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G Proteins Part 1

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

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G Proteins Part 1

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  1. G ProteinsPart 1 Biochemistry 4000 Dr. Ute Kothe

  2. Background Reading Textbooks: Biochemistry, Voet, Chapter 19-2., p 673 – 680 Molecular Cell Biology, Lodish, 5th 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 Gta, Gia, Gsa 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 inactive Switch ON Switch OFF active

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

  6. Consensus sequences • P-loop: GXXXXGK(S/T) contacts a- & b-phosphates of guanine nucleotide • Switch I: Contains conserved T involved in Mg2+ coordination • Switch II: DXXG links subsites for binding of Mg2+ and g-phosphate of GTP • NKXD - recognizes guanine ring • (T/G)(C/S)A buttresses the guanine base recognition site

  7. Structural States • “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 g-phosphate • upon GTP hydrolysis and release of the g-phosphate, the switch • regions relax into their GDP-specific conformations Vetter & Wittinghofer, Science 2001

  8. Guanine nucleotide exchange factors (GEF) • 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 Bos et al., Cell 2007

  9. GEF - Mechanism • Diverse Structures – similar mechanisms: • interact with Switch I and II • induce conformational changes P loop => release of phosphates • sterically occlude Mg2+ binding site => weakens nucleotide binding Bos et al., Cell 2007

  10. Ras-RasGAP Structure • Individual Structures of Ras and RasGAP known • Only transient interaction terminated by GTP hydrolysis • Stabilized by transition state analog found biochemically: GDP + AlF3 = mimics GTP in transition state - AlF3 occupies position of g-phosphate - but is already further apart from the b-phosphate than in the ground state Features of the crystal structure: • 2.5 Å resolution • 81 % Completeness • Solved by molecular replacement using individual structures • Rcryst = 23.3 % • Rfree = 32.3 % Scheffzek et al., Science 1997

  11. Ras-RasGAP structure • Contacts between: • P-loop, Switch I & II, helix a3 in Ras • a6c, a7c, L1c (finger loop), L6c (variable loop) in RasGAP • weak van der Waals interactions (yellow) and • several polar interactions (red) Scheffzek et al., Science 1997

  12. Ras activation Attacking H2O molecule in H-bonding distance to carbonyl group of Gln81 and Thr35 main chain AlF3 in contact with Mg2+, Thr35, Lys16, Gln61 (Ras) & Arg 789 (RasGAP) Catalytic Arginine finger provided in trans by RasGAP Scheffzek et al., Science 1997

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

  14. Catalytic Mechanisms - Repetition Voet, Chapter 15-1, p 496ff

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

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

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