Structure, function and mechanisms of G-Proteins Oliver Daumke MDC-Berlin, House 31.2 (Flachbau), R0225

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 Ribose Phosphates α  Guanosine EsterAnhydride 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 Effector: Interacts stably with the GTP-bound form GEF: Guanine nucleotide Exchange Factor GAP: GTPase Activating Protein To allow switch function: high affinity 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: k cat = min -1 ) S N 2 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, )

Enzymatic strategies for GTP hydrolysis 1)Counteracting of negative charge at phosphates - P-loop (GxxxxGKS), hydrogen bonds and lysine - Mg 2+ 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 10 5 – 10 6 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) P i  GAP G-Protein (GDP) GAP G-Protein (GDP) + GAP k1k2 k3 k4 k5 PiPi

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 (K M, k cat )

Monitoring the GAP-catalysed reaction G-Protein (GTP) + GAP G-Protein (GTP)  GAP G-Protein (GDP) P i  GAP G-Protein (GDP) GAP G-Protein (GDP) + GAP k1k2 k3 k4 k5 PiPi

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 k 1, k 2, K D, …

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)

AlF 3 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

100 nM RapGAP 800 µM Rap1(GTP)

Rap1GAP stimulates intrinsic Rap1 reaction fold k cat = 6 s -1 K m = 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 K d = 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 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 ConstrictaseEffector

Stowell et al., Nat Cell Biol (1999) 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-Dynamin streptavidin – polysterene bead

EHD = Eps15 homology domain containing protein Highly conserved in all higher eukaryotes, but not in yeast and bacteria Four paralogues in human, % 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)

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 ( 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)