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GTP-binding proteins as molecular switches

Alfred Wittinghofer, Max-Planck Institute for Molecular Physiology

Growth control by Ras (Rat sarcoma)

Uncontrolled growth = Cancer

Effect of (Ras-like) Rho proteins
quiescent cell Rho(G14V)

Rac(G12V)
Hall, A. Science (1998) 279: 509–514

Cdc42(G12V)

How to make molecular ON-OFF switches
More than 38000 GTP-binding (G) Proteins in 1383 Genomes (Dec. 2010)

General switch mechanism for Ras-like proteins Effector .

General switch mechanism for Ras-like G proteins kcat = koff kdiss = kon kon [G-GTP] ~ —— koff .

How are G proteins recognized? Sequence Motifs Structure Biochemistry .

G4 sAk.Conserved sequence motifs GxxxxGKS/T. G1 xTx. G binding. G binding. Switch I. G5 . P loop. G3 N/TKxD. Switch II. G2 DxxGq/h.

Succ-CoA Synthetase. PEP Carboxykinase . FtsZ Metabolic Enzymes (very few) such as Adenylosuccinate Synthetase.Not all GTP-binding proteins have a G domain fold GTP-binding proteins with different folds: Tubulin.

invaluable for understanding the biochemistry and biology of your favorite system .3D Structures.

Some protein crystals .

The X-Ray experiment X-Rays → Crystal → Detector .

build your model .Try yourself.

Correct!!!!!! .

a typical α.The G domain.β Fold .

Sequence motifs and topology G1-G5 Motifs are located in loops .

Sequence motifs around the nucleotide binding site .

G1 .The P-loop. GxxxxGKS/T. the most frequent sequence motif in the database P loop.

GxxxxGKS/T.The polyanion hole P loop. G1 .

Comparing different G proteins .

Ras superfamily of GTP-binding proteins Rab33 .

. 209-214 (1989) 2008: > 400 deposits in the pdb data base 31 complexes with effectors 14 complexes with GEFs 8 complexes with GAPs 4 complexes with toxins 2011: >500 deposits .Rapid increase in 3D knowledge 1989: (Correct) Structure of Ras-GppNHp Pai et al. Nature 341.

Very similar structures Ras-5p21 RhoA-1a2b Arl2-1ksg Rap-3rap Cdc42-2qrz Rab33B-1z06 .

The interacting surfaces make the difference Ras-5p21 RhoA-1a2b Arl2-1ksg Rap-3rap Cdc42-2qrz Rab33B-1z06 .

How does the switch work? .

Kiel .The loaded-spring mechanism design: C.

Conformations of the switch regions in Ras .

The Ras-switch in action .

Surface of Ras during the transition (a simulation) .

The C-terminal end of Ran .

The C-terminal switch of Ran .

The N-terminal switch of Arl/Arf the canonical γphosphate binding site is too far away in the GDP-bound form .

The N-terminal switch of Arl/Arf .

Conserved switch mechanism between GTP and ATP-binding P-loop proteins Motor Protein G Protein .

Some biochemical properties (in particular of small G proteins) • • • • • • High affinitity (pM to nM Kd) Slow dissociation of nucleotide Mg2+ dependent affinity High specificity Slow GTPase Mg2+ dependent GTPase .

G4 sAk.Binding of the guanine base N/TKxD. G5 .

The essential Mg2+ ion .

Reverse HPLC of purified Protein GMP GDP GTP Control Sample .

Value of using EDTA to exchange nucleotide + EDTA .EDTA Ras•GDP + [3H]GTP ⇌ Ras• [3H]GTP + GDP .

The magic bullet: mGXP Fluorescent reporter group mant-GXP or mGDP/GTP. wl Em: 440 nm .

Ras and mGDP/mGTP more than 100 % fluorescence increase from water to protein .

Intrinsic versus catalyzed GDP release in real time .Sos + Sos Ras•mGDP + GDP ⇌ Ras•GDP + mGDP .

Multi-domain G Proteins .

SRP-receptor (SR) Septins Many small subfamilies . Gα Ras superfamily proteins Dynamin superfamily SRP. ie EF-Tu Heterotrimeric G proteins.The most important G protein (super) families • • • • • • • Translation factors.

Extra domains as additional .

EF-Tu. additional domains Domain2 Domain3 .

Conformational change of EF-Tu .

β structure Work by a canonical switch mechanism Are specific for guanine nucleotides Have a slow intrinsic nucleotide exchange Have a slow intrinsic GTPase • Are regulated by GEFs and GAPs .Conclusions • • • • • • G proteins are universal switch molecules Their G domain has a typical α.