A Concerted Mechanism for Opening the GDP Binding Pocket and Release of the Nucleotide in Hetero-Trimeric G-Proteins Maxime Louet 1 , David Perahia 2 , Jean Martinez 1 and Nicolas Floquet 1 ⁎ 1 Institut des Biomolécules Max Mousseron (IBMM), CNRS UMR5247, Université Montpellier 1–Université Montpellier 2, Faculté de Pharmacie, 15 Avenue Charles Flahault, BP 14491, 34093 Montpellier Cedex 05, France 2 Laboratoire de Biologie et Pharmacologie Appliquée (LBPA), CNRS UMR8113, École Normale Supérieure de Cachan, 61 Avenue du Président-Wilson, 94235 Cachan Cedex, France Received 3 December 2010; received in revised form 14 May 2011; accepted 20 May 2011 Available online 30 May 2011 Edited by D. Case Keywords: G-proteins; normal mode analysis; molecular dynamics; ligand escape; activation mechanism G-protein hetero-trimers play a fundamental role in cell function. Their dynamic behavior at the atomic level remains to be understood. We have studied the Gi hetero-trimer through a combination of molecular dynamics simulations and normal mode analyses. We showed that these big proteins could undergo large-amplitude conformational changes, without any energy penalty and with an intrinsic dynamics centered on their GDP binding pocket. Among the computed collective motions, one of the modes (mode 17) was particularly able to significantly open both the base and the phosphate sides of the GDP binding pocket. This mode describing mainly a motion between the Ras-like and the helical domains of G α was in close agreement with some available X-ray data and with many other biochemical/biophysical observations including the kink of helix α5. The use of a new protocol, which allows extraction of the GDP ligand along the computed normal modes, supported that the exit of GDP was largely coupled to an opening motion along mode 17. We propose for the first time a “concerted mechanism” model in which the opening of the GDP pocket and the kink of the α5 helix occur concomitantly and favor GDP release from G αβγ complexes. This model is discussed in the context of the G- protein-coupled receptor/G-protein interaction close to the cell membrane. © 2011 Elsevier Ltd. All rights reserved. Introduction On the cytoplasmic side of biological membranes, hetero-trimeric G-proteins are involved in signal transduction, through a direct interaction with seven transmembrane receptors, also known as G-protein- coupled receptors (GPCRs). Available X-ray struc- tures in the Protein Data Bank (PDB) show that hetero-trimeric G-proteins include (1) an α subunit (G α ) constituted by two “ras-like” and α-helical domains and (2) a high-affinity complex comprising β and γ subunits (G βγ ). Both G α and G γ possess modified amino acids that allow their anchoring to the cell membrane: an N-myristoyl-Gly and an S- palmitoyl-Cys at positions 2 and 3 of G α and an S- geranylgeranyl-Cys at position 68 of G γ . Activation of G-proteins is mediated by their interactions with GPCRs, triggering the GDP/GTP exchange in the G α subunit and promoting the molecular dissociation of G α from both the receptor and the G βγ complex. This *Corresponding author. E-mail address: nicolas.floquet@univ-montp1.fr. Abbreviations used: GPCR, G-protein-coupled receptor; PDB, Protein Data Bank; MD, molecular dynamics; NMA, normal mode analysis; NM, normal mode; RMSF, root- mean-square fluctuation; MRMS, mass-weighted root- mean-square; PCA, principal component analysis; 3D, three-dimensional; 2D, two-dimensional; BRET, bioluminescence resonance energy transfer; EPR, electron paramagnetic resonance. doi:10.1016/j.jmb.2011.05.034 J. Mol. Biol. (2011) 411, 298–312 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.