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

doi:10.1006/jmbi.1994.1320

Journal of Molecular Biology

Volume 238, Issue 4, 12 May 1994, Pages 630-634

https://doi.org/10.1006/jmbi.1994.1320 Get rights and content

Abstract

Several different crystal forms of Giα1 have been grown and analyzed. Crystals of native protein containing bound GTPγS belong to space group P 3₁21 or P3₂21 with cell dimensions a,b = 80·6 Å and c = 106·3 Å and diffract to a resolution of 1·9 Å using synchroton radiation. Crystals of native protein containing bound GDP belong to space group I4 with cell dimensions a,b = 121·3 Å, and c = 67?7 Å and diffract to 3·0 Å. Data sets from crystals grown using mutant proteins have also been obtained and characterized.

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2017, Journal of Molecular Graphics and Modelling
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Such effect is supposed to result of a Gαq with impaired autohydrolysis (hydrolysis of α subunit bound GTP to GDP) and impaired inactivation [6]. In fact, substitution of cysteine at the R183 position was shown to result in reduced hydrolysis of GTP to GDP, a key step required for G-protein inactivation [24,25]. On the other hand, the inactivation of G protein-activated signaling pathways was shown to be dependent on re-association of heterotrimeric G protein subunits and their binding to the receptor, which occurs as a result of intrinsic GTPase activity of Gα subunit [15,26,27].
Somatic activating mutations in the GNAQ have been recently associated with several congenital genetic disorders and tumors; however, the molecular mechanism/etiology that leads to GNAQ somatic mosaic mutation are unknown. Here, we reported a case of Sturge-Weber Syndrome (SWS) manifesting cutaneous vascular malformations (hemifacial Port-wine stain), cerebral and ocular vascular abnormalities (including epilepsy and glaucoma) and harboring a c.548G > A (p.R183Q) somatic mosaic mutation in GNAQ. Computational modeling studies were performed to assistant with the comprehension of the functional impact of p.R183Q and p.Q209L mutations in GNAQ, which encodes a G protein subunit alpha q (Gαq). The p.R183Q mutation was predicted to abolish hydrogen bonds between R183 residue and GDP molecule, destabilizing the inactive GDP-bound conformation of the Gαq mutants. Furthermore, replacement of R183 by Q183 residue was predicted to promote conformation changes in protein surface features affecting the switch I region, a key region that undergoes conformational changes triggered by receptor binding during signal transduction. In addition, replacement of Q209 by L209 residue was predicted to affect the molecular interaction between Gαq and Gβ subunit, impairing formation of the inactive heterotrimeric complex. These findings, in association with PPI network analysis, indicate that p.R183Q and p.Q209L mutations result in the over-activation of different downstream effectors, which in turn will determine the distinct cell responses and phenotype. These findings bring new insights on molecular etiology of vascular malformations associated to SWS and on different mechanisms underlying hyperactivation of downstream pathways to Gαq.
Structural Evidence for a Sequential Release Mechanism for Activation of Heterotrimeric G Proteins
2009, Journal of Molecular Biology
Heptahelical G-protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors couple to heterotrimeric G proteins to relay extracellular signals to intracellular signaling networks, but the molecular mechanism underlying guanosine 5′-diphosphate (GDP) release by the G protein α-subunit is not well understood. Amino acid substitutions in the conserved α5 helix of G_i, which extends from the C-terminal region to the nucleotide-binding pocket, cause dramatic increases in basal (receptor-independent) GDP release rates. For example, mutant Gα_i1-T329A shows an 18-fold increase in basal GDP release rate and, when expressed in culture, it causes a significant decrease in forskolin-stimulated cAMP accumulation. The crystal structure of Gα_i1-T329A·GDP shows substantial conformational rearrangement of the switch I region and additional striking alterations of side chains lining the catalytic pocket that disrupt the Mg⁺² coordination sphere and dislodge bound Mg⁺². We propose a “sequential release” mechanism whereby a transient conformational change in the α5 helix alters switch I to induce GDP release. Interestingly, this mechanistic model for heterotrimeric G protein activation is similar to that suggested for the activation of the plant small G protein Rop4 by RopGEF8.
Evidence for two distinct Mg<sup>2+</sup> binding sites in G<inf>sα</inf> and G<inf>iα1</inf> proteins
2008, Biochemical and Biophysical Research Communications
Citation Excerpt :
Fig. 3 shows that the high affinity Mg2+ binding site is primarily populated as the α subunit is first titrated with Mg2+ while population of the low affinity site requires higher concentrations of Mg2+ relative to the α subunit concentration. The low affinity Mg2+ binding site has not been observed in Gsα (Fig. 4A) and Giα1 X-ray structures [3–8]; however, we can speculate that the low affinity site is located near the guanine nucleotide binding pocket based on the X-ray structure of the monomeric G protein ral that shows two Mg2+ bound to GppNHp (a non-hydrolyzable GTP analog) [12] (Fig. 4B). This is a reasonable assumption since the guanine nucleotide binding P loop motif is highly conserved among G proteins [1].
The function of guanine nucleotide binding (G) proteins is Mg²⁺ dependent with guanine nucleotide exchange requiring higher metal ion concentration than guanosine 5′-triphosphate hydrolysis. It is unclear whether two Mg²⁺ binding sites are present or if one Mg²⁺ binding site exhibits different affinities for the inactive GDP-bound or the active GTP-bound conformations. We used furaptra, a Mg²⁺-specific fluorophore, to investigate Mg²⁺ binding to α subunits in both conformations of the stimulatory (G_sα) and inhibitory (G_iα1) regulators of adenylyl cyclase. Regardless of the conformation or α protein studied, we found that two distinct Mg²⁺ sites were present with dissimilar affinities. With the exception of G_sα in the active conformation, cooperativity between the two Mg²⁺ sites was also observed. Whereas the high affinity Mg²⁺ site corresponds to that observed in published X-ray structures of G proteins, the low affinity Mg²⁺ site may involve coordination to the terminal phosphate of the nucleotide.
Competition between lithium and magnesium ions for the G-protein transducin in the guanosine 5<sup>′</sup>-diphosphate bound conformation
2004, Journal of Inorganic Biochemistry
Citation Excerpt :
By inhibiting the release of Gt from SROS membranes, Li+ may be able to modulate the activity of Gt. Considering the homology of the high-affinity Mg2+ binding site among all G-proteins [16–21], and the present study, it is unlikely that Li+/Mg2+ competition can occur at this site. By contrast, sequence and structural analyses of the low-affinity Mg2+ binding site are unavailable.
Li⁺ 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⁺ competes with Mg²⁺ for the Mg²⁺ binding sites on guanine-nucleotide binding proteins (G-proteins). Using ⁷Li T₁ relaxation measurements and fluorescence spectroscopy with the Mg²⁺ fluorophore furaptra, we detected Li⁺/Mg²⁺ 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⁺ in the presence of fixed amounts of Mg²⁺, the apparent Li⁺ binding constant decreased due to Li⁺/Mg²⁺ competition. Whereas for SROS the competition mechanism was monophasic, for G_t, the competition was biphasic, suggesting that in G_t, Li⁺/Mg²⁺ competition occurred with different affinities for Mg²⁺ in two types of Mg²⁺ binding sites. Moreover, as [Li⁺] increased, the fluorescence excitation spectra of both ROS-T and G_t were blue shifted, indicating an increase in free [Mg²⁺] compatible with Li⁺ displacement of Mg²⁺ from two low affinity Mg²⁺ binding sites of G_t. G_t release from ROS-T membrane was also inhibited by Li⁺ addition. In summary, we found evidence of Li⁺/Mg²⁺ competition in G_t-containing preparations.
Clinical implications of genetic defects in G proteins: Oncogenic mutations in Gα(s) as the molecular basis for the McCune-Albright syndrome
1999, Archives of Medical Research
Signal-transducing guanine nucleotide-binding proteins (G proteins) couple extracellular receptor proteins to intracellular effector enzymes and ion channels, and therefore are critical mediators of cellular responses to external stimuli. G proteins are comprised of three subunits (α, β, γ), each encoded by many different genes. The multiplicity of G protein subunits facilitates great combinatorial variability, which, in part, accounts for the ability of G proteins to interact with many different receptor and effector proteins. Hundreds of G protein-coupled receptors have been identified, and their unique patterns of expression among a restricted number of cell types contributes greatly to the apparent specificity of hormone action. Mutations that either activate or inactivate some of these receptors account for a number of highly specific syndromes, which affect a limited number of target tissues. By contrast, most G proteins are widely expressed in many tissues. Accordingly, mutations in these signaling molecules would be expected to produce a more generalized pattern of hormone dysfunction. Activating mutations in the gene (GNAS1) that encode the α subunit of the G protein that stimulates adenylyl cyclase (AC) have been identified in many endocrine neoplasms and diverse tissues of patients with McCune-Albright syndrome. The McCune-Albright syndrome is characterized by autonomous endocrine function, hyperpigmented skin lesions, and fibrous dysplasia of bone—effects which reflect the ability of CAMP to stimulate cell function and proliferation in a wide variety of tissues. The unusual features of the McCune-Albright syndrome are explained by the mosaic distribution of cells bearing the mutant allele, an observation that is most consistent with postzygotic mutation of GNAS1. Experimental analysis of this syndrome has extended our understanding of the clinical and biochemical consequences of dysfunctional G protein action and has provided a bench-to-bedside demonstration of the critical role that G proteins play in transmembrane signal transduction in humans.
Structure of G(iα1)·GppNHp, autoinhibition in a Gα protein-substrate complex
1999, Journal of Biological Chemistry
The structure of the G protein G_iα1 complexed with the nonhydrolyzable GTP analog guanosine-5′-(βγ-imino)triphosphate (GppNHp) has been determined at a resolution of 1.5 Å. In the active site of G_iα1·GppNHp, a water molecule is hydrogen bonded to the side chain of Glu⁴³ and to an oxygen atom of the γ-phosphate group. The side chain of the essential catalytic residue Gln²⁰⁴ assumes a conformation which is distinctly different from that observed in complexes with either guanosine 5′-O-3-thiotriphosphate or the transition state analog GDP·AlF₄⁻. Hydrogen bonding and steric interactions position Gln²⁰⁴ such that it interacts with a presumptive nucleophilic water molecule, but cannot interact with the pentacoordinate transition state. Gln²⁰⁴ must be released from this auto-inhibited state to participate in catalysis. RGS proteins may accelerate the rate of GTP hydrolysis by G protein α subunits, in part, by inserting an amino acid side chain into the site occupied by Gln²⁰⁴, thereby destabilizing the auto-inhibited state of Gα.

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Journal of Molecular Biology

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

Abstract

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