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  • 1 GTP-Binding Proteins As Molecular Switches Wittinghofer

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    Oli.Talty
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    This document discusses GTP-binding proteins and how they function as molecular switches. It describes how Ras proteins control cell growth and proliferation and how mutations can lead to uncontrolled growth and cancer. GTP-binding proteins use a conserved switch mechanism involving conformational changes between GTP-bound and GDP-bound states. There are over 38,000 GTP-binding proteins across genomes that are regulated by guanine nucleotide exchange factors and GTPase activating proteins. X-ray crystallography has provided insights into the structures of GTP-binding domains and switch regions across different protein families.

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    1 GTP-Binding Proteins as Molecular Switches Wittinghofer

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    This document discusses GTP-binding proteins and how they function as molecular switches. It describes how Ras proteins control cell growth and proliferation and how mutations can lead to uncontrolled growth and cancer. GTP-binding proteins use a conserved switch mechanism involving conformational changes between GTP-bound and GDP-bound states. There are over 38,000 GTP-binding proteins across genomes that are regulated by guanine nucleotide exchange factors and GTPase activating proteins. X-ray crystallography has provided insights into the structures of GTP-binding domains and switch regions across different protein families.

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    162 tayangan48 halaman

    1 GTP-Binding Proteins As Molecular Switches Wittinghofer

    Diunggah oleh

    Oli.Talty
    This document discusses GTP-binding proteins and how they function as molecular switches. It describes how Ras proteins control cell growth and proliferation and how mutations can lead to uncontrolled growth and cancer. GTP-binding proteins use a conserved switch mechanism involving conformational changes between GTP-bound and GDP-bound states. There are over 38,000 GTP-binding proteins across genomes that are regulated by guanine nucleotide exchange factors and GTPase activating proteins. X-ray crystallography has provided insights into the structures of GTP-binding domains and switch regions across different protein families.

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    Attribution Non-Commercial (BY-NC)
    Unduh sebagai PDF, TXT atau baca online dari Scribd
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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: 509514

    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

    Conserved sequence motifs


    GxxxxGKS/T, P loop, G1
    xTx, Switch I, G2

    DxxGq/h, Switch II, G3 N/TKxD, G binding, G4 sAk, G binding, G5

    Not all GTP-binding proteins have a G domain fold


    GTP-binding proteins with different folds: Tubulin, FtsZ Metabolic Enzymes (very few) such as Adenylosuccinate Synthetase, Succ-CoA Synthetase, PEP Carboxykinase

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

    Some protein crystals

    The X-Ray experiment

    X-Rays Crystal

    Detector

    Try yourself, build your model

    Correct!!!!!!

    The G domain, a typical , Fold

    Sequence motifs and topology

    G1-G5 Motifs are located in loops

    Sequence motifs around the nucleotide binding site

    The P-loop, the most frequent sequence motif in the database

    P loop, GxxxxGKS/T, G1

    The polyanion hole

    P loop, GxxxxGKS/T, G1

    Comparing different G proteins

    Ras superfamily of GTP-binding proteins

    Rab33

    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

    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?

    The loaded-spring mechanism

    design: C.Kiel

    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 phosphate 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

    Binding of the guanine base

    N/TKxD, G4

    sAk, G5

    The essential Mg2+ ion

    Reverse HPLC of purified Protein


    GMP

    GDP

    GTP

    Control

    Sample

    Value of using EDTA to exchange nucleotide


    + EDTA

    - EDTA

    RasGDP + [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

    RasmGDP + GDP

    RasGDP + mGDP

    Multi-domain G Proteins

    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

    Extra domains as additional

    EF-Tu, additional domains


    Domain2 Domain3

    Conformational change of EF-Tu

    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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