Structure, function and mechanisms of G-Proteins

Structure, function and mechanisms of G-Proteins

Oliver Daumke

MDC-Berlin, House 31.2 (Flachbau), R0225

oliver.daumke@mdc-berlin.de

1994 Nobel Prize in Medicine, Alfred Gilman and Martin Rodbell, for their „discovery of G-Proteinsand the role of these proteins in signal transduction in cells.“

G-Protein = Guanine-nucleotide binding protein(GNBD)

12

5

43

Guanine

Ribose

Phosphates

α

1

3

42

65 7

89

Guanosine

EsterAnhydride

Guanosine-triphosphate - GTP

G-Protein families

• Heterotrimeric G-Proteins (Transducin, Gi, Gq …), 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

Effector: Interacts stably with the GTP-bound form GEF: Guanine nucleotide Exchange FactorGAP: GTPase Activating Protein

To allow switch function: highaffinity for nucleotide required pMol

Vetter and Wittinghofer, Science (2001)

The switch regions

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

G-Protein (GTP)GAP

G-Protein (GDP) Pi GAP

G-Protein (GDP) GAP

G-Protein (GDP) + GAP

k1 k2

k3

k4

k5

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

G-Protein (GTP)GAP

G-Protein (GDP) Pi GAP

G-Protein (GDP) GAP

G-Protein (GDP) + GAP

k1 k2

k3

k4

k5

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 increase:

complex formation

Fluorescence decrease:

GTP hydrolysis

Fluorescence stopped-flow to monitor the GAP reaction

Ras(mantGTP) vs. RasGAP

Ahmadian et al., Nature Structure Biology (1997)

Ras(mantGTP) vs. RasGAP

An arginine residue in RasGAPs is essential for GAP activity

Ahmadian et al., Nature Structure Biology (1997)

AlF3 promotes formation of a transition state complex

Mittal et al., Science (1994)

Scheffzek et al., Science (1997)

The RasGAP-Ras complex

• 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

Rap1

0 100 200 300 400 5000

20000

40000

60000

80000

100000

120000

140000

160000

180000c

ou

nts

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 terminalKosaka and Ikeda, J Neurobiol., 1982

shibire 30°C Drosophila nerve terminalKosaka and Ikeda, J Neurobiol., 1982

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-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 ?

Sever et al., Nature (1999)N&V by T. Kirchhausen

Constrictase Effector

Stowell et al., Nat Cell Biol (1999)www.endocytosis.org

No Dynamin GTP--S GDP

Is Dynamin a popase ?

Is Dynamin working as a twistase ?

Roux et al., Nature (2006)Dynamin, no nucleotide

Roux et al., Nature (2006)Dynamin, addition GTP

Roux et al., Nature (2006)Dynamin, addition GTP

Biotin-Dynaminstreptavidin – polysterene bead

• 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

The EHD family

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 WittinghoferVetter 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)