Structure, function and mechanisms of G-Proteins

1. Structure, function and mechanisms of G-Proteins Oliver Daumke MDC-Berlin, House 31.2 (Flachbau), R0225 oliver.daumke@mdc-berlin.de

2. 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.“

3. 6 7 1 5 Anhydride Ester 2 4 9 8 3 Guanosine 5   α 4 1 2 3 Guanosine-triphosphate - GTP G-Protein = Guanine-nucleotide binding protein(GNBD) Guanine Phosphates Ribose

4. 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)

5. The G-domain Mixed - protein 5 conserved motifs (G1-G5) involved in nucleotide binding Pai et al., Nature (1989)

6. 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

7. The switch regions Vetter and Wittinghofer, Science (2001)

8. 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)

9. 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

10. Non-hydrolysable GTP analogues Abbreviations GTP--S GMPPCP GMPPNP

11. Transition state mimicks of GTP hydrolysis

12. 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 ?

13. 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

14. 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)

15. 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

16. 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, …

17. The mechanism of RasGAP Scheffzek et al., Nature (1996)

18. 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)

19. An arginine residue in RasGAPs is essential for GAP activity Ras(mantGTP) vs. RasGAP Ahmadian et al., Nature Structure Biology (1997)

20. AlF3 promotes formation of a transition state complex Mittal et al., Science (1994)

21. The RasGAP-Ras complex Scheffzek et al., Science (1997)

23. 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

24. 100 nM RapGAP 800 µM Rap1(GTP)

25. Rap1GAP stimulates intrinsic Rap1 reaction 100.000 fold kcat= 6 s-1 Km = 50 µM Brinkmann et al., JBC (2001)

26. No arginine finger is involved in catalysis Brinkmann et al, JBC (2001)

28. The Rap1GAP Dimer Daumke et al., Nature (2004)

29. The catalytic domain of Rap1GAP has a G-domain fold Ras Rap1GAP cat

32. Rap1-Rap1GAP reaction followed by fluorescence stopped-flow

33. R286 is not essential for the GAP reaction

34. His287 is involved in binding to Rap1

35. Rap1GAP provides a catalytic Asn, the „Asn thumb“, for catalysis Daumke et al., Nature (2004)

36. Asn290 is a purely catalytic residue and not involved in binding to Rap1 Kd = 4 M

37. Rap1GAP-Rap1 complex indicates that Asn thumb positions attacking water molecule Scrima et al., EMBOJ (2008)

38. The Dynamin-family of GTPases

39. The shibire fly Bing Zhang, UT Austin

40. Wt 30°C Drosophila nerve terminal Kosaka and Ikeda, J Neurobiol., 1982

41. shibire 30°C Drosophila nerve terminal Kosaka and Ikeda, J Neurobiol., 1982

42. 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

43. 1000 x stimulation of Dynamin‘s GTPase reaction by lipid tubule binding Stowell et al., Nat Cell Biol (1999)

44. What is the mechanism of Dynamin ? Constrictase Effector Sever et al., Nature (1999) N&V by T. Kirchhausen

45. Is Dynamin a popase ? No Dynamin GTP--S GDP Stowell et al., Nat Cell Biol (1999) www.endocytosis.org

46. Is Dynamin working as a twistase ? Roux et al., Nature (2006) Dynamin, no nucleotide

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

48. Biotin-Dynamin streptavidin – polysterene bead Dynamin, addition GTP Roux et al., Nature (2006)

49. The EHD family • EHD = Eps15 homologydomain containing protein • Highly conserved in all higher eukaryotes, but not in • yeast and bacteria • Four paralogues in human, 70 - 80% amino acid identity

50. 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)