Identification of Two Distinct Inactive Conformations of the 2-Adrenergic Receptor Reconciles Structural and Biochemical ObservationsRon Dror, Daniel Arlow,David Borhani, Morten Jensen,Stefano Piana, and David Shaw D. E. Shaw Research

Adrenergic signaling 101

Adrenergic signaling 101

Adrenergic signaling 101

Adrenergic signaling 101Adrenaline

Adrenergic signaling 101

Adrenergic signaling 101

Adrenergic signaling 101P Scheerer et al. Nature 455, 497-502 (2008)GDPa

Adrenergic signaling 101

Adrenergic signaling 101

Adrenergic signaling 101

Adrenergic signaling 101

Adrenergic signaling 101

Adrenergic signaling 101

Adrenergic signaling 101

Adrenergic signaling 101

Adrenergic signaling 101

GPCR crystal structuresRhodopsin(2000)1AR(2008)A2AAR(2008)2AR(2007)T4LT4LRasmussen et al., 2007Cherezov et al., 2007Palczewski et al., 2000Li et al., 2004Jaakola et al., 2008Warne et al., 2008

Broken ionic lock in 2AR crystals Rhodopsin2ARextracellularintracellular

GPCR crystal structuresRhodopsin(2000)1AR(2008)A2AAR(2008)2AR(2007)T4LT4LRasmussen et al., 2007Cherezov et al., 2007Palczewski et al., 2000Li et al., 2004Jaakola et al., 2008Warne et al., 2008

Broken ionic lock presents a puzzleBiochemical data suggests that lock stabilizes inactive state of 2AR and other GPCRs (Ballesteros et al., 2001; Yao et al., 2006)Hypotheses for broken lock in inactive 2AR crystal structures:Lock is typically broken in 2AR (Rosenbaum et al., 2007; Warne et al., 2008)Broken lock reflects particular ligand properties(Lefkowitz et al., 2008; Audet & Bouvier, 2008)Crystals capture one of multiple inactive conformations (Rasmussen et al. 2007; Ranganathan, 2007)

Molecular dynamics simulations: inactive 2AR T4L

Molecular dynamics simulations: inactive 2AR

All-atom simulations performed in Desmond with CHARMM force field

Ionic lock forms

Ionic lock formsHelices 3 and 6 move together, adopting a rhodopsin-like conformation

Ionic lock formsHelices 3 and 6 move together, adopting a rhodopsin-like conformation

Lock shows broken/formed equilibriumIn four similar simulations, lock formed 91% of time on average

T4L fusion biases equilibrium toward broken lock stateT4L removed, carazolol-boundNo ligand% time lock formedReconstructed intracellular loop 3InactiveActive-likeT4L fusion protein*

Intracellular loop 2 folds into a helix, matching 1AR structure

Intracellular loop 3 foldsIntracellular loop 3 is absent from 2AR crystal structures. It was reconstructed for this simulation.

ConclusionsInactive 2AR appears to be in equilibrium between major conformation with ionic lock formed and minor conformation with lock brokenExplains biochemical observationsCrystal structures may represent minor conformationSecondary structure elements form, some of which match 1AR structure.

AcknowledgmentsAcknowledgments: Michael Eastwood, Justin Gullingsrud, Kresten Lindorff-Larsen, Paul Maragakis, and Kim Palmo and other colleagues at D. E. Shaw Research

Questions? Dan.Arlow@DEShawResearch.com, Ron.Dror@DEShawResearch.com Paper in press at PNASDesmond available for free for non-commercial use: www.DEShawResearch.com

Ill be focusing on the beta-2 adrenergic receptor, which is one of the best studied and most clinically important GPCRs.The GPCRs or G-protein coupled receptors are the largest class of membrane proteins in humans, and the largest class of drug targets. Roughly 50% of drugs on the market target GPCRs. Beta-2 is a target for a variety of drugs, particularly cardiac and asthma drugs; even if youve never heard of the beta-2 adrenergic receptor, youve almost certainly heard of beta blockers.As youd expect, GPCRs have been studied very extensively in the laboratory, but obtaining structures has proven very difficult.Until late 2007, the structure for only one GPCR was available: bovine rhodopsin.Rhodopsin is an unusual GPCR: its activated by absorption of light, whereas the great majority of GPCRs are activated by the binding of a diffusible ligand (e.g., a drug).The last year and a half has seen breakthroughs in GPCR crystallography. The structure of beta-2 was solved in late 2007, followed by the beta-1 adrenergic receptor and the adenosine 2A receptor in 2008. Various ingenious tricks were required to stabilize these GPCRs for crystallization. The beta-2 structure shown here has a rigid protein called T4 lysozyme fused onto it.For the most part, these structures are similar to rhodopsin, but there are certain differences. One surprising difference involved something called the ionic lock a set of salt bridges which is intact in rhodopsin but broken in all the other recent GPCR structures.

Mention structures of multiple forms of rhodopsinHeres a more detailed comparison of the rhodopsin and beta-2 structures. The ionic lock in rhodopsin involves a salt bridge between a negatively charged glutamate in transmembrane helix 6 and a negatively charged arginine in transmembrane helix 3. Theres an additional negatively charged residue next to the arginine that helps restrain its motion.This is important because its believed to play a role in the activation process. When rhodopsin absorbs light, it undergoes a conformational change (called activation) that transmits a signal to an intracellular G protein. The largest motion involved in the activation process is that helices 3 and 6 move apart by some 5 angstroms, breaking this ionic lock. The ionic lock helps stabilize the inactive state.Beta-2 has homologous residues, and is believed to activate in a similar way that is, to undergo similar conformational change when a ligand binds to it. But in the structure, theres no contact between the glutamate residue on helix 6 and the arginine on helix 3. The two helices are also further apart. This is despite the fact that the structure was solved in complex with carazolol, which is an inverse agonist (should stabilize the inactive conformation). The same phenomenon was also observed in another beta-2 structure that was stabilize with an antibody fragment instead of through fusion to a lysozyme. It was also osbserved in the beta-1 and adenosine-2A structures, as mentioned previously.As youd expect, GPCRs have been studied very extensively in the laboratory, but obtaining structures has proven very difficult.Until late 2007, the structure for only one GPCR was available: bovine rhodopsin.Rhodopsin is an unusual GPCR: its activated by absorption of light, whereas the great majority of GPCRs are activated by the binding of a diffusible ligand (e.g., a drug).The last year and a half has seen breakthroughs in GPCR crystallography. The structure of beta-2 was solved in late 2007, followed by the beta-1 adrenergic receptor and the adenosine 2A receptor in 2008. Various ingenious tricks were required to stabilize these GPCRs for crystallization. The beta-2 structure shown here has a rigid protein called T4 lysozyme fused onto it.For the most part, these structures are similar to rhodopsin, but there are certain differences. One surprising difference involved something called the ionic lock a set of salt bridges which is intact in rhodopsin but broken in all the other recent GPCR structures.

Mention structures of multiple forms of rhodopsinThis broken lock has engendered a great deal of discussion and speculation ever since the first beta-2 structure came out all the more so because theres a good deal of biochemical evidence on beta-2 specifically suggesting that it is formed in the inactive state and that it stabilizes the inactive state. This includes mutation studies and fluorescence studies. In fact, term ionic lock was coined in studies of beta-2.A number of different explanations for the crystallographic observations have been put forth in the literature. They fall into three categories:1) The lock may always be broken in in beta-2, even if its not bound to any ligand or bound to a ligand that suppresses activity. If that were the case, the ionic lock wouldnt play the role in beta-2 that it does in rhodopsin, and the biochemical evidence to the contrary would have somehow been misleading.2) A second hypothesis is that the ionic lock is formed in the unliganded state, and also formed in the presence of most inactivating ligands, but broken specifically by the ligand with which is was cocrystallized, carazolol. This hypothesis was suggested by the fact that carazolol and certain similar ligands have been discovered recently to have unique signaling properties they prevent signaling through G-proteins, but promote signaling through other pathways.3) A third hypothesis is that the crystal structures dont tell the whole story. Beta-2 may visit multiple conformations in its inactive state, and the ionic lock may be formed in some but not in others.We decided to use molecular dynamics to figure out which of these explanations is the correct one.

Yet biochemical evidence suggests that intact ionic lock stabilizes inactive state of GPCRs, especially 2ARMutation studies (Ballesteros et al., 2001, and more)Fluorescence quenching studies (Yao et al, 2006)In fact, the term ionic lock was coined in studies of beta-2.We start with the 2.4-A resolution crystal structure of beta-2, which has a large intracellular loop replaced by T4 lysozyme. This lysozyme may interfere with the dynamics, particularly because it connects to the protein next to the helix-6 part of the ionic lock. We handled this in various ways in various simulations sometimes we left it on, sometimes we replaced it by a model of the loop, but in most cases, we just cut it away. Theres evidence that you can cut this loop in various places without affecting activity of the protein.We embedded the protein in a lipid bilayer with explicit solvent and simulated it using Desmond, a software package we developed that offers high parallel performance on a standard linux cluster. We performed a total of more than 10 microseconds of simulation.* In the absence of T4 lysozyme, we see the ionic lock form within a few hundred nanoseconds.

Which trajectory is shown in movie?Here are snapshots from the beginning of the simulation, showing the ionic lock broken, and about 300 nanoseconds into the simulation, showing the lock formed. You can also see that the helices have moved together. If we superimpose both these images on the rhodopsin crystal structure (shown here in purple), we can see that the helices are adopting a conformation like that of inactive rhodopsin.The plot on the bottom shows in red the distance between the C-alpha backbone atoms of the glutamate on helix 6 and the aspartate on helix 3, as a function of simulation time. In blue is the distance between the closest nitrogen and oxygen side-chain atoms of these two residues. The shaded area shows the times at which the ionic lock is formed and the helices are close together.If we run a longer simulation this one is two microseconds we see that once the lock closes, it stays closed most of the time, but not all the time. Once in a while, it opens occasionally for tens of nanoseconds or even a hundred nanoseconds at a time.In this simulation, the lock is formed 90% of the time. We performed three other similar simulations, and on average across all of them it was formed 91% of the time.Next, we performed a set of simulations of different constructs, and measured the fraction of time the lock was formed, with the helices close together in a rhodopsin-like conformation.The simulations I showed previously had carazolol bound, as in the crystal structures. When we remove carazolol, we get similar results.Likewise, if instead of simply removing the T4 lysozyme we replace it by a model of intracellular loop 3, which it replaced in the crystal structure, we get similar results.However, if we simulate the original crystal structure with the T4L intact, we find that the ionic lock is closed most of the time.Likewise, when we simulated two mutants that are believed to disrupt the ionic lock and that have been shown experimentally to lead to increased activity in the absence of a ligand, we see similar results.Indeed, T4L mutant displays CAM-like properties.

Therefore there appears to be an equilibrium between a conformational state with the lock formed and the helices close together, and one with the lock broken and the helices further apart.Independent of the ionic lock, we also observe several secondary structure formation events during our simulations.One of the most interesting is the formation of a helix in intracellular loop 2, which is an unstructured coil in the beta-2 crystal structures.

A number of side chain rearrangements are required for the ionic lock to form. One of these is a motion of Tyr219, of helix 5, from one side of helix 6 to the other. This tyrosine is in different positions in the two beta-2 crystal structures, and in our simulation, it moves from one of these positions to the other. We believe the starting position may reflect an artifact of the T4L-fusion, and we believe it may be associated with constitutive activity.

position of this tyrosine in the starting crystal structure is

Tyr219 moves from one side of helix 6 to another, matching inactive structure of rhodopsin [dicsuss role in activation, and also Fab-bound structure]