Chapter 3: Comparison

However, to gain simply a general knowledge of the structure was not Hattori and Gouaux’s.  They wanted to discover the changes that occur in the structure when it binds ATP compared to its apo state. Only this comparison would allow them to fully characterise the ATP binding site and propose a mechanism of activation.

They first decided to compare the structural differences between a single apo monomer and an ATP bound monomer but they found that the conformational differences between the two were fairly small. This must have been a disconcerting discovery since we often assume that ligand binding triggers significant conformational changes.

 Next, they superimposed the images of the apo and ATP-bound trimers together. In contrast to the two monomers they found  the two trimers differed largely in their conformations. Clearly, when ATP binds each monomer undergoes a small conformational change that cumulatively translates into a large conformational change in the trimer.

 For your ease we have collated the various comparisons Hattori and Gouaux made and the conformational changes they observed in the extracellular and transmembrane domains into the tables below.

Comparison of the extracellular domains:

Receptor	Upper body domain	Lower body domain (form walls of extracellular vestibule)	Central pathway through the EC domains	Lateral fenestrations (openings) leading into  the EC vestibule
Apo	Close contact between the 3 upper body domains	Two lower body walls are separated from each other by 15 angstroms	Too narrow for ion passage	Too narrow for ion passage
ATP-bound	Close contact retained, no large conformational difference, thought to act as a rigid scaffold about which other conformational changes occur	Outward flexing of the lower body domains, two lower body domains are now separated by 25.5angstroms	Too narrow for ion passage	Wide open

The most exciting change observed in the ATP bound receptor was that the location and fold of the ATP binding clefts could clearly be observed. Hattori and Gouaux dedicate a large chunk of their paper to characterisation of these sites and we as narrators can only follow their lead by dedicating the whole of the next chapter to them.

Comparison of the transmembrane domains:

Receptor	Ion channel pore diameter	Transmembrane domain rotations	Transmembrane domain interactions
Apo	-pore entrance is 10 angstroms -this narrows to ~0.5 angstroms, widens to 2 angstroms then narrows again to ~0.5 angstroms across a set of 7 residues (the gate) -pore exit is ~5 angstroms	-tm helices form the arrangement indicated in the image of the apo structure. -all rotations are measured relative to helical positions in the apo state	-interactions between 2 leucine residues and an alanine residue stabilise the closed pore state
ATP-bound	-pore entrance is 10 angstroms -this narrows to ~3.5 angstroms across a set of 3 residues (the gate) then widens again -pore exit is 10 angstroms -this suggests that the pore gate has decreased in length and expanded in diameter by ~3angstroms	- helix 1 rotates 10 angstroms anticlockwise about an axis perpendicular to the membrane -helix 2 rotates 55 angstroms anticlockwise about the same axis and forms a kink at flexible glysine residue -gaps form between helices of adjacent subunits -the overall effect is of the iris of an eye opening	-leu/leu/ala interactions are disrupted -new contacts between leu and ile stabilise the open pore state -it is proposed that in the native environment helices form new interactions with membrane lipids and allosteric modulators that occupy the newly formed gaps between adjacent subunits

Here is a great video that compares the structural arrangement of the transmembrane domains between first the apo and the ATP bound state. From this video you can start to how by putting all the data from their comparisons together Hattori and Gouaux could begin to piece together the steps of activation.

Supplementary movie 1, 'Molecular mechanism of ATP binding and ion channel activation in P2X receptors', Motoyuki Hattori and Eric Gouaux, 2012, Nature