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

128

187

The guanine nucleotide-binding protein Ras exists in solution in two different conformational states when complexed with different GTP analogs such as GppNHp or GppCH 2 p. State 1 has only a very low affinity to effectors and seems to be recognized by guanine nucleotide exchange factors, whereas state 2 represents the high affinity effector binding state. In this work we investigate Ras in complex with the physiological nucleoside triphosphate GTP. By polarization transfer 31 P NMR experiments and effector binding studies we show that Ras(wt)⅐Mg 2؉ ⅐GTP also exists in a dynamical equilibrium between the weakly populated conformational state 1 and the dominant state 2. At 278 K the equilibrium constant between state 1 and state 2 of C-terminal truncated wild-type Ras(1-166) K 12 is 11.3. K 12 of full-length Ras is >20, suggesting that the C terminus may also have a regulatory effect on the conformational equilibrium. The exchange rate (k ex ) for Ras(wt)⅐Mg 2؉ ⅐GTP is 7 s ؊1 and thus 18-fold lower compared with that found for the Ras⅐GppNHp complex. The intrinsic GTPase activity substantially increases after effector binding for the switch I mutants Ras(Y32F), (Y32R), (Y32W), (Y32C/ C118S), (T35S), and the switch II mutant Ras(G60A) by stabilizing state 2, with the largest effect on Ras(Y32R) with a 13-fold increase compared with wild-type. In contrast, no acceleration was observed in Ras(T35A). Thus Ras in conformational state 2 has a higher affinity to effectors as well as a higher GTPase activity. These observations can be used to explain why many mutants have a low GTPase activity but are not oncogenic.The guanine nucleotide-binding protein Ras is involved in cellular signal transduction pathways inducing proliferation, differentiation, or apoptosis of cells. It functions as a molecular switch, cycling between an inactive GDP-bound state and an active GTP-bound state. In the active conformation Ras is able to bind different effector proteins such as Raf kinase or RalGDS with nanomolar affinity by interacting with its switch I region. Thus signals can be transmitted resulting in the corresponding cellular response.When bound to guanosine triphosphates Ras exists in two different conformational states, defined as states 1 and 2, which are in chemical equilibrium in solution (1-3). These conformational states are actually only defined for Ras with guanosine triphosphate bound (T), a state that may be different from a state with guanine diphosphate bound (D). Therefore, we will denote the two states in the following as state 1(T) and 2(T) whenever the nucleotide ligand is of concern. The equilibrium between the two states is strongly influenced by the nature of the guanine nucleotide bound to Ras and can be shifted by interaction with effector proteins or regulators such as GTPase activation proteins (GAPs).2 It was found that GTP analogs GppNHp and GppCH 2 p partially shift the equilibrium toward state 1. For the complex of wild-type Ras with physiological GTP itself the existence of the two states could not be...

supporting

confidence: 67%

Conformational States of Human Rat Sarcoma (Ras) Protein Complexed with Its Natural Ligand GTP and Their Role for Effector Interaction and GTP Hydrolysis

Spoerner

Hozsa

Poetzl

et al. 2010

128

187

“…This structure has been postulated to resemble the transition state for GTP hydrolysis. When AlF 4 Ϫ binds to the GDP-bound form of ␣ T, there is an increase in the intrinsic tryptophan fluorescence due to a conformational change in the Switch 2 region, one of three regions of ␣ T that change conformation upon binding GTP (29). As expected, a robust increase in tryptophan fluorescence was observed upon addition of AlF 4 Ϫ to the wild-type ␣ T * subunit (Fig.…”

Section: The Purified Recombinant ␣ T *(S43n) Mutant Is Isolated Withsupporting

confidence: 50%

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

Ramachandran

Cerione

2011

Self Cite

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...

“…alanine, does not have a large effect on the hydrolysis rate (e.g. Cdc42 (42,43)), whereas in GTPases with a slow hydrolysis the tyrosine is close to the ␥-phosphate (e.g. Rab6a, which is approximately 4 times slower than Ran⅐GTP and 8 times slower than Ran⅐GTP⅐RanBD1) (44).…”

Section: Volume 290 • Number 40 • October 2 2015mentioning

confidence: 99%

Catalysis of GTP Hydrolysis by Small GTPases at Atomic Detail by Integration of X-ray Crystallography, Experimental, and Theoretical IR Spectroscopy

Rudack

Jenrich

Brucker

et al. 2015

Background:The magnesium position in the active site of GTPases varies among x-ray structures of Ran and related GTPases. Results: The magnesium in both Ras and Ran is always coordinated to the ␤-and ␥-phosphate. Conclusion: Other factors like steric hindrance by the Tyr-39 side chain influence the hydrolysis rate in Ran. Significance: The new information allows an improved view on the catalytic mechanism of GTPases.