Structure, function and

mechanisms of G-Proteins
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 7
1 5

Anhydride Ester 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
Pi
G-Protein (GDP) GAP

k5

G-Protein (GDP) + GAP

 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
Pi
G-Protein (GDP) GAP

k5

G-Protein (GDP) + GAP

 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

120000

100000
counts

80000

60000

40000

20000

0
0 100 200 300 400 500
sec

100 nM RapGAP
800 µM Rap1(GTP)
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 PRD

• 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
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