Lecture 9: Armstrong and Gouaux, 2000 Flashcards
What is the apo state of a receptor?
When there is no ligand bound
What are the similarities and differences between the glutamate receptor agonists?
- All have overall negative charge (except glycine)
- Have two neg carboxy groups and one positive amine group
-AMPA and kainate have pentameric rings - kainate has positive charge in ring, AMPA has negative charge in ring
What type of antagonist is DNQX?
It is a competitive antagonist, binding over glutamate and preventing current flow
What is the structure of DNQX?
Have two nitro groups. Also has two carbonyl groups and nitrogen groups that mimic a carboxyl on one end
What does higher Kd mean?
Lower affinity
What is a competition assay?
Saturated receptor with AMPA, then added kainate, glutamate, or DNQX
What is the IC50?
Inhibitory current 50, concentration at which you displace 50% of bound receptors
What were the IC50s for kainate, glutamate, and DNQX
glutamate: 821 nanomolar, kainate: 14.5 micromolar, DNQX: 998 nM
What did the binding assays and competition assays show the authors?
It showed them that the receptor construct functioned the same as the endogenous receptor
The N-termini of which helices point towards the interdomain crevice?
D, F, H, I
How is DNQX oriented in the binding pocket?
carbonyl oxygens point towards D1, nitro groups point towards D2
What is the difference between the apo bound and DNQX-bound structure?
DNQX shows 2.5˚ more closure than apo form. E705 and K730 make salt-bridge interactions
What are the interactions between DNQX and GluR2?
Major difference is E705: in apo it is forming salt bridge with K, with DNQX it flips 135˚ and is pointing towards aromatic rings, breaking salt bridge.
Forms pi-stacking interactions with Y450.
H-bonds with Y732, T686, T707, P478, R485, T480
Sulfate hydrogen bonds to 2H20s, S654, and T655 of F helix positive dipole
What are the interactions between glutamate and the receptor?
-carboxylic acid group (up): R485 (salt-bridge), T480 (H-bond), S654 (H-bond)
-Amide group: P478 (H-bond), Y450 (cation-pi), E705 (salt-bridge), T480 (H-bond)
- carboxylic acid group (down): S654 (H-bond), T655 (H-bond), H-bond with water molecule
What are the interactions between kainate and the receptor?
Basically the same as glutamate, but I650 moves closer with glutamate compared to kainate –> there is less closure of the clam shell with kainate, domain 2 does not move as close
What are the different degrees of closure between APO and KA and APO and AMPA?
APO-KA 12˚, APO-AMPA 20˚. The F-helix in AMPA is a lot more closed in
How do the degrees of closure compare between different ligands?
Apo is least closed (2˚), then DNQX (5˚), then KA (12˚), then GLU and AMPA are similar at around 20˚ of closure. Full agonists cause more closure than partial agonists. DNQX stabilizes structure in apo state.
What is the proposed mechanism of glutamate binding?
- Glutamate binds to residues in D1: R485, T480, P478, Y450 and E705. E705 breaks a salt bridge with K730 and flips 135˚
- D1 and 2 close in together
- Glutamate is further stabilized by D2: S654 and T655 of helix F. Then K730 salt bridges with D728.
In resting state, clam shell is open and stabilized by E705 salt bridge with L730, and H bonding with T655
