Post on 14-Dec-2015
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