The importance of being patient

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

Elegant switches in an E. Coli ion channel

Molecular dynamics simulations comprise one of the most important tools in the armamentarium of chemists and biologists. Initially a curiosity for theoretical scientists, MD is now an explanatory and predictive tool in chemistry, biology, materials science, engineering and even weather prediction. In the field of biology, some masters such as Martin Karplus have honed this tool to the status of an art. While great leaps have been made in the context of methods, hardware, software and applications in this field, much remains to be still done. One of the reasons is that even now, running microsecond MD is computationally quite expensive. But many important biological events involving biomacromolecules take place on this time scale, thus making the achievement of efficient microsecond MD simulations important.

Writing in Science, scientists from D. E. Shaw company, Columbia and the Hebrew University of Jerusalem have a lovely paper documenting the application of the new and innovative MD program Desmond to the dynamics of a bacterial ion channel that transports Na+ ions using the electromotive force generated by proton transport. Desmond is supposed to enable efficient microsecond MD. It is going to be interfaced with the Maestro interface developed by Schrodinger and is due to be released this year I believe. Currently the fastest MD program on a single processor is GROMACS. While head to head comparisons of GROMACS and Desmond have not been reported to my knowledge, Desmond is supposed to be very fast on multiple processors, a facility that many can now afford to have.

In the Science paper, the researchers apply Desmond to understand the transport mechanism of the Na+/H+ antiport ion channel in E. Coli. This protein is crucial for E. Coli to survive harsh conditions of pH, alkalinity, and ionic lithium environments. The authors basically focus on the protonation state of certain key aspartates and find something pretty interesting- two crucial aspartates essentially act as switches that decide whether Na+ ions would be transported to the cytoplasm or to the periplasm. Using many long MD simulations involving different protonation states, the authors discovered that one of the carboxylates always has to be protonated. This acts like a "master aspartate" switch. Once this switch's state is set, it's the state of the other switch that decides the direction of transport- protonated leads to expulsion of the Na+ into the periplasm, while deprotonated leads to expulsion into the cytoplasm.

The observation reminded me of a high-school "staircase lighting" electricity experiment. A master switch had to be always on for the assembly to work. The On/Off state of another switch would then govern whether current flowed or not.

Using this discovery as the basis for exploring further conformational changes related to it, the authors come up with an elegant stepwise mechanism for the transport of Na+ and H+ ions that accounts for the observed stoichiometry of one Na+ ion for every two H+ transported. Using free energy perturbation binding affinity calculations, the authors also rationalize the channel's observed selectivity for Na+ over K+, and slightly for Li+ over Na+.

There is also a a very intriguing explanation for the pH sensitivity of this ion channel. The crystal structure of the channel is solved at pH 4, and it is inactive at this pH. How does the channel get activated at higher pH? To explore this, the authors do something simple but quite clever. They first identify all the key aspartates lining the channel and determine their pKa values. Perhaps not surprisingly, the pKa values of these are abnormally high- not an uncommon observation for amino acids in the unusual environments in protein interiors. They then selectively deprotonate one aspartate keeping all others protonated and do MD on the resulting structures. If there is a key "pH sensing" aspartate, its protonation state will likely govern a conformational change from inactive-active. Indeed, one aspartate, D133, turns out to modulate a conformational change involving two helices when it is deprotonated. Crucially, this results in the "master aspartate" noted above to move away from the Na+ entry/exit pathways. Thus it can no longer bind the ion, resulting in an inactive channel. Mutagenesis studies also support the observations.

A neat conclusion from a beautiful set of experiments. A fascinating example of how nature essentially and surprisingly uses high-school chemistry to modulate movements in complex proteins. And a highly successful and inspiring example of how efficient, long MD simulations can shed light on these crucial processes.

Arkin, I.T., Xu, H., Jensen, M.O., Arbely, E., Bennett, E.R., Bowers, K.J., Chow, E., Dror, R.O., Eastwood, M.P., Flitman-Tene, R., Gregersen, B.A., Klepeis, J.L., Kolossvary, I., Shan, Y., Shaw, D.E. (2007). Mechanism of Na+/H+ Antiporting. Science, 317(5839), 799-803. DOI: 10.1126/science.1142824