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Structure, function and mechanisms of G-Proteins

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Structure, function and mechanisms of G-Proteins Oliver Daumke MDC-Berlin, House 31.2 (Flachbau), R0225 oliver.daumke_at_mdc-berlin.de G-Protein = Guanine-nucleotide ... – PowerPoint PPT presentation

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Title: Structure, function and mechanisms of G-Proteins


1
Structure, function and mechanisms of G-Proteins
  • Oliver Daumke
  • MDC-Berlin, House 31.2 (Flachbau), R0225
  • oliver.daumke_at_mdc-berlin.de

2
1994 Nobel Prize in Medicine, Alfred Gilman and
Martin Rodbell, for their discovery of
G-Proteins and the role of these proteins in
signal transduction in cells.
3
G-Protein Guanine-nucleotide binding
protein(GNBD)
Guanine
Phosphates
Ribose
4
G-Protein families
  • Heterotrimeric G-Proteins (Transducin, G?i, G?q
    ), in 7-TM receptor signalling
  • Initiation, elongation, termination factors in
    protein synthesis (IF1, EF-Tu, EF-TS)
  • Signal recognition particle (SRP) and its
    receptor, translocation of nascent polypeptide
    chains in the ER
  • Ras-like GTPases (Ras, Rap, Rho, Ran, Rab, Arf,
    Arl, Sar), molecular switches in signal
    transduction
  • Dynamin superfamily of GTPases, remodelling of
    membranes
  • 60 further distinct families
  • Leipe et al., JMB (2002)

5
The G-domain
Mixed ?-? protein 5 conserved motifs (G1-G5)
involved in nucleotide binding
Pai et al., Nature (1989)
6
Ras-like G-Proteins are molecular switches
To allow switch function high affinity for
nucleotide required ? pMol
Effector Interacts stably with the GTP-bound
form GEF Guanine nucleotide Exchange
Factor GAP GTPase Activating Protein
7
The switch regions
Vetter and Wittinghofer, Science (2001)
8
The GTPase reaction
  • Intrinsic GTPase rates of small G-Proteins are
    slow (range kcat10-2 - 10-3 min-1)
  • SN2 nucleophilic attack with trigonal bipyramidal
    transition state
  • Phosphate hydrolysis reaction is
    thermodynamically highly favourable but
    kinetically very slow (Westheimer FH (1987), Why
    nature chose phosphates, Science 235, 1173-1178)

9
Enzymatic strategies for GTP hydrolysis
  • Counteracting of negative charge at phosphates
  • - P-loop (GxxxxGKS), hydrogen bonds and lysine
  • - Mg2 ion, essential for nucleotide binding
    and hydrolysis
  • - catalytic arginine (and lysine residues)
  • Positioning of attacking nucleophile
  • - catalytic glutamine

10
Non-hydrolysable GTP analogues
Abbreviations
GTP-?-S
GMPPCP
GMPPNP
11
Transition state mimicks of GTP hydrolysis
12
GTPase Activating Proteins
  • Accelerate intrinsic GTPase by a factor of 105
    106
  • Ras, Rap, Rho, Rab, Ran have completely unrelated
    GAPs
  • High affinity binding to the GTP-bound form, low
    affinity interaction with the GDP-bound form
  • Mechanism of GTP hydrolysis ?

13
Monitoring the GAP-catalysed reaction
  • G-Protein (GTP) GAP
  • G-Protein (GTP)?GAP
  • G-Protein (GDP) Pi ? GAP
  • G-Protein (GDP) GAP
  • G-Protein (GDP) GAP

k1
k2
k3
k4
Pi
k5
14
Multiple-turnover assays
  • Monitors several rounds of GAP catalysed
    G-Protein (GTP) hydrolysis
  • G-Protein (GTP) as substrate, in excess, e.g. 200
    µM
  • GAP in catalytic amounts, e.g. 100 nM
  • Determine initial rates of GTP hydrolysis by
  • HPLC (ratio GDP, GTP)
  • Thin layer chromatography using radioactively
    labelled GTP
  • Phosphate release (colorimetric assay,
    radioactive assays)
  • Vary concentration of G-Protein to determine
    Michaelis-Menten parameters (KM, kcat)

15
Monitoring the GAP-catalysed reaction
  • G-Protein (GTP) GAP
  • G-Protein (GTP)?GAP
  • G-Protein (GDP) Pi ? GAP
  • G-Protein (GDP) GAP
  • G-Protein (GDP) GAP

k1
k2
k3
k4
Pi
k5
16
Single-turnover assays
  • Analysis of a single cycle of GTP hydrolysis
  • Often monitored by fluorescence stopped-flow
  • Typically 1 2 µM fluorescently labelled
    G-Protein (GTP) in one cell, excess of GAP in the
    other cell
  • Vary concentration of GAP ? multiparameter fit
    allows determination of k1, k2, KD,

17
The mechanism of RasGAP
Scheffzek et al., Nature (1996)
18
Fluorescence stopped-flow to monitor the GAP
reaction
Ras(mantGTP) vs. RasGAP
Fluorescence increase complex formation
Fluorescence decrease GTP hydrolysis
Ahmadian et al., Nature Structure Biology (1997)
19
An arginine residue in RasGAPs is essential for
GAP activity
Ras(mantGTP) vs. RasGAP
Ahmadian et al., Nature Structure Biology (1997)
20
AlF3 promotes formation of a transition state
complex
Mittal et al., Science (1994)
21
The RasGAP-Ras complex
Scheffzek et al., Science (1997)
22
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23
Rap1
  • Involved in various signalling pathways, e.g.
    integrin activation
  • close Ras homologue
  • BUT No catalytic glutamine residue
  • own set of GAPs with no sequence homology to
    RasGAPs

24
100 nM RapGAP 800 µM Rap1(GTP)
25
Rap1GAP stimulates intrinsic Rap1 reaction
100.000 fold
kcat 6 s-1 Km 50 µM
Brinkmann et al., JBC (2001)
26
No arginine finger is involved in catalysis
Brinkmann et al, JBC (2001)
27
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28
The Rap1GAP Dimer
Daumke et al., Nature (2004)
29
The catalytic domain of Rap1GAP has a G-domain
fold
Ras
Rap1GAP cat
30
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31
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32
Rap1-Rap1GAP reaction followed by fluorescence
stopped-flow
33
R286 is not essential for the GAP reaction
34
His287 is involved in binding to Rap1
35
Rap1GAP provides a catalytic Asn, the Asn
thumb, for catalysis
Daumke et al., Nature (2004)
36
Asn290 is a purely catalytic residue and not
involved in binding to Rap1
Kd 4 ?M
37
Rap1GAP-Rap1 complex indicates that Asn thumb
positions attacking water molecule
Scrima et al., EMBOJ (2008)
38
The Dynamin-family of GTPases
39
The shibire fly
Bing Zhang, UT Austin
40
Wt 30C Drosophila nerve terminal Kosaka and
Ikeda, J Neurobiol., 1982
41
shibire 30C Drosophila nerve terminal Kosaka and
Ikeda, J Neurobiol., 1982
42
The family of Dynamin-related GTPases
  • Classical Dynamins Dyn1, Dyn2, Dyn3
  • Dynamin-related proteins Mx, Mitofusin
  • GBP-related proteins GBPs, Atlastins
  • Bacterial Dynamins

GTPase Middle PH GED
PRD
Common features - Low affinity for
nucleotide - Template induced self-oligomerisatio
n - Assembly-stimulated GTP hydrolysis
43
1000 x stimulation of Dynamins GTPase reaction
by lipid tubule binding
Stowell et al., Nat Cell Biol (1999)
44
What is the mechanism of Dynamin ?
Constrictase
Effector
Sever et al., Nature (1999) NV by T. Kirchhausen
45
Is Dynamin a popase ?
No Dynamin
GTP-?-S
GDP
Stowell et al., Nat Cell Biol (1999) www.endocytos
is.org
46
Is Dynamin working as a twistase ?
Roux et al., Nature (2006)
Dynamin, no nucleotide
47
Dynamin, addition GTP
Roux et al., Nature (2006)
48
Biotin-Dynamin streptavidin polysterene bead
Dynamin, addition GTP
Roux et al., Nature (2006)
49
The EHD family
  • EHD Eps15 homology domain containing protein
  • Highly conserved in all higher eukaryotes, but
    not in
  • yeast and bacteria
  • Four paralogues in human, 70 - 80 amino acid
    identity

50
Biochemical features
  • Binds to adenine and not guanine nucleotides with
    affinity in the low micromolar range
  • Binds to negatively charged liposomes
  • Liposome-stimulated ATP hydrolysis (very slow)

PS liposomes
EHD2
Daumke et al., Nature (2007)
51
Daumke et al., Nature (2007)
52
Lipid binding site of EHD2
53
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54
Implications for membrane remodelling
  • Factors involved in membrane remodelling /
    destabilisation
  • Oligomer formation into rings around a lipid
    template
  • Insertion of hydrophobic residues into outer
    membrane
  • bilayer
  • Interaction of highly curved membrane
    interaction site
  • perpendicular to curvature of lipid
    tubule
  • Conformational changes upon ATP hydrolysis

55
Acknowledgements / References
  • Alfred Wittinghofer
  • Vetter and Wittinghofer The Guanine nucleotide
    binding switch in three dimensions. Science
    (2001)
  • Bos, Rehmann, Wittinghofer GEFs and GAPs
    critical elements in the control of G-Proteins.
    Cell (2007)
  • A. Wittinghofer, H. Waldmann. Ras - A molecular
    switch involved in tumor formation. Angew. Chem.
    Int. Ed. (2000)
  • Scheffzek, Ahmadian, Kabsch, Wiesmuller,
    Lautwein, Schmitz Wittinghofer The Ras-RasGAP
    complex structural basis for GTPase activation
    and its loss in oncogenic Ras mutants. Science
    (1997)
  • Harvey McMahon (www.endocytosis.org)
  • Praefcke, McMahon, The dynamin superfamily
    universal membrane tubulation and fission
    molecules? Nat Rev Mol Cell Biology (2004)
  • McMahon, Gallop, Membrane curvature and
    mechanisms of dynamic cell membrane remodelling,
    Nature (2005)
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