Crystallization and Preliminary Crystallographic Studies of Giα1 and Mutants of Giα1 in the GTP and GDP-bound States

Coleman, David E.; Lee, Ethan; Mixon, Mark B.; Linder, Maurine E.; Berghuis, Albert M.; Gilman, Alfred G.; Sprang, Stephen R.

doi:10.1006/jmbi.1994.1320

Cited by 50 publications

(40 citation statements)

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“…Using these NMR methods based on 7 binding site among all G-proteins [16][17][18][19][20][21], and the present study, it is unlikely that Li þ /Mg 2þ competition can occur at this site. By contrast, sequence and structural analyses of the low-affinity Mg 2þ binding site are unavailable.…”

Section: Discussionmentioning

confidence: 95%

“…Additionally, the ROS membrane, in which G t is normally embedded, can also be quickly and easily isolated, allowing the construction of a reconstituted membrane system [15]. Considering the fact that the amino acids involved in high-affinity Mg 2þ coordination are essentially the same in most of the proteins in the G-protein superfamily [16][17][18][19][20][21], whereas those involved in low-affinity Mg 2þ binding are unknown, it is important to determine whether the metal ion competition results obtained in our study may be extended to other G-proteins.…”

Section: Introductionmentioning

confidence: 99%

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Competition between lithium and magnesium ions for the G-protein transducin in the guanosine 5 ′ -diphosphate bound conformation

Srinivasan

Toon

Amari

et al. 2004

Journal of Inorganic Biochemistry

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Liþ is the most effective drug used to treat bipolar disorder; however, its exact mechanism of action has yet to be elucidated. One hypothesis is that Li þ competes with Mg 2þ for the Mg 2þ binding sites on guanine-nucleotide binding proteins (G-proteins). Using 7 Li T 1 relaxation measurements and fluorescence spectroscopy with the Mg 2þ fluorophore furaptra, we detected Li þ /Mg 2þ competition in three preparations: the purified G-protein transducin (G t ), stripped rod outer segment membranes (SROS), and SROS with purified G t reattached (ROS-T). When purified ROS-T, SROS or transducin were titrated with Li þ in the presence of fixed amounts of Mg 2þ , the apparent Li þ binding constant decreased due to Li þ /Mg 2þ competition. Whereas for SROS the competition mechanism was monophasic, for G t , the competition was biphasic, suggesting that in G t , Li þ /Mg 2þ competition occurred with different affinities for Mg 2þ in two types of Mg 2þ binding sites. Moreover, as [Li þ ] increased, the fluorescence excitation spectra of both ROS-T and G t were blue shifted, indicating an increase in free [Mg 2þ ] compatible with Li þ displacement of Mg 2þ from two low affinity Mg 2þ binding sites of G t . G t release from ROS-T membrane was also inhibited by Li þ addition. In summary, we found evidence of Li þ /Mg 2þ competition in G t -containing preparations.

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Section: Discussionmentioning

confidence: 95%

Section: Introductionmentioning

confidence: 99%

Competition between lithium and magnesium ions for the G-protein transducin in the guanosine 5 ′ -diphosphate bound conformation

Srinivasan

Toon

Amari

et al. 2004

Journal of Inorganic Biochemistry

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“…Crystallographic (4)(5)(6)(7)(8)(9), biochemical (10), and biophysical (11-13) studies have elucidated details of the conformational states of the Gα subunit during the GTPase cycle. The Gα subunit has two structural domains, a nucleotide-binding domain (the Ras-like domain) and a helical domain (the α-H domain) that partially occludes the bound nucleotide (Fig.…”

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confidence: 99%

Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding

Goricanec

Stehle

Egloff

et al. 2016

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

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Heterotrimeric G proteins play a pivotal role in the signal-transduction pathways initiated by G-protein-coupled receptor (GPCR) activation. Agonist-receptor binding causes GDP-to-GTP exchange and dissociation of the Gα subunit from the heterotrimeric G protein, leading to downstream signaling. Here, we studied the internal mobility of a G-protein α subunit in its apo and nucleotide-bound forms and characterized their dynamical features at multiple time scales using solution NMR, small-angle X-ray scattering, and molecular dynamics simulations. We find that binding of GTP analogs leads to a rigid and closed arrangement of the Gα subdomain, whereas the apo and GDPbound forms are considerably more open and dynamic. Furthermore, we were able to detect two conformational states of the Gα Ras domain in slow exchange whose populations are regulated by binding to nucleotides and a GPCR. One of these conformational states, the open state, binds to the GPCR; the second conformation, the closed state, shows no interaction with the receptor. Binding to the GPCR stabilizes the open state. This study provides an in-depth analysis of the conformational landscape and the switching function of a G-protein α subunit and the influence of a GPCR in that landscape.H eterotrimeric G proteins are localized at the inner leaflet of the plasma membrane where they convey signals from cellsurface receptors to intracellular effectors (1). Heterotrimeric G proteins consist of two functional units, an α subunit (Gα) and a tightly associated βγ complex. The Gα subunit harbors the guanine nucleotide-binding site. In the inactive GDP-bound state, the Gα subunit is associated with the βγ complex. Exchange of GDP for GTP on the Gα subunit, triggered by interaction with the agonist-bound G-protein-coupled receptor (GPCR), results in a conformational change leading to GDP release and ultimately to GTP binding and subunit dissociation. The complexity of the mechanism by which a GPCR activates the Gα subunit based on available crystal structures has been discussed recently (2, 3). Both the Gα subunit and the βγ subunit interact with downstream effectors and regulate their activity. The intrinsic GTP hydrolysis of the Gα subunit returns the protein to the GDP-bound state, thereby increasing its affinity for the Gβγ subunit, and the subunits reassociate (Fig. 1A), ready for interaction with the agonist-bound GPCR. Throughout this cycle, the Gα subunit is engaged in specific interactions with the GPCR and/or the βγ subunit that stabilize the flexible parts of the protein, e.g., its switch regions. Only the GTP-bound form is stable enough to mediate downstream signaling.Crystallographic (4-9), biochemical (10), and biophysical (11-13) studies have elucidated details of the conformational states of the Gα subunit during the GTPase cycle. The Gα subunit has two structural domains, a nucleotide-binding domain (the Ras-like domain) and a helical domain (the α-H domain) that partially occludes the bound nucleotide (Fig. 1A). Because of this steric considerati...

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“…The crystal structures of the active (GTP␥S 1 and GDP AlF 4 Ϫ -bound) (8 -10) and inactive (GDP-bound) (11,12) forms of the ␣ subunits of transducin (G t ) and G i1 have been reported. Analysis of the two crystal forms has established the nature of the conformational change induced by the exchange of GTP for GDP and the switch mechanism by which the presence or absence of the ␥-phosphate defines the active or inactive state of the ␣ t subunit (9,11).…”

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confidence: 99%

Interaction of Transducin with Light-activated Rhodopsin Protects It from Proteolytic Digestion by Trypsin

Mazzoni

Hamm

1996

Journal of Biological Chemistry

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The tryptic cleavage pattern of transducin (G t ) in solution was compared with that in the presence of phospholipid vesicles, rod outer segment (ROS) membranes kept in the dark, or ROS membranes containing lightactivated rhodopsin, metarhodopsin II (Rh*). When G t was in the high affinity complex with Rh*, the ␣ t subunit was almost completely protected from proteolysis. , only a few angstroms away from the carboxyl terminus of ␣ t , which is known to directly bind to Rh*, is likely to also be a part of the Rh* binding site. This is in agreement with other studies and has implications for the mechanism by which receptors catalyze GDP release from G proteins. The protection of Lys 18 in the presence of phospholipid vesicles suggests that the amino-terminal region is in contact with the membrane, consistent with the crystal structure of the heterotrimer (Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319).Certain extracellular signals, including hormones, neurotransmitters, neuromodulators, chemokines, odorants, and light, activate a class of receptors that initiate cellular effects via activation of heterotrimeric G proteins. Agonist binding to the G protein-coupled receptors leads to conformational changes that promote a tighter interaction with specific G proteins, catalysis of GDP release, and subsequent G protein activation. In the absence of guanine nucleotides, agonist binding to the receptor is stabilized by the bound G protein. The structural basis of the ternary complex among agonist, receptor, and heterotrimeric G protein is an active area of study. Extensive mutagenesis experiments, as well as peptide competition investigations for a variety of G protein-coupled receptors, have led to an understanding that the second and third cytoplasmic loops and, in some circumstances, the putative fourth loop, as well as portions of ␣ helices VI and VII, are important in recognition of cognate G proteins (1-3). It has been shown that the heterotrimeric G protein, rather than just the ␣ or ␤␥ subunits, is required for the interaction, but studies pointing out the importance of specific regions are thus far limited to the ␣ subunits (4 -7).The crystal structures of the active (GTP␥S 1 and GDP AlF 4 Ϫ -bound) (8 -10) and inactive (GDP-bound) (11, 12) forms of the ␣ subunits of transducin (G t ) and G i1 have been reported. Analysis of the two crystal forms has established the nature of the conformational change induced by the exchange of GTP for GDP and the switch mechanism by which the presence or absence of the ␥-phosphate defines the active or inactive state of the ␣ t subunit (9, 11). The high resolution crystal structures of the heterotrimeric G proteins, G t and G i1 , provide a fundamental context for understanding how a heterotrimer interacts with the membrane and with activated receptors (13,14). The molecular mechanisms involved in the conformational changes of the ␣ subunit and the nucleotide exchange induced by the heterotrimeric G protein inter...

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Crystallization and Preliminary Crystallographic Studies of Giα1 and Mutants of Giα1 in the GTP and GDP-bound States

Cited by 50 publications

References 0 publications

Competition between lithium and magnesium ions for the G-protein transducin in the guanosine 5 ′ -diphosphate bound conformation

Competition between lithium and magnesium ions for the G-protein transducin in the guanosine 5 ′ -diphosphate bound conformation

Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding

Interaction of Transducin with Light-activated Rhodopsin Protects It from Proteolytic Digestion by Trypsin

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