Molecular Dynamics Simulations of the Effect of the G-Protein and Diffusible Ligands on the β2-Adrenergic Receptor Angela Goetz 1,2 , Harald Lanig 2 , Peter Gmeiner 1 and Timothy Clark 2,3 ⁎ 1 Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich Alexander University, Schuhstrasse 19, 91052 Erlangen, Germany 2 Department of Chemistry and Pharmacy, Computer Chemistry Center, Friedrich Alexander University, Naegelsbachstrasse 25, 91052 Erlangen, Germany 3 Centre for Molecular Design, University of Portsmouth, Mercantile House, Portsmouth PO1 2EG, UK Received 18 May 2011; received in revised form 4 October 2011; accepted 11 October 2011 Available online 20 October 2011 Edited by D. Case Keywords: GPCR; β2-adrenergic receptor; molecular dynamics simulations; protein–receptor interactions; 7TM receptor activation G-protein-coupled receptors have extraordinary therapeutic potential as targets for a broad spectrum of diseases. Understanding their function at the molecular level is therefore essential. A variety of crystal structures have made the investigation of the inactive receptor state possible. Recently released X-ray structures of opsin and the β 2 -adrenergic receptor (β 2 AR) have provided insight into the active receptor state. In addition, we have contributed to the crystal structure of an irreversible agonist–β 2 adreno- ceptor complex. These extensive studies and biophysical investigations have revealed that agonist binding leads to a low-affinity conformation of the active state that is suggested to facilitate G-protein binding. The high- affinity receptor state, which promotes signal transduction, is only formed in the presence of both agonist and G-protein. Despite numerous crystal structures, it is not yet clear how ligands tune receptor dynamics and G- protein binding. We have now used molecular dynamics simulations to elucidate the distinct impact of agonist and inverse agonist on receptor conformation and G-protein binding by investigating the influence of the ligands on the structure and dynamics of a complex composed of β 2 AR and the C-terminal end of the Gα s subunit (GαCT). The simulations clearly showed that the agonist isoprenaline and the inverse agonist carazolol influence the ligand-binding site and the interaction between β 2 AR and GαCT differently. Isoprenaline induced an inward motion of helix 5, whereas carazolol blocked the rearrangement of the extracellular part of the receptor. Moreover, in the presence of isoprenaline, β 2 AR and GαCT form a stable interaction that is destabilized by carazolol. © 2011 Elsevier Ltd. All rights reserved. Introduction The β 2 -adrenergic receptor (β 2 AR), which repre- sents an important target for cardiac and asthma drugs, is an extensively studied model system for the large superfamily of G-protein-coupled recep- tors (GPCRs). In 2007, the receptor was crystallized in its inactive conformation bound to the partial inverse agonist carazolol (1). 1–3 Like rhodopsin, 4 *Corresponding author. Computer-Chemie-Centrum, Friedrich-Alexander-Universität Erlangen-Nürnberg, Nägelsbachstraße 25, 91052 Erlangen, Germany. E-mail address: Tim.Clark@chemie.uni-erlangen.de. Abbreviations used: GPCR, G-protein-coupled receptor; β 2 AR, β 2 -adrenergic receptor; GαCT, C-terminal end of the Gα subunit; TM, transmembrane helix; IL, intracellular loop; EL, extracellular loop; MD, molecular dynamics; DOPC, dioleoylphospathidylcholine; PDB, Protein Data Bank; GAFF, general all-atom AMBER force field. doi:10.1016/j.jmb.2011.10.015 J. Mol. Biol. (2011) 414, 611–623 Contents lists available at www.sciencedirect.com Journal of Molecular Biology journal homepage: http://ees.elsevier.com.jmb 0022-2836/$ - see front matter © 2011 Elsevier Ltd. All rights reserved.