1 GTP-Binding Proteins as Molecular Switches Wittinghofer

8/2/2019 1 GTP-Binding Proteins as Molecular Switches Wittinghofer

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GTP-binding proteins

as molecular switches

Alfred Wittinghofer,Max-Planck Institute

for Molecular Physiology


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Growth control by Ras (Rat sarcoma)

Uncontrolled growth = Cancer


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Effect of (Ras-like) Rho proteinsquiescent cell Rho(G14V)

Rac(G12V) Cdc42(G12V)

Hall, A. Science (1998) 279: 509514


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How to make molecular ON-OFF switches

More than

38000

GTP-binding

(G) Proteins

in 1383

Genomes

(Dec. 2010)


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General switch mechanism for Ras-like

proteins

Effector


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General switch mechanism for Ras-like

G proteins

kon

[G-GTP] ~

kdiss= konkcat =

koff


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How are G proteins recognized?

Sequence Motifs

Structure

Biochemistry


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Conserved sequence motifs

xTx, Switch I, G2

DxxGq/h, Switch II, G3

GxxxxGKS/T, P loop, G1

sAk, G binding, G5

N/TKxD, G binding, G4


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GTP-binding proteins with different folds:

Tubulin, FtsZ

Metabolic Enzymes (very few)

such asAdenylosuccinate Synthetase,

Succ-CoA Synthetase,PEP Carboxykinase

Not all GTP-binding proteins have a

G domain fold


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3D Structures,

invaluable for

understandingthe biochemistry

and biology of

your favorite system


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Some protein crystals


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The X-Ray experiment

X-Rays Crystal Detector


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Try yourself, build your model


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Correct!!!!!!


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Sequence motifs and topology

G1-G5 Motifs are

located in loops


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Sequence motifs around the

nucleotide binding site


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The P-loop, the most frequent

sequence motif in the database

P loop, GxxxxGKS/T, G1


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The polyanion hole

P loop, GxxxxGKS/T, G1


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Comparing differentG proteins


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Ras superfamily of GTP-binding proteins

Rab33


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Rapid increase in 3D knowledge

1989: (Correct) Structure of Ras-GppNHp

Pai et al., Nature 341, 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


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Ras-5p21 RhoA-1a2b

Rap-3rap Cdc42-2qrz Rab33B-1z06

Arl2-1ksg

Very similar structures


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Rap-3rap Cdc42-2qrz Rab33B-1z06

Ras-5p21 RhoA-1a2b

The interacting surfaces make

the difference

Arl2-1ksg


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How does the

switch work?


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The loaded-spring mechanism

design: C.Kiel


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Conformations of the switch regions in Ras


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The Ras-switch in action


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Surface of Ras during the transition

(a simulation)


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The C-terminal end of Ran


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The C-terminal switch of Ran


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The N-terminal switch of Arl/Arf

the canonical -phosphate

binding site istoo far away inthe GDP-bound

form


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The N-terminal switch of Arl/Arf


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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


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Binding of the guanine base

sAk, G5N/TKxD, G4


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The essential Mg2+ ion


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Reverse HPLC of purified Protein

Control Sample

GMP

GDP

GTP


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Value of using EDTA

to exchange nucleotide

RasGDP + [3H]GTP Ras [3H]GTP +GDP

- EDTA

+ EDTA


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The magic bullet: mGXP

mant-GXP or mGDP/GTP, wl Em: 440 nm

Fluorescent

reporter group


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Ras and mGDP/mGTP

more than 100 % fluorescence increase

from water to protein


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Intrinsic versus catalyzed GDP releasein real time

RasmGDP + GDP RasGDP +mGDP

+ Sos

- Sos


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Multi-domain G Proteins


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The most important G protein (super)

families

Translation factors, ie EF-Tu

Heterotrimeric G proteins, G

Ras superfamily proteins

Dynamin superfamily

SRP, SRP-receptor (SR)

Septins Many small subfamilies


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Extra domains as additional


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Conformational change of EF-Tu


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Conclusions

G proteins are universal switch molecules

Their G domain has a typical , 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

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1 GTP-Binding Proteins as Molecular Switches Wittinghofer