Rho GTPases in cell migration

Question 1

What is a small GTPase?

Answer 1

• A specific kind of G protein that functions independently as a hydrolase enzyme to bind and hydrolyse GTP (active) to GDP (inactive)

Question 2

Give some examples of small GTPases in the Ras superfamily

Answer 2

Rab: Endosomal trafficking
Ras: Cell proliferation
Arf: Membrane budding
Rho: cytoskeleton/migration

Question 3

How does GTPase signalling depend on the bound nucleotide?

Answer 3

Often the GTPase will signal in its GTP form but will then hydrolyse one of the phosphate to be in its GDP form switching off its signalling activity

Question 4

What are the features of GTPases?

Answer 4

• P loop (phosphate coordinating loop) binding and stabilising bound nucleotide
• Magnesium ion- brings in a 2+ charge which is important because the phosphate has a negative charge so this balance is essential for nucleotide binding activity
• Switch regions (Switch 1 and 2) bind to downstream effectors. Switch regions change conformation between the signalling active and the signalling inactive forms

Question 5

What is the importance of Glutamine 61 in small GTPases?

Answer 5

• Glutamine-61 positions water so that it can attack that end phosphate and see the release of it
• If we were to substitute this Glutamine in mutation, the water would always be there and the GTP would always be in its active form as hydrolysis wouldn’t occur

Question 6

What is another way that GTPase catalyses hydrolysis?

Answer 6

P loop will counteract the negative charge in the phosphates through lysine and hydrogen which pulls some of the negative charge away from the phosphate

Question 7

What are GEFs and what do they do?

Answer 7

• Enzymes which stabilise the transition from GDP to GTP
• Can increase the transition from 10-10^7 times faster
• Once in active form it will bind to downstream effectors
• Switches itself off by hydrolysing GTP on its own but also with a GTPase activating protein (GAPs)
• Stabilising the nucleotide-free form where the GTPase is MG2+ free

Question 8

What do GAPs do?

Answer 8

• Catalyse GTP to GDP
• Restricting freedom of water molecule and lowering the energy barrier making it easier for hydrolysis to occur
• Has molecules with positive charge which draw negative charge away from the oxygen that links this together -> weak bond so water can attack and break the bond so the phosphate can be moved