A Transient Interaction between the Phosphate Binding Loop and Switch I Contributes to the Allosteric Network between Receptor and Nucleotide in Gαi1

Thaker, Tarjani; Sarwar, Maruf; Preininger, Anita M.; Hamm, Heidi E.; Iverson, T.M.

doi:10.1074/jbc.m113.539064

Cited by 7 publications

(10 citation statements)

References 79 publications

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“…In addition, the structure of Rasmussen and co-workers (7) together with mass spectrometry results also confirm that both N-terminal ␤1 strand and C-terminal ␣5 helix are major interaction sites for receptors. This supports the previously suggested role of these regions in coupling receptor binding and nucleotide dissociation activities (11)(12)(13)(14)(15)(16)(17). Despite these advances, critical questions remain unanswered: How do the distinct conformations evident in the accumulated structures interconvert?…”

Section: And Ref 3)supporting

confidence: 68%

Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins

Yao¹,

Malik

Griggs

et al. 2016

Journal of Biological Chemistry

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G protein ␣ subunits cycle between active and inactive conformations to regulate a multitude of intracellular signaling cascades. Important structural transitions occurring during this cycle have been characterized from extensive crystallographic studies. However, the link between observed conformations and the allosteric regulation of binding events at distal sites critical for signaling through G proteins remain unclear. Here we describe molecular dynamics simulations, bioinformatics analysis, and experimental mutagenesis that identifies residues involved in mediating the allosteric coupling of receptor, nucleotide, and helical domain interfaces of G␣ i . Most notably, we predict and characterize novel allosteric decoupling mutants, which display enhanced helical domain opening, increased rates of nucleotide exchange, and constitutive activity in the absence of receptor activation. Collectively, our results provide a framework for explaining how binding events and mutations can alter internal dynamic couplings critical for G protein function.Heterotrimeric G proteins are key mediators of intracellular signaling pathways that control diverse cellular processes ranging from movement and division to differentiation and neuronal activity (1). G proteins consist of three subunits: G␣, G␤, and G␥. Bound with GDP, G␣ forms an inactive complex with its G␤␥ subunit partners. Interaction with activated receptor (GPCR) 3 promotes the exchange of GDP for GTP on G␣ and its separation from G␤␥. Both isolated G␣ and G␤␥ can then bind and activate or inhibit downstream effectors. GTP hydrolysis deactivates G␣, which subsequently reassociates with G␤␥ completing the cycle. This cycle is further regulated by two classes of additional proteins called regulators of G protein signaling. These function as either GTPase-activating proteins (which promote GTP hydrolysis) or GDP dissociation inhibitors (GDIs, which hinder exchange of GDP for GTP) (2). Important conformational transitions occurring at each stage of this regulated cycle have been characterized from extensive crystallographic studies. These include GDP, GTP analogue, G␤␥, GTPase-activating protein, GDI and most recently GPCR bound complex structures of G␣. However, the link between the observed conformations and the atomic level mechanisms involved in coupling receptor association, G protein activation, and effector interaction remain unclear.All G␣ proteins consist of a catalytic GTP binding Ras-like domain (termed RasD) and a heterotrimeric G protein specific helical domain (HD). Recent principal component analysis (PCA) of 53 available G␣ crystallographic structures identified three major conformationally distinct groups ( Fig. 1 and Ref. 3). These groups correspond to structures with bound GTP analogues, GDP, and GDI (red, green, and blue points in Fig. 1a, respectively). The major variation in the accumulated structures is the concerted displacements of three nucleotide-binding site loops termed the switch regions (SI, SII, and SIII), as well as a relatively small...

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Section: And Ref 3)supporting

confidence: 68%

Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins

Yao¹,

Malik

Griggs

et al. 2016

Journal of Biological Chemistry

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“…The presented model could not be deduced from a number of Gα i1 crystal structures that were solved with sulfur-or nitrogen-substituted, nonhydrolyzable GTP analogs. Interestingly the position that is occupied by Arg178 in our simulations of intrinsic Gα i1 is identical to the position of the O→S substitution at the γ-phosphate in various structures (2,(45)(46)(47)(48). Sulfur substitution causes altered biochemical and biophysical properties of the γ-GTP group (e.g., an increased van der Waals radius), probably hindering the Arg178 binding.…”

Section: Discussionmentioning

confidence: 73%

Mechanism of the intrinsic arginine finger in heterotrimeric G proteins

Mann

Teuber

Tennigkeit

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Heterotrimeric G proteins are crucial molecular switches that maintain a large number of physiological processes in cells. The signal is encoded into surface alterations of the Gα subunit that carries GTP in its active state and GDP in its inactive state. The ability of the Gα subunit to hydrolyze GTP is essential for signal termination. Regulator of G protein signaling (RGS) proteins accelerates this process. A key player in this catalyzed reaction is an arginine residue, Arg178 in Gα i1 , which is already an intrinsic part of the catalytic center in Gα in contrast to small GTPases, at which the corresponding GTPase-activating protein (GAP) provides the arginine "finger." We applied time-resolved FTIR spectroscopy in combination with isotopic labeling and site-directed mutagenesis to reveal the molecular mechanism, especially of the role of Arg178 in the intrinsic Gα i1 mechanism and the RGS4-catalyzed mechanism. Complementary biomolecular simulations (molecular mechanics with molecular dynamics and coupled quantum mechanics/molecular mechanics) were performed. Our findings show that Arg178 is bound to γ-GTP for the intrinsic Gα i1 mechanism and pushed toward a bidentate α-γ-GTP coordination for the Gα i1 ·RGS4 mechanism. This movement induces a charge shift toward β-GTP, increases the planarity of γ-GTP, and thereby catalyzes the hydrolysis.GTPase | FTIR spectroscopy | QM/MM calculations | arginine finger | reaction mechanism

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“…The yeast Gpa2 homology model was built using Swiss-Model (http://swissmodel.expasy.org) 4 by threading the Gpa2 primary sequence, obtained from the Saccharomyces Genome Database (http://www.yeastgenome. org), 4 onto the template structure corresponding to chain A of the PDB identifier 4N0D (Rattus norvegicus G␣ i1 subunit) (104).…”

Section: Structural Informaticsmentioning

confidence: 99%

Coordinated regulation of intracellular pH by two glucose-sensing pathways in yeast

Isom

Page

Collins

et al. 2018

Journal of Biological Chemistry

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The yeast employs multiple pathways to coordinate sugar availability and metabolism. Glucose and other sugars are detected by a G protein-coupled receptor, Gpr1, as well as a pair of transporter-like proteins, Rgt2 and Snf3. When glucose is limiting, however, an ATP-driven proton pump (Pma1) is inactivated, leading to a marked decrease in cytoplasmic pH. Here we determine the relative contribution of the two sugar-sensing pathways to pH regulation. Whereas cytoplasmic pH is strongly dependent on glucose abundance and is regulated by both glucose-sensing pathways, ATP is largely unaffected and therefore cannot account for the changes in Pma1 activity. These data suggest that the pH is a second messenger of the glucose-sensing pathways. We show further that different sugars differ in their ability to control cellular acidification, in the manner of inverse agonists. We conclude that the sugar-sensing pathways act via Pma1 to invoke coordinated changes in cellular pH and metabolism. More broadly, our findings support the emerging view that cellular systems have evolved the use of pH signals as a means of adapting to environmental stresses such as those caused by hypoxia, ischemia, and diabetes.

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A Transient Interaction between the Phosphate Binding Loop and Switch I Contributes to the Allosteric Network between Receptor and Nucleotide in Gαi1

Cited by 7 publications

References 79 publications

Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins

Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins

Mechanism of the intrinsic arginine finger in heterotrimeric G proteins

Coordinated regulation of intracellular pH by two glucose-sensing pathways in yeast

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