibroma. NF1iscausedbyalterationsofthe
NF1 gene, encoding the Ras specific GTPase activating protein Neu-
rofibromin, which participates in several major signaling pathways.
A structural proteomics approach recently led to the discovery of an
unpredicted pleckstrin homology (PH)- and a Sec14-like domain.
In this thesis I have investigated the biochemical properties of the
NF1-SecPH module. NF1-SecPH can bind glycerophospholipids with
a preference for phosphatidylethanolamine and -glycerol (PtdEtn,
-Gro), of which PtdEtn is abundant in Neurofibromin containing cells
and thus a likely physiological ligand. It was furthermore possible to
crystallize NF1-SecPH in complex with glycerophospholipids which is
the first structure of a CRAL Trio domain bound to such ligands and
shows that PtdEtn binds to the interior of the Nf1-Sec portion. Lipid
exchange experiments revealed that PtdEtn and PtdGro are readily
exchanged, but phosphatidylcholine, -serine and -inositol (PtdCho,
-Ser,-Ins)areonlyincorporatedtoaminordegree. Thelipidexchange
activity can be modulated by soluble headgroups of phosphorylated
PtdIns derivatives (PIPs), which is consistent with a regulatory in-
teraction between Nf1-Sec and NF1-PH. While some patient derived
mutants show significant structural alterations compared to the cellu-
lar NF1-SecPH module, their properties with respect to lipid content
and PIP binding are only affected slightly. Localization studies in the
presence and absence of stimuli did not reveal a specific compartment
association compared to other PH domain containing proteins.
Taken together, PtdEtn is probably a physiological ligand of NF1-
SecPH, which seems able to incorporate membrane derived lipids in a
regulated fashion.
81 - Abstracts
1.2 Zusammenfassung
NeurofibromatoseTypI(NF1)isteineneurokutaneErbkrankheit, die
bei einem von 3500 Neugeborenen auftritt. H¨aufige Symptome sind
Pigmentanomalien, Lisch-Knoten und verschiedene Tumore, wie Neu-
rofibrome. DieErkrankungwirddurchMutationenimNF1Genverur-
sacht, das mit dem Ras spezifischen GTPase aktivierenden Protein
Neurofibromin einen Regulator zentraler Signalwege kodiert. Durch
strukturbezogene Proteomik wurde kurzlic¨ h eine unerwartete Pleck-
strin Homologie- (PH) und eine Sec14-¨ahnliche Dom¨ane entdeckt.
IndervorliegendenArbeituntersuchteichdiebiochemischenEigen-
schaften des NF1-SecPH Moduls. NF1-SecPH bindet Glycerophos-
pholipide – im Besonderen Phosphatidylethanolamin und -glycerol
(PtdEtn, -Gro) – was zusammen mit seiner H¨aufigkeit in Neurofi-
bromin enthaltenden Zellen fur¨ PtdEtn als wahrscheinlichen physi-
ologischen Liganden spricht. Zudem konnte NF1-SecPH im Komplex
mitGlycerophospholipidenkristallisiertwerden,wasdieersteStruktur
einer CRAL Trio Dom¨ane mit solchen Liganden darstellt und zeigt,
dass PtdEtn im Inneren von NF1-Sec bindet. W¨ahrend PtdEtn und
PtdGro leicht austauschbar sind, werden Phosphatidylcholin, -serin
und -inositol (PtdCho, -Ser, -Ins) nur schlecht inkorporiert. Die Aus-
tauschreaktionkannzudemmithohenKonzentrationenl¨oslicherKopf-
gruppen phosphorylierter PtdIns Derivate (PIPs) moduliert werden,
waskonsistentmiteinerregulatorischenInteraktionzwischenNF1-Sec
und NF1-PH ist. Obwohl die Strukturen einiger Mutanten von Pa-
¨tientensignifikanteAnderungenzumzellul¨arenNF1-SecPHaufweisen,
sind hinsichtlich gebundener Lipide oder der Bindung von PIPs kaum
Unterschiede feststellbar. Lokalisierungsstudien bei An- oder Abwe-
senheit von Stimuli zeigten keine Assoziierung mit bestimmten Zell-
bereichen, im Gegensatz zu anderen Proteine mit PH Dom¨anen.
Zusammenfassend ist PtdEtn ein wahrscheinlicher physiologischer
LigandvonNF1-SecPH,dasanscheinendaufregulierteArtundWeise
Membranlipide inkorporieren kann.
92 - Abbreviations
2 Abbreviations
AC adenylyl cylcase
α-TTP α-tocopherol transfer protein
AMP adenosine monophosphate
ArA arachidonic acid
Arp2/3 actin related proteins 2/3
BEACH Beige and Ch´ediak-Higashi domain
Bp base pair
BSA bovine serum albumin
cAMP cyclic adenosine monophosphate
CASK calcium/calmodulin-dependent serine protein kinase
CBD caveolin binding domain
CD circular dicroism
CNS central nervous system
CRAL Trio domain type named after CRALBP and Trio
CRALBP cellular retinaldehyde binding protein
CREB cAMP response element binding
CSRD cysteine and serine rich domain
CRMP-2 collapsin response mediator protein 2
Dab disabled
DNA deoxyribonucleic acid
D-GD3 disiaganglioside-GD3
ECL enhanced chemiluminescence
ECM extracellular matrix
EDTA ethylenediaminetetraacetic acid
EGF epidermal growth factor
EM electron microscopy
Ena enabled
ER endoplasmatic reticulum
ESI electrospray ionisation
EVH1 enabled / VASP homology 1
EVI2A ecotropic viral integration site 2A
EVI2B eic viral inon site 2B
E.coli Escherichia coli
FFDM fat free dry milk powder
GAP GTPase activating protein
GDP guanosine diphosphate
GEF guanosine nucleotide exchange factor
GFP green fluorescent protein
GPCR G-protein coupled receptor
GRD GAP related domain
GRP1 general receptor for phosphoinositides isoform 1
GST glutathion-S-transferase
GTP guanosine triphosphate
10