Probing Gαi1 protein activation at single–amino acid resolution

We present comprehensive maps at single–amino acid resolution of the residues stabilizing the human Gαi1 subunit in nucleotide- and receptor-bound states. We generated these maps by measuring the effects of alanine mutations on the stability of Gαi1 and the rhodopsin–Gαi1 complex. We identified stabilization clusters in the GTPase and helical domains responsible for structural integrity and the conformational changes associated with activation. In activation cluster I, helices α1 and α5 pack against strands β1–β3 to stabilize the nucleotide-bound states. In the receptor-bound state, these interactions are replaced by interactions between α5 and strands β4–β6. Key residues in this cluster are Y320, which is crucial for the stabilization of the receptor-bound state, and F336, which stabilizes nucleotide-bound states. Destabilization of helix α1, caused by rearrangement of this activation cluster, leads to the weakening of the interdomain interface and release of GDP.

[1]  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.

[2]  Shahab M. Danesh,et al.  Recognition of the activated states of Galpha13 by the rgRGS domain of PDZRhoGEF. , 2008, Structure.

[3]  Gebhard F. X. Schertler,et al.  The structural basis of agonist-induced activation in constitutively active rhodopsin , 2011, Nature.

[4]  A. J. Venkatakrishnan,et al.  Universal allosteric mechanism for Gα activation by GPCRs , 2015, Nature.

[5]  A. Fersht,et al.  Mapping the transition state and pathway of protein folding by protein engineering , 1989, Nature.

[6]  R. Stevens,et al.  Structural Basis for Allosteric Regulation of GPCRs by Sodium Ions , 2012, Science.

[7]  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.

[8]  O. Lichtarge,et al.  Receptor and betagamma binding sites in the alpha subunit of the retinal G protein transducin. , 1997, Science.

[9]  H. Hamm,et al.  Site of G protein binding to rhodopsin mapped with synthetic peptides from the alpha subunit. , 1988, Science.

[10]  Edward A. Dratz,et al.  NMR structure of a receptor-bound G-protein peptide , 1997, Nature.

[11]  Ned Van Eps,et al.  Mechanism of the receptor-catalyzed activation of heterotrimeric G proteins , 2006, Nature Structural &Molecular Biology.

[12]  H. Hamm,et al.  A Conserved Phenylalanine as a Relay between the α5 Helix and the GDP Binding Region of Heterotrimeric Gi Protein α Subunit* , 2014, The Journal of Biological Chemistry.

[13]  T. Sakmar,et al.  Rapid Activation of Transducin by Mutations Distant from the Nucleotide-binding Site , 2001, The Journal of Biological Chemistry.

[14]  Jeremy C. Smith,et al.  Functional interactions in bacteriorhodopsin: a theoretical analysis of retinal hydrogen bonding with water. , 1995, Biophysical journal.

[15]  Olivier Lichtarge,et al.  Receptor and βγ Binding Sites in the α Subunit of the Retinal G Protein Transducin , 1997, Science.

[16]  Ben M. Webb,et al.  Comparative Protein Structure Modeling Using MODELLER , 2007, Current protocols in protein science.

[17]  Gebhard F. X. Schertler,et al.  Crystallization Scale Preparation of a Stable GPCR Signaling Complex between Constitutively Active Rhodopsin and G-Protein , 2014, PloS one.

[18]  H. Bourne,et al.  Transducin‐alpha C‐terminal mutations prevent activation by rhodopsin: a new assay using recombinant proteins expressed in cultured cells. , 1995, The EMBO journal.

[19]  S. Sprang,et al.  Tertiary and Quaternary Structural Changes in Giα1 Induced by GTP Hydrolysis , 1995, Science.

[20]  Virgil L. Woods,et al.  Conformational changes in the G protein Gs induced by the β2 adrenergic receptor , 2011, Nature.

[21]  T. Sakmar,et al.  Disruption of the alpha5 helix of transducin impairs rhodopsin-catalyzed nucleotide exchange. , 2002, Biochemistry.

[22]  E. Weiss,et al.  The Effect of Carboxyl-terminal Mutagenesis of G on Rhodopsin and Guanine Nucleotide Binding (*) , 1995, The Journal of Biological Chemistry.

[23]  Alan M. Jones,et al.  Heterotrimeric G protein signalling in the plant kingdom , 2013, Open Biology.

[24]  Michael F. Brown,et al.  Two protonation switches control rhodopsin activation in membranes , 2008, Proceedings of the National Academy of Sciences.

[25]  S R Sprang,et al.  G proteins, effectors and GAPs: structure and mechanism. , 1997, Current opinion in structural biology.

[26]  D. Higgins,et al.  Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega , 2011, Molecular systems biology.

[27]  K Schulten,et al.  Molecular dynamics study of the nature and origin of retinal's twisted structure in bacteriorhodopsin. , 2000, Biophysical journal.

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

[29]  H. Hamm,et al.  Heterotrimeric G protein activation by G-protein-coupled receptors , 2008, Nature Reviews Molecular Cell Biology.

[30]  Heidi E. Hamm,et al.  The 2.2 Å crystal structure of transducin-α complexed with GTPγS , 1993, Nature.

[31]  S. Sprang,et al.  Structural and biochemical characterization of the GTPgammaS-, GDP.Pi-, and GDP-bound forms of a GTPase-deficient Gly42 --> Val mutant of Gialpha1. , 1997, Biochemistry.

[32]  Alan M. Jones,et al.  The Crystal Structure of a Self-Activating G Protein α Subunit Reveals Its Distinct Mechanism of Signal Initiation , 2011, Science Signaling.

[33]  Ben M. Webb,et al.  Comparative Protein Structure Modeling Using Modeller , 2006, Current protocols in bioinformatics.

[34]  W. Liu,et al.  The helical domain of a G protein alpha subunit is a regulator of its effector. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[35]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

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

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

[38]  Ron O. Dror,et al.  Structural basis for nucleotide exchange in heterotrimeric G proteins , 2015, Science.

[39]  H. Hamm,et al.  Energetic analysis of the R*-G complex links the α5 helix to GDP release and domain opening , 2013, Nature Structural &Molecular Biology.

[40]  N. Oppenheimer,et al.  Structure and mechanism , 1989 .

[41]  P B Sigler,et al.  The 2.2 A crystal structure of transducin-alpha complexed with GTP gamma S. , 1994, Nature.

[42]  A. Gilman,et al.  G proteins: transducers of receptor-generated signals. , 1987, Annual review of biochemistry.

[43]  So Iwata,et al.  Methods and Results in Crystallization of Membrane Proteins , 2003 .

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

[45]  S. Sprang,et al.  Tertiary and quaternary structural changes in Gi alpha 1 induced by GTP hydrolysis. , 1995, Science.

[46]  Dawei Sun,et al.  AAscan, PCRdesign and MutantChecker: A Suite of Programs for Primer Design and Sequence Analysis for High-Throughput Scanning Mutagenesis , 2013, PloS one.

[47]  S. Sprang,et al.  Structures of active conformations of Gi alpha 1 and the mechanism of GTP hydrolysis. , 1994, Science.

[48]  Alexander D. MacKerell,et al.  All-atom empirical potential for molecular modeling and dynamics studies of proteins. , 1998, The journal of physical chemistry. B.

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

[50]  Heidi E. Hamm,et al.  Structural determinants for activation of the α-subunit of a heterotrimeric G protein , 1994, Nature.

[51]  Michel Bouvier,et al.  Probing the activation-promoted structural rearrangements in preassembled receptor–G protein complexes , 2006, Nature Structural &Molecular Biology.