1. G Proteins Part 1 Biochemistry 4000 Dr. Ute Kothe

2. Background Reading Textbooks: Biochemistry, Voet, Chapter 19-2., p 673 – 680 Molecular Cell Biology, Lodish, 5 th Edition, Chapters 13.3 & 13.4 Reviews: Vetter & Wittinghofer, Science 2001 Bos et al., Cell 2007 Research Publications: Scheffzek et al., Science 1997 – crystal structure of Ras-RasGAP complex Tesmer et al., Cell 1997 – crystal structure heterotrimeric G protein

3. G protein families Small G proteins Ras, Rho, Rab, Arf, Ran families Heterotrimeric G proteins G t , G i , G s  Translation Factors EF-Tu, EF-G, IF2 Others SRP & SR (SRP receptor) hGBP (human guanylate binding protein) etc. Variety of Functions: sensual perception protein synthesis transport cell growth differentiation etc.

4. G protein = Molecular switch GDP: Guanosine diphosphate GTP: Guanosine triphosphate Pi: inorganic phosphate GAP: GTPase activating protein GEF: Guanine nucleotide- exchange factor GDI: Guanine nucleotide- dissociation inhibitor active inactive Switch ON Switch OFF

5. G domain Vetter & Wittinghofer, Science 2001 Universal structure: Ras: example of minimal G domain 20 – 25 kD Mixed 6-stranded  sheet 5  helices on both sides  domain

6. Consensus sequences 1.P-loop: GXXXXGK(S/T) contacts  - &  -phosphates of guanine nucleotide 2.Switch I: Contains conserved T involved in Mg 2+ coordination 3.Switch II: DXXG links subsites for binding of Mg 2+ and  -phosphate of GTP 4.NKXD - recognizes guanine ring 5.(T/G)(C/S)A buttresses the guanine base recognition site

7. Structural States Vetter & Wittinghofer, Science 2001 “Loaded-Spring Mechanism”: in GTP form, both switch regions are held in place by contacts of Thr in Switch I and Gly in Switch II to the  -phosphate upon GTP hydrolysis and release of the  -phosphate, the switch regions relax into their GDP-specific conformations

8. Guanine nucleotide exchange factors (GEF) Bos et al., Cell 2007 Switch ON, i.e. activate G proteins necessary since G proteins bind guanine nucleotides tightly (KDs in nM – pM range), i.e. dissociation is slow on its own (hours) accelerate dissociation of guanine nucleotides without altering the equilibrium “compete” with guanine nucleotide for binding in vivo [GTP] = 10 x [GDP], i.e. typically GDP is replaced by GTP

9. GEF - Mechanism Bos et al., Cell 2007 Diverse Structures – similar mechanisms: interact with Switch I and II induce conformational changes P loop => release of phosphates sterically occlude Mg 2+ binding site => weakens nucleotide binding

10. Ras-RasGAP Structure Features of the crystal structure: 2.5 Å resolution 81 % Completeness Solved by molecular replacement using individual structures R cryst = 23.3 % R free = 32.3 % Scheffzek et al., Science 1997 Individual Structures of Ras and RasGAP known Only transient interaction terminated by GTP hydrolysis  Stabilized by transition state analog found biochemically: GDP + AlF 3 = mimics GTP in transition state - AlF 3 occupies position of  -phosphate - but is already further apart from the  -phosphate than in the ground state

11. Ras-RasGAP structure Scheffzek et al., Science 1997 Contacts between: P-loop, Switch I & II, helix  3 in Ras  6c,  7c, L1c (finger loop), L6c (variable loop) in RasGAP weak van der Waals interactions (yellow) and several polar interactions (red)

12. Scheffzek et al., Science 1997 Catalytic Arginine finger provided in trans by RasGAP Attacking H 2 O molecule in H- bonding distance to carbonyl group of Gln81 and Thr35 main chain AlF 3 in contact with Mg 2+, Thr35, Lys16, Gln61 (Ras) & Arg 789 (RasGAP) Ras activation

13. Scheffzek et al., Science 1997 Ras activation Activation by RasGAP: 1.Stabilization of the Switch II region containing Gln61 2.Providing of a catalytic residue (Arginine finger) in trans

14. Catalytic Mechanisms - Repetition 1. 2. 3. 4. 5. 6. Voet, Chapter 15-1, p 496ff

15. Catalysis of GTP hydrolysis associative mechanism of phosphoryl transfer: negative charge develops on  -phosphate, pentavalent phosphorous intermediate stabilization of the transition state: Arg finger shields developping negative charges on  - phosphate Scheffzek et al., Science 1997

16. Mechanisms of GTPase activation Bos et al., Cell 2007 diverse GAP structures diverse mechanisms of GTPase activation Common Features: 1.Stabilization of intrinsically mobile catalytic machinery 2.Insertion of a catalytic residue in trans (not in heterotrimeric G proteins)