New Insights into the Role of Conserved, Essential Residues in the GTP Binding/GTP Hydrolytic Cycle of Large G Proteins

Majumdar, Sharmistha; Ramachandran, Sekar; Cerione, Richard A.

doi:10.1074/jbc.m513837200

Cited by 26 publications

(27 citation statements)

References 34 publications

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“…The buffer was exchanged on a PD-10 column (Amersham Biosciences) equilibrated with HMA. Nucleotide content was confirmed by high pressure liquid chromatography analysis, using a previously published protocol (18).…”

Section: Methodsmentioning

confidence: 99%

Effector Proteins Exert an Important Influence on the Signaling-active State of the Small GTPase Cdc42

Phillips

Calero

Chan

et al. 2008

Journal of Biological Chemistry

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GTP-binding (G) proteins regulate the flow of information in cellular signaling pathways by alternating between a GTPbound "active" state and a GDP-bound "inactive" state. Cdc42, a member of the Rho family of Ras-related small G-proteins, plays key roles in the regulation of cell shape, motility, and growth. Here we describe the high resolution x-ray crystal structure for Cdc42 bound to the GTP analog guanylyl ␤,␥-methylenediphosphonate (GMP-PCP) (i.e. the presumed signaling-active state) and show that it is virtually identical to the structures for the signaling-inactive, GDP-bound form of the protein, contrary to what has been reported for Ras and other G-proteins. Especially surprising was that the GMP-PCP-and GDP-bound forms of Cdc42 did not show detectable differences in their Switch I and Switch II loops. Fluorescence studies using a Cdc42 mutant in which a tryptophan residue was introduced at position 32 of Switch I also showed that there was little difference in the Switch I conformation between the GDP-and GMP-PCPbound states (i.e. <10%), which again differed from Ras where much larger changes in Trp-32 fluorescence were observed when comparing these two nucleotide-bound states (>30%). However, the binding of an effector protein induced significant changes in the Trp-32 emission specifically from GMP-PCPbound Cdc42, as well as in the phosphate resonances for GTP bound to this G-protein as indicated in NMR studies. An examination of the available structures for Cdc42 complexed to different effector proteins, versus the x-ray crystal structure for GMP-PCP-bound Cdc42, provides a possible explanation for how effectors can distinguish between the GTP-and GDPbound forms of this G-protein and ensure that the necessary conformational changes for signal propagation occur.Cdc42 is a member of the Rho family of Ras-related small G-proteins and is an essential protein found in all eukaryotic organisms, including yeast, flies, and mammals (1-3). Like Ras, the founding member of the small G-protein family (4), Cdc42 undergoes a GTP-binding/GTP-hydrolytic cycle that enables it to act as a molecular switch in cells. It is activated to undergo GDP-GTP exchange by members of the Dbl family of guanine nucleotide exchange factors (5-7). GTP-bound Cdc42 can bind and/or activate over 20 downstream effector proteins that are responsible for mediating a diversity of cellular functions, including actin cytoskeletal remodeling, cell polarity, intracellular trafficking, epidermal growth factor receptor degradation, and cell cycle progression (1)(2)(3)8). These different signals are terminated when Cdc42 is deactivated through its ability to hydrolyze GTP, a reaction that is catalyzed by GTPase-activating proteins (GAPs) 2 (9). Structural studies of a number of GTP-binding proteins, beginning with the bacterial elongation factor Ef-Tu, and including H-Ras and the ␣ subunits of various members of the family of large G-proteins, have shown that a conserved architecture exists for GTP binding and hydrolytic activity, comprising five...

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Section: Methodsmentioning

confidence: 99%

Effector Proteins Exert an Important Influence on the Signaling-active State of the Small GTPase Cdc42

Phillips

Calero

Chan

et al. 2008

Journal of Biological Chemistry

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“…It is well known that glutamine-205 of G␣ i2 is essential for GTPase activity. This residue is conserved throughout the GTPase superfamily and is exchanged frequently for leucine to obtain the corresponding constitutively active G proteins (25,26). To demonstrate that deamidation of glutamine-205 results in inhibition of GTPase activity of G␣ i2 , we changed this residue by site-directed mutagenesis to glutamic acid and studied the RGS-stimulated GTPase activity.…”

Section: Deamidation Of Recombinant G␣i2 After Coexpression With Pmt Inmentioning

confidence: 99%

Pasteurella multocida toxin activation of heterotrimeric G proteins by deamidation

Orth

Preuß

Fester

et al. 2009

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

103

123

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Pasteurella multocida toxin is a major virulence factor of Pasteurella multocida, which causes pasteurellosis in men and animals and atrophic rhinitis in rabbits and pigs. The Ϸ145 kDa protein toxin stimulates various signal transduction pathways by activating heterotrimeric G proteins of the G␣ q, G␣i, and G␣12/13 families by using an as yet unknown mechanism. Here, we show that Pasteurella multocida toxin deamidates glutamine-205 of G␣ i2 to glutamic acid. Therefore, the toxin inhibits the intrinsic GTPase activity of G␣ i and causes persistent activation of the G protein. A similar modification is also evident for G␣ q, but not for the closely related G␣ 11, which is not a substrate of Pasteurella multocida toxin. Our data identify the ␣-subunits of heterotrimeric G proteins as the direct molecular target of Pasteurella multocida toxin and indicate that the toxin does not act like a protease, which was suggested from its thiol protease-like catalytic triad, but instead causes constitutive activation of G proteins by deamidase activity.bacterial protein toxin ͉ Gi protein ͉ posttranslational modification ͉ transglutaminase

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“…In addition, residues 244 and 247 were changed back to the original amino acids in ␣ T , yielding what we refer to as ␣ T *. The recombinant ␣ T * subunits, both wild-type and S43N, were expressed in BL21 (DE3) supercompetent cells and purified as described (11,13). The proteins were further purified by ion exchange chromatography as outlined for the ␤1␥1 complex.…”

Section: Methodsmentioning

confidence: 99%

“…The final yield of chimeric ␣ T * was typically ϳ1-2 mg of pure protein/liter of bacterial culture. Nucleotide occupancy of the purified recombinant ␣ T * and ␣ T *(S43N) was determined using HPLC (11).…”

Section: Methodsmentioning

confidence: 99%

A Dominant-negative Gα Mutant That Traps a Stable Rhodopsin-Gα-GTP-βγ Complex

Ramachandran

Cerione

2011

Journal of Biological Chemistry

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Residues comprising the guanine nucleotide-binding sites of the ␣ subunits of heterotrimeric (large) G-proteins (G␣ subunits), as well as the Ras-related (small) G-proteins, are highly conserved. This is especially the case for the phosphate-binding loop (P-loop) where both G␣ subunits and Ras-related G-proteins have a conserved serine or threonine residue. Substitutions for this residue in Ras and related (small) G-proteins yield nucleotide-depleted, dominant-negative mutants. Here we have examined the consequences of changing the conserved serine residue in the P-loop to asparagine, within a chimeric G␣ subunit (designated ␣ T *) that is mainly comprised of the ␣ subunit of the retinal G-protein transducin and a limited region from the ␣ subunit of Gi1. The ␣ T *(S43N) mutant exhibits a significantly higher rate of intrinsic GDP-GTP exchange compared with wild-type ␣ T *, with light-activated rhodopsin (R*) causing only a moderate increase in the kinetics of nucleotide exchange on ␣ T *(S43N). The ␣ T *(S43N) mutant, when bound to either GDP or GTP, was able to significantly slow the rate of R*-catalyzed GDP-GTP exchange on wild-type ␣ T *. Thus, GTP-bound ␣ T *(S43N), as well as the GDP-bound mutant, is capable of forming a stable complex with R*. ␣ T *(S43N) activated the cGMP phosphodiesterase (PDE) with a dose-response similar to wild-type ␣ T *. Activation of the PDE by ␣ T *(S43N) was unaffected if either R* or ␤1␥1 alone was present, whereas it was inhibited when R* and the ␤1␥1 subunit were added together. Overall, our studies suggest that the S43N substitution on ␣ T * stabilizes an intermediate on the G-protein activation pathway consisting of an activated G-protein-coupled receptor, a GTPbound G␣ subunit, and the ␤1␥1 complex. G protein-coupled receptors (GPCR)2 are one of the largest families of membrane proteins and are involved in various physiological functions. In the past several years, significant advances have been made in the determination of structures at an atomic level of GPCRs (1, 2), their cognate G-proteins, and their downstream targets (3). In addition, structures have been solved for complexes of G-proteins with their downstream targets as well as their regulators (e.g. the regulators of G-protein signaling (RGS) proteins) (3). However, one of the central unresolved questions in this field involves the mechanism utilized by a GPCR to catalyze the release of GDP from its cognate G-protein (4, 5).The x-ray crystal structures of various G␣ subunits have shown that they are composed of two distinct domains: one that highly resembles the GTPase domain of the small G-protein Ras, and a second domain which is mainly ␣-helical in content and thus referred to as the helical domain. The guanine nucleotide is nestled between these two domains (4, 5). The binding of GTP to ␣ T induces structural changes within 3 regions of the GTPase domain, designated as Switches 1, 2, and 3.The vertebrate visual system in rod cells has provided an excellent model system for understanding how GPCRs activate he...

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New Insights into the Role of Conserved, Essential Residues in the GTP Binding/GTP Hydrolytic Cycle of Large G Proteins

Cited by 26 publications

References 34 publications

Effector Proteins Exert an Important Influence on the Signaling-active State of the Small GTPase Cdc42

Effector Proteins Exert an Important Influence on the Signaling-active State of the Small GTPase Cdc42

Pasteurella multocida toxin activation of heterotrimeric G proteins by deamidation

A Dominant-negative Gα Mutant That Traps a Stable Rhodopsin-Gα-GTP-βγ Complex

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