Download Structure, function and mechanism of G

Structure, function and
mechanisms of G-Proteins
Oliver Daumke
MDC-Berlin, House 31.2 (Flachbau), R0225
[email protected]
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.“
G-Protein = Guanine-nucleotide binding protein
(GNBD)
Guanine
6
1
Anhydride Ester
7
5
2
4
9
8
3
5


Guanosine
α
4
1
3
2
Phosphates
Ribose
Guanosine-triphosphate - GTP
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)
The G-domain
Mixed - protein
5 conserved motifs (G1-G5)
involved in nucleotide binding
Pai et al., Nature (1989)
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
The switch regions
Vetter and Wittinghofer, Science (2001)
The GTPase reaction
• Intrinsic GTPase rates of small G-Proteins are slow
(range: kcat=10-2 - 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)
Enzymatic strategies for GTP hydrolysis
1) 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)
2) Positioning of attacking nucleophile
- catalytic glutamine
Non-hydrolysable GTP analogues
Abbreviations
GTP--S
GMPPCP
GMPPNP
Transition state mimicks of GTP hydrolysis
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 ?
Monitoring the GAP-catalysed reaction
G-Protein (GTP) + GAP
k1
k2
G-Protein (GTP)GAP
k3
G-Protein (GDP) Pi  GAP
k4
G-Protein (GDP) GAP
k5
G-Protein (GDP) + GAP
Pi
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)
Monitoring the GAP-catalysed reaction
G-Protein (GTP) + GAP
k1
k2
G-Protein (GTP)GAP
k3
G-Protein (GDP) Pi  GAP
k4
G-Protein (GDP) GAP
k5
G-Protein (GDP) + GAP
Pi
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, …
The mechanism of RasGAP
Scheffzek et al., Nature (1996)
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)
An arginine residue in RasGAPs is essential
for GAP activity
Ras(mantGTP) vs. RasGAP
Ahmadian et al., Nature Structure Biology (1997)
AlF3 promotes formation of a transition state
complex
Mittal et al., Science (1994)
The RasGAP-Ras complex
Scheffzek et al., Science (1997)
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
180000
160000
140000
counts
120000
100000
80000
60000
40000
20000
0
0
100
200
300
sec
100 nM RapGAP
800 µM Rap1(GTP)
400
500
Rap1GAP stimulates intrinsic Rap1 reaction
100.000 fold
kcat= 6 s-1
Km = 50 µM
Brinkmann et al., JBC (2001)
No arginine finger is involved in catalysis
Brinkmann et al, JBC (2001)
The Rap1GAP Dimer
Daumke et al., Nature (2004)
The catalytic domain of Rap1GAP has a
G-domain fold
Ras
Rap1GAP cat
Rap1-Rap1GAP reaction followed by
fluorescence stopped-flow
R286 is not essential for the GAP reaction
His287 is involved in binding to Rap1
Rap1GAP provides a catalytic Asn,
the „Asn thumb“, for catalysis
Daumke et al., Nature (2004)
Asn290 is a purely catalytic residue and not
involved in binding to Rap1
Kd = 4 M
Rap1GAP-Rap1 complex indicates that Asn
thumb positions attacking water molecule
Scrima et al., EMBOJ (2008)
The Dynamin-family of GTPases
The shibire fly
Bing Zhang, UT Austin
Wt 30°C Drosophila nerve terminal
Kosaka and Ikeda, J Neurobiol., 1982
shibire 30°C Drosophila nerve terminal
Kosaka and Ikeda, J Neurobiol., 1982
The family of Dynamin-related GTPases
• Classical Dynamins: Dyn1, Dyn2, Dyn3
GTPase
Middle
PH
GED
• Dynamin-related proteins: Mx, Mitofusin
• GBP-related proteins: GBPs, Atlastins
• Bacterial Dynamins
Common features:
- Low affinity for nucleotide
- Template induced self-oligomerisation
- Assembly-stimulated GTP hydrolysis
PRD
1000 x stimulation of Dynamin‘s GTPase
reaction by lipid tubule binding
Stowell et al., Nat Cell Biol (1999)
What is the mechanism of Dynamin ?
Constrictase
Effector
Sever et al., Nature (1999)
N&V by T. Kirchhausen
Is Dynamin a popase ?
No Dynamin
GTP--S
GDP
Stowell et al., Nat Cell Biol (1999)
www.endocytosis.org
Is Dynamin working as a twistase ?
Dynamin, no nucleotide
Roux et al., Nature (2006)
Dynamin, addition GTP
Roux et al., Nature (2006)
Biotin-Dynamin
streptavidin – polysterene bead
Dynamin, addition GTP
Roux et al., Nature (2006)
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
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)
Daumke et al., Nature (2007)
Lipid binding site of EHD2
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
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)