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Ron Dror, Daniel Arlow, David Borhani, Morten Jensen, Stefano Piana, and David Shaw

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.

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Ron Dror, Daniel Arlow, David Borhani, Morten Jensen, Stefano Piana, and David Shaw

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  1. 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

  2. Adrenergic signaling 101

  3. Adrenergic signaling 101

  4. Adrenergic signaling 101

  5. Adrenergic signaling 101 Adrenaline

  6. Adrenergic signaling 101

  7. Adrenergic signaling 101

  8. Adrenergic signaling 101  a GDP P Scheerer et al.Nature455, 497-502 (2008)

  9. Adrenergic signaling 101

  10. Adrenergic signaling 101

  11. Adrenergic signaling 101

  12. Adrenergic signaling 101

  13. Adrenergic signaling 101

  14. Adrenergic signaling 101

  15. Adrenergic signaling 101

  16. Adrenergic signaling 101

  17. Adrenergic signaling 101

  18. GPCR crystal structures Rhodopsin (2000) β2AR (2007) β1AR (2008) A2AAR (2008) T4L T4L Palczewski et al., 2000 Li et al., 2004 Rasmussen et al., 2007 Cherezov et al., 2007 Warne et al., 2008 Jaakola et al., 2008

  19. Broken ionic lock in β2AR crystals Rhodopsin β2AR extracellular intracellular

  20. Ionic lock broken Ionic lock broken Ionic lock broken Ionic lock formed GPCR crystal structures Rhodopsin (2000) β2AR (2007) β1AR (2008) A2AAR (2008) T4L T4L Palczewski et al., 2000 Li et al., 2004 Rasmussen et al., 2007 Cherezov et al., 2007 Warne et al., 2008 Jaakola et al., 2008

  21. 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)

  22. Molecular dynamics simulations: inactive β2AR T4L

  23. Molecular dynamics simulations: inactive β2AR

  24. All-atom simulations performed in Desmond with CHARMM force field

  25. Ionic lock forms

  26. Ionic lock forms Helices 3 and 6 move together, adopting a rhodopsin-like conformation

  27. Ionic lock forms Helices 3 and 6 move together, adopting a rhodopsin-like conformation

  28. Lock shows broken/formed equilibrium In four similar simulations, lock formed 91% of time on average

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

  30. Intracellular loop 2 folds into a helix, matching β1AR structure

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

  32. 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.

  33. 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 PNAS Desmond available for free for non-commercial use: www.DEShawResearch.com

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