Identification of Two Distinct Inactive Conformations of the β 2 -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

Adrenaline

Adrenergic signaling 101

P Scheerer et al. Nature 455, (2008) GDP  

Adrenergic signaling 101

GPCR crystal structures Rhodopsin (2000) β 1 AR (2008) A 2A AR (2008) β 2 AR (2007) T4L Rasmussen et al., 2007 Cherezov et al., 2007 Palczewski et al., 2000 Li et al., 2004 Jaakola et al., 2008Warne et al., 2008

Broken ionic lock in β 2 AR crystals Rhodopsinβ 2 AR extracellular intracellular

GPCR crystal structures Rhodopsin (2000) β 1 AR (2008) A 2A AR (2008) β 2 AR (2007) Ionic lock formed Ionic lock broken Ionic lock broken Ionic lock broken T4L Rasmussen et al., 2007 Cherezov et al., 2007 Palczewski et al., 2000 Li 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 β 2 AR and other GPCRs (Ballesteros et al., 2001; Yao et al., 2006) Hypotheses for broken lock in inactive β 2 AR crystal structures: –Lock is typically broken in β 2 AR (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 β 2 AR T4L

Molecular dynamics simulations: inactive β 2 AR

All-atom simulations performed in Desmond with CHARMM force field

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

T4L removed, carazolol-bound No ligand T4L fusion biases equilibrium toward broken lock state % time lock formed Reconstructed intracellular loop 3 Inactive Active-likeT4L fusion protein *

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

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

Conclusions Inactive β 2 AR 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 β 1 AR structure.

Acknowledgments Acknowledgments: Michael Eastwood, Justin Gullingsrud, Kresten Lindorff-Larsen, Paul Maragakis, and Kim Palmo and other colleagues at D. E. Shaw Research Questions? Paper in press at PNAS Desmond available for free for non-commercial use: