GDP Release Preferentially Occurs on the Phosphate Side in Heterotrimeric G-proteins

After extra-cellular stimulation of G-Protein Coupled Receptors (GPCRs), GDP/GTP exchange appears as the key, rate limiting step of the intracellular activation cycle of heterotrimeric G-proteins. Despite the availability of a large number of X-ray structures, the mechanism of GDP release out of heterotrimeric G-proteins still remains unknown at the molecular level. Starting from the available X-ray structure, extensive unconstrained/constrained molecular dynamics simulations were performed on the complete membrane-anchored Gi heterotrimer complexed to GDP, for a total simulation time overcoming 500 ns. By combining Targeted Molecular Dynamics (TMD) and free energy profiles reconstruction by umbrella sampling, our data suggest that the release of GDP was much more favored on its phosphate side. Interestingly, upon the forced extraction of GDP on this side, the whole protein encountered large, collective motions in perfect agreement with those we described previously including a domain to domain motion between the two ras-like and helical sub-domains of Gα.

[1]  S. Rasmussen,et al.  Crystal Structure of the β2Adrenergic Receptor-Gs protein complex , 2011, Nature.

[2]  Oliver P. Ernst,et al.  Crystal structure of opsin in its G-protein-interacting conformation , 2008, Nature.

[3]  H. Hamm,et al.  Interaction of a G protein with an activated receptor opens the interdomain interface in the alpha subunit , 2011, Proceedings of the National Academy of Sciences.

[4]  D. Pérahia,et al.  A concerted mechanism for opening the GDP binding pocket and release of the nucleotide in hetero-trimeric G-proteins. , 2011, Journal of Molecular Biology.

[5]  KumarShankar,et al.  The weighted histogram analysis method for free-energy calculations on biomolecules. I , 1992 .

[6]  Alexander D. MacKerell,et al.  Extending the treatment of backbone energetics in protein force fields: Limitations of gas‐phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations , 2004, J. Comput. Chem..

[7]  Junmei Wang,et al.  Development and testing of a general amber force field , 2004, J. Comput. Chem..

[8]  C. Zhan,et al.  Reaction pathway and free energy profile for prechemical reaction step of human butyrylcholinesterase-catalyzed hydrolysis of (-)-cocaine by combined targeted molecular dynamics and potential of mean force simulations. , 2010, Journal of Physical Chemistry B.

[9]  S. Sprang,et al.  The structure of the G protein heterotrimer Gi alpha 1 beta 1 gamma 2. , 1995, Cell.

[10]  H. Hamm,et al.  How do receptors activate G proteins? , 2007, Advances in protein chemistry.

[11]  D. Siderovski,et al.  Two Gα i1 Rate-Modifying Mutations Act in Concert to Allow Receptor-Independent, Steady-State Measurements of RGS Protein Activity , 2009, Journal of biomolecular screening.

[12]  Robert Schulz,et al.  Functional Rotation of the Transporter AcrB: Insights into Drug Extrusion from Simulations , 2010, PLoS Comput. Biol..

[13]  W. L. Jorgensen,et al.  Comparison of simple potential functions for simulating liquid water , 1983 .

[14]  P. Krüger,et al.  Targeted molecular dynamics: a new approach for searching pathways of conformational transitions. , 1994, Journal of molecular graphics.

[15]  H. Hamm,et al.  The 2.0 Å crystal structure of a heterotrimeric G protein , 1996, Nature.

[16]  S. Sprang,et al.  The structure of the G protein heterotrimer Giα1 β 1 γ 2 , 1995, Cell.

[17]  N. Mantel.  The detection of disease clustering and a generalized regression approach. , 1967, Cancer research.

[18]  R. Abagyan,et al.  Conserved binding mode of human beta2 adrenergic receptor inverse agonists and antagonist revealed by X-ray crystallography. , 2010, Journal of the American Chemical Society.

[19]  A. Gilman,et al.  The effect of GTP and Mg2+ on the GTPase activity and the fluorescent properties of Go. , 1987, The Journal of biological chemistry.

[20]  Prahlad T. Ram,et al.  G Protein Pathways , 2002, Science.

[21]  K Schulten,et al.  VMD: visual molecular dynamics. , 1996, Journal of molecular graphics.

[22]  J. Sondek,et al.  Structural determinants for GoLoco-induced inhibition of nucleotide release by Gα subunits , 2002, Nature.

[23]  T. Frimurer,et al.  Identification of a novel site within G protein alpha subunits important for specificity of receptor-G protein interaction. , 2004, Molecular pharmacology.

[24]  R. Cerione,et al.  A Dominant-negative Gα Mutant That Traps a Stable Rhodopsin-Gα-GTP-βγ Complex* , 2011, The Journal of Biological Chemistry.

[25]  H. Berendsen,et al.  Essential dynamics of proteins , 1993, Proteins.

[26]  R. Swendsen,et al.  THE weighted histogram analysis method for free‐energy calculations on biomolecules. I. The method , 1992 .

[27]  Bernard Maigret,et al.  Collective motions in glucosamine-6-phosphate synthase: influence of ligand binding and role in ammonia channelling and opening of the fructose-6-phosphate binding site. , 2009, Journal of molecular biology.

[28]  Laxmikant V. Kalé,et al.  Scalable molecular dynamics with NAMD , 2005, J. Comput. Chem..

[29]  Tong Liu,et al.  Structural flexibility of the Gαs α-helical domain in the β2-adrenoceptor Gs complex , 2011, Proceedings of the National Academy of Sciences.

[30]  R. Neubig,et al.  Interdomain interactions regulate GDP release from heterotrimeric G proteins. , 1999, Biochemistry.

[31]  P. Kollman,et al.  Automatic atom type and bond type perception in molecular mechanical calculations. , 2006, Journal of molecular graphics & modelling.

[32]  T Darden,et al.  New tricks for modelers from the crystallography toolkit: the particle mesh Ewald algorithm and its use in nucleic acid simulations. , 1999, Structure.

[33]  David Perahia,et al.  Activation of the ghrelin receptor is described by a privileged collective motion: a model for constitutive and agonist-induced activation of a sub-class A G-protein coupled receptor (GPCR). , 2010, Journal of molecular biology.

[34]  R. Stevens,et al.  High-Resolution Crystal Structure of an Engineered Human β2-Adrenergic G Protein–Coupled Receptor , 2007, Science.