Structural Biochemistry/G Protein Mechanism

Introduction

G-protein

G-Protein was first discovered in 1994 by Alfred Gilman and Martin Rodbell who later receive Nobel Prize in Medicine. G-Protein stands for “Guanine-nucleotide binding protein”. Martin Rodbell and Alfred Gilman used genetic and biochemical techniques to identify and purify the G protein. They found that a transducer provided the link between the hormone receptor and the amplifier. They used lymphoma cells that normally can be activated by a receptor to form cyclic AMP. A mutated lymphoma cell was usually found to contain a normal receptor and a normal cyclic AMP-generating enzyme but was yet unable to respond because it lacked the transducer. This was a good system to assay purified G proteins. A G-protein could be isolated from normal brain tissue and inserted in the mutated cell, thereby restoring its function. Besides, G proteins are a key to the chemical switches. They bind the guanine nucleotides GDP and GTP. Also, they are heterotrimers that are associated with the inner surface of the plasma membrane.

G-Protein Family

G-Domain

Some G-Protein families include:

1. heterotrimetic G-protein in 7-TM receptor signaling
2. initiation, elongation, termination factors in protein synthesis (IF1, EF-Tu, EF-TS)
3. signal recognition particle and its receptor, translocation of nascent polypeptide chains in the ER
4. Ras-like GTPases (Ras, Rap, Rho, Ran, Rab, Arf, Arl, Sar), molecular switches in signal transduction
5. dynamin superfamily of GTPases, remodeling of membranes, etc. The family of Dynamin-related GTPases are classical dynamins: Dyn1, Dyn2 and Dyn3.

The dynamin-related proteins are Mx and Mitofusin; GBP-related proteins: GBPs and atlastins and bacterial dynamics. The common features are:

1. low affinity for nucleotide
2. template induced self-oligomerisation
3. assembly stimulated GTP hydrolysis.

Ras-like G-Protein

Ras-like G-Protein

Ras-like G-Protein: molecular switches
Effector: interacts stably with the GTP-bound form
GEF: guanine nucleotide exchange factor
GAP: GTPase Activating protein

Switch Forms

The switch regions in two forms:

1. GTP form
2. GDP form

GTPase Reaction

The GTPase reaction intrinsic GTPase rates of small G-Proteins are slow in a range of Kcal=10^(-2) to 10^(-3) min^(-1). Then, the reaction performs the Sn2 nucleophilic attack with trigonal bipyramidal transition state. The phosphate hydrolysis reaction is thermodynamically highly favorable but kinetically very slow.
There are mainly two enzymatic strategies for GTP hydrolysis involved:

1. counteracting negative charge at phosphates with arginine as the catalyst
2. positing of attacking nucleophile with the catalyst of glutamine.

The non-hydrolysable GTP analogues:

1. GTP-y-S
2. GMPPCP
3. GMPPNP

The GTPase Activating Proteins accelerate intrinsic GTPase by a factor of 10^5 to 10^6. Ras, Rap, Rho, Rab, Ran have completely unrelated GAPs. High affinity binding to the GTP-bound form, low affinity interaction with the GDP-bound form.

Turnover Assays

Single turnover

1) Multiple turnover assays:

The monitors several rounds of GAP catalysed G-Protein hydrolysis. G-Protein as substrate, GAP in catalytic amounts. Vary concentration of G-protein to determine Michaelis-Menten parameters.

2) Single turnover assays:

The analysis of a single cycle of GTP hydrolysis often monitored by fluorescently labeled G-Protein in one cell, excess of GAP in the other cell. It vary concentration of GAP is multiparameter firt allows determination of K1, K2, KD, etc. The Biochemical features are such binds to adenine and not guanine nucleotides with affinity in the low micromolar range and binds to negatively charged liposome stimulated ATP hydrolysis.

Membrane Remodeling

The implications for membrane remodeling factors involved in membrane remodeling/ destabilization: the oligomer formation intro rings around a lipid template; insertion of hydrophobic residues into outer membrane bilayer; interaction of highly curved membrane interaction site perpendicular to curvature of lipid tubule; conformational changes upon ATP hydrolysis.

Reference

Alfred Wittinghofer

1. Vetter and Wittinghofer "The Guanine nucleotide binding switch in three dimensions." Science (2001)
2. Bos, Rehmann, Wittinghofer "GEFs and GAPs critical elements in the control of G-Proteins." Cell (2007)
3. A. Wittinghofer, H. Waldmann. "Rad-A molecular switch involved in tumor formation." Angew.Chem.Int.Ed (2000)
4. Scheffzek,Ahmadian,Kabsch,Wiesmuller,Lautwein,Schmitz& Wittinghofer "The Rass-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants." Science (1997)

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/G/G_Proteins.html

Harvey McMahon

1. Prafcke, McMahon. "The dynamin superfamily: universal membrane tubulation and fission molecules?" Nat Rev Mol cell Biology (2004)
2. McMahon, Gallop "Membrane curvature and mechanisms of dynamic cell membrane remodelling" Nature (2005)