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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: - PowerPoint PPT Presentation
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G ProteinsPart 1
Biochemistry 4000
Dr. Ute Kothe
Background ReadingTextbooks:
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
G protein families
Small G proteinsRas, Rho, Rab, Arf, Ran families
Heterotrimeric G proteinsGt, Gi, Gs
Translation Factors
EF-Tu, EF-G, IF2
OthersSRP & SR (SRP receptor)
hGBP (human guanylate binding protein)
etc.
Variety of Functions:
• sensual perception
• protein synthesis
• transport
• cell growth
• differentiation
• etc.
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
inactiveSwitch
ON
SwitchOFF
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
Consensus sequences
1. P-loop: GXXXXGK(S/T)
contacts - & -phosphates of guanine nucleotide
2. Switch I: Contains conserved T involved in Mg2+ coordination
3. Switch II: DXXG
links subsites for binding of Mg2+ and -phosphate of GTP
4. NKXD - recognizes guanine ring
5. (T/G)(C/S)A buttresses the guanine base
recognition site
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
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
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 Mg2+ binding site => weakens nucleotide binding
Ras-RasGAP Structure
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
• 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 -phosphate
- but is already further apart from the -phosphate
than in the ground state
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)
Scheffzek et al., Science 1997Catalytic Arginine finger provided in trans by RasGAP
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)
Ras activation
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
Catalytic Mechanisms - Repetition
1.
2.
3.
4.
5.
6.
Voet, Chapter 15-1, p 496ff
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
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)