G Proteins Part 1

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)