Chapter 4: The ATP-binding site

From the closed state structure only vague predictions had been possible regarding the nature of the 3 ATP binding sites.

Imagine Hattori and Gouaux’s excitement when they realised that their new structure allowed clear characterisation and analysis of these mysterious binding sites where the previous apo structure had failed.

They observed three equivalent, novel ATP binding sites found at all 3 subunit interfaces (ie between adjacent subunits) in the extracellular region of the trimer. From herein the site will be described with reference to subunit 1 and 2, but this could refer to any of the subunit interfaces in the trimer which are all equivalent.

Regions from both subunits at the interface combine to form the ATP binding cleft. You can see from our pymol structures below that, looking into the cleft the back wall and top of the ATP binding site is formed by the left flipper, upper body and head domains from subunit 1 and the base of the cleft is formed by the lower body and dorsal fin from subunit 2.

ATP is held in place in a U-shape in the cleft via electrostatic interactions with the upper body (subunit 1), the lower body (subunit 2) and to a lesser extent by some contacts with the head domain, dorsal domain and left flipper. The most stabilising interactions are made to the triphosphate group.

Several water molecules (replaced with glycerol in the x-ray structures) act to bridge interactions between ATP and cleft residues.

In the table below we have summarised some of the key interactions that hold the ATP in place.

Residue	Residue location in cleft	α phosphate salt bridge interactions?	β phosphate salt bridge interactions?	Ɣ phosphate salt bridge interactions?	Adenine interactions?	Deoxyribose interactions?
Lys 70	Base	yes	yes	yes	no	no
Asn 269	Back wall	no	yes	no	no	no
Lys 316	Roof	no	yes	yes	no	no
Lys 72	Roof	no	no	yes	no	no
Threo 189	Deep in base	no	no	no	yes (H bonds)	no
Leu 217	Base	no	no	no	no	Yes (hydrophobic)

An aside on site specificity

Receptors are not activated by ADP or AMP as 

these ligands do not have gamma or beta phosphates 

so cannot form many of the necessary stabilising interactions

 so binding is not favourable. CTP, GTP and UTP cannot 

activate P2X4 channels. The base of CTP is not large 

enough to form an essential H bond whilst GTP and UTP

 do not have the correct orientation of donor/acceptor 

groups to form the necessary H bonds. The exquisite specificity 

for ATP-like structural and chemical properties will be an

 important consideration in designing P2X4 channel blockers.