The determination of the ß-adrenergic receptor GPCR structure in 2007 was a breakthrough in structural biology. Combined with the earlier structure of rhodopsin, this provided a template for structure-based design for GPCRs. However, there was a lurking mystery in the structure, a mystery which was not always discussed but which has started to come to light recently.
Th mystery is exemplified by a recent paper in which authors from D E Shaw Research in New York use extremely long molecular dynamics simulations to uncover a peculiar conformational characteristic of the ß2 AR. The original structure was crystallized bound to an inverse agonist named carazolol. The receptor as crystallized was thought to be in an inactive state. In this state, two helices of the receptor were at some distance from each other. However, this observation did not square with biochemical experiments that indicated proximity of the two helices mediated by a crucial ionic lock, a salt bridge between a glutamate and arginine. This lock however was absent in the crystal structure, raising questions about the exact role of the lock in activating the receptor and the nature of the inactive state.
In the present study, the authors used extremely long, microsecond MD simulations on the crystal structure. They used the DESMOND program recently introduced by Schrodinger and D E Shaw to perform simulations of the GPCR in a lipid bilayer.
All they really had to do was wait.
The first 150 ns were not very interesting from the perspective of the salt bridge. However, the salt bridge spontaneously formed after 150 ns and then stayed put like a fly on fly paper. Notice the N-O distance (blue) and how it stabilizes after 150 ns.
The bridge also involved local movement of the helices and some important residues. The authors also did the simulation in the presence and absence of the ligand and found that this lock forms irrespective of the presence of the ligand. They follow up with some mutagenesis experiments that reconcile the conformational changes with experimental observations. Interestingly, they mutate an aspartate that is also proximal to the arginine in the salt bridge. Mutation of this aspartate would be expected to "free up" the arginine and further encourage its interaction with the glutamate. However, the opposite seemed to happen, indicating an interesting role for the aspartate as a something of lock itself in holding the aspartate fixed.
The overall conclusion is that there are probably two inactive states, one in which the salt bridge is formed (the dominant one) and one in which it's broken (not highly populated) and the receptor recycles between the two. The less populated conformation is nonetheless the one that is crystallized which is interesting. This kind of observation is clearly important for further structure-based design since it implies that one could encourage GPCR activation if the "right" conformation of the receptor could be preferentially stabilized.
The thing to note here is the time. Nothing interesting would have been observed had the simulation been run for less than 150 ns. Researchers who ran the simulation for less than 150 ns may not have had something worth reporting. 150 ns is a reasonably long amount of time for any MD program or simulation. The fact that such simulations can be run for microseconds attests to the rapid development of hardware and software exemplified by D E Shaw's program DESMOND and their processor named ANTON.
Sometimes simply waiting long enough can lead to productive results. Echoing an unpleasant man's ominous pronouncement, "Quantity has a quality of its own".
Dror, R., Arlow, D., Borhani, D., Jensen, M., Piana, S., & Shaw, D. (2009). Identification of two distinct inactive conformations of the ß2-adrenergic receptor reconciles structural and biochemical observations Proceedings of the National Academy of Sciences, 106 (12), 4689-4694 DOI: 10.1073/pnas.0811065106