Identification of Two Distinct Inactive Conformations of the β2-Adrenergic Receptor Reconciles Structural

and Biochemical Observations

Ron 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 101

Adrenaline

Adrenergic signaling 101

Adrenergic signaling 101

Adrenergic signaling 101

P Scheerer et al. Nature 455, 497-502 (2008)

GDP

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 structures

Rhodopsin(2000)

β1AR(2008)

A2AAR(2008)

β2AR(2007)

T4LT4L

Rasmussen et al., 2007Cherezov et al., 2007

Palczewski et al., 2000Li et al., 2004 Jaakola et al., 2008Warne et al., 2008

Broken ionic lock in β2AR crystals

Rhodopsin β2ARextracellular

intracellular

GPCR crystal structures

Rhodopsin(2000)

β1AR(2008)

A2AAR(2008)

β2AR(2007)

Ionic lock formed

Ionic lock broken

Ionic lock broken

Ionic lock broken

T4LT4L

Rasmussen et al., 2007Cherezov et al., 2007

Palczewski et al., 2000Li et al., 2004 Jaakola et al., 2008Warne et al., 2008

Broken ionic lock presents a puzzle

• Biochemical data suggests that lock stabilizes inactive state of β2AR and other GPCRs

(Ballesteros et al., 2001; Yao et al., 2006)

• Hypotheses for broken lock in inactive β2AR crystal structures:– Lock is typically broken in β2AR

(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 β2AR

T4L

Molecular dynamics simulations: inactive β2AR

All-atom simulations performed in Desmond with CHARMM force field

Ionic lock forms

QuickTime™ and a decompressor

are needed to see this picture.

Ionic lock forms

Helices 3 and 6 move together, adopting a rhodopsin-like conformation

Ionic lock forms

Helices 3 and 6 move together, adopting a rhodopsin-like conformation

Lock shows broken/formed equilibrium

In four similar simulations, lock formed 91% of time on average

41%

92%

91%

91%

0% 20% 40% 60% 80% 100%

T4L removed, carazolol-bound

No ligand

T4L fusion biases equilibrium toward broken lock state

% time lock formed

Reconstructed intracellular loop 3

Inactive

Active-like T4L fusion protein*

Intracellular loop 2 folds into a helix, matching β1AR structure

QuickTime™ and a decompressor

are needed to see this picture.

Intracellular loop 3 folds

QuickTime™ and a decompressor

are needed to see this picture.

Intracellular loop 3 is absent from β2AR crystal structures. It was reconstructed for this simulation.

Conclusions

• Inactive β2AR appears to be in equilibrium between major conformation with ionic lock formed and minor conformation with lock broken– Explains biochemical observations– Crystal structures may represent minor conformation

• Secondary structure elements form, some of which match β1AR structure.

Acknowledgments• Acknowledgments: 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