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

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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.
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G-Protein Guanine-nucleotide binding
protein(GNBD)
Guanine
Phosphates
Ribose
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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)

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The G-domain
Mixed ?-? protein 5 conserved motifs (G1-G5)
involved in nucleotide binding
Pai et al., Nature (1989)
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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
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The switch regions
Vetter and Wittinghofer, Science (2001)
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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)
  

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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

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Non-hydrolysable GTP analogues
Abbreviations
GTP-?-S
GMPPCP
GMPPNP
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Transition state mimicks of GTP hydrolysis
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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 ?

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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
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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)

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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
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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,

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The mechanism of RasGAP
Scheffzek et al., Nature (1996)
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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)
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An arginine residue in RasGAPs is essential for
GAP activity
Ras(mantGTP) vs. RasGAP
Ahmadian et al., Nature Structure Biology (1997)
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AlF3 promotes formation of a transition state
complex
Mittal et al., Science (1994)
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The RasGAP-Ras complex
Scheffzek et al., Science (1997)
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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

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100 nM RapGAP 800 µM Rap1(GTP)
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Rap1GAP stimulates intrinsic Rap1 reaction
100.000 fold
kcat 6 s-1 Km 50 µM
Brinkmann et al., JBC (2001)
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No arginine finger is involved in catalysis
Brinkmann et al, JBC (2001)
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The Rap1GAP Dimer
Daumke et al., Nature (2004)
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The catalytic domain of Rap1GAP has a G-domain
fold
Ras
Rap1GAP cat
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Rap1-Rap1GAP reaction followed by fluorescence
stopped-flow
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R286 is not essential for the GAP reaction
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His287 is involved in binding to Rap1
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Rap1GAP provides a catalytic Asn, the Asn
thumb, for catalysis
Daumke et al., Nature (2004)
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Asn290 is a purely catalytic residue and not
involved in binding to Rap1
Kd 4 ?M
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Rap1GAP-Rap1 complex indicates that Asn thumb
positions attacking water molecule
Scrima et al., EMBOJ (2008)
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The Dynamin-family of GTPases
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The shibire fly
Bing Zhang, UT Austin
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Wt 30C Drosophila nerve terminal Kosaka and
Ikeda, J Neurobiol., 1982
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shibire 30C Drosophila nerve terminal Kosaka and
Ikeda, J Neurobiol., 1982
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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
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1000 x stimulation of Dynamins GTPase reaction
by lipid tubule binding
Stowell et al., Nat Cell Biol (1999)
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What is the mechanism of Dynamin ?
Constrictase
Effector
Sever et al., Nature (1999) NV by T. Kirchhausen
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Is Dynamin a popase ?
No Dynamin
GTP-?-S
GDP
Stowell et al., Nat Cell Biol (1999) www.endocytos
is.org
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Is Dynamin working as a twistase ?
Roux et al., Nature (2006)
Dynamin, no nucleotide
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Dynamin, addition GTP
Roux et al., Nature (2006)
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Biotin-Dynamin streptavidin polysterene bead
Dynamin, addition GTP
Roux et al., Nature (2006)
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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

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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)
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Daumke et al., Nature (2007)
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Lipid binding site of EHD2
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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

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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)