Conformational States of Ras Complexed with the GTP Analogue GppNHp or GppCH<sub>2</sub>p:  Implications for the Interaction with Effector Proteins

Spoerner, Michael; Nuehs, Andrea; Ganser, Petra; Herrmann, Christian; Wittinghofer, A.; Kalbitzer, Hans Robert

doi:10.1021/bi0488000

Cited by 87 publications

(175 citation statements)

References 37 publications

Supporting

Mentioning

149

Contrasting

Unclassified

Order By: Relevance

“…The observation that A117V shows a doublet on SDS-PAGE gel may suggest a conformational change. This is consistent with the effect of the known nucleotide-dependent changes in conformations of G-proteins (Vetter and Wittenghofer 2001;Spoerner et al 2005). A117 is conserved in E. coli and T. aquaticus EF-Tu and in human eEF1A, whereas R164 is conserved only in yeast and in human eEF1A.…”

Section: Discussionsupporting

confidence: 85%

Unique Classes of Mutations in the Saccharomyces cerevisiae G-Protein Translation Elongation Factor 1A Suppress the Requirement for Guanine Nucleotide Exchange

et al. 2006

View full text Add to dashboard Cite

G-proteins play critical roles in many cellular processes and are regulated by accessory proteins that modulate the nucleotide-bound state. Such proteins, including eukaryotic translation elongation factor 1A (eEF1A), are frequently reactivated by guanine nucleotide exchange factors (GEFs). In the yeast Saccharomyces cerevisiae, only the catalytic subunit of the GEF complex, eEF1Ba, is essential for viability. The requirement for the TEF5 gene encoding eEF1Ba can be suppressed by the presence of excess substrate, eEF1A. These cells, however, have defects in growth and translation. Two independent unbiased screens performed to dissect the cause of these phenotypes yielded dominant suppressors that bypass the requirement for extra eEF1A. Surprisingly, all mutations are in the G-protein eEF1A and cluster in its GTP-binding domain. Five mutants were used to construct novel strains expressing only the eEF1A mutant at normal levels. These strains show no growth defects and little to no decreases in total translation, which raises questions as to the evolutionary expression of GEF complexity and other potential functions of this complex. The location of the mutations on the eEF1A-eEF1Ba structure suggests that their mechanism of suppression may depend on effects on the conserved G-protein elements: the P-loop and NKXD nucleotide-binding element.

show abstract

Section: Discussionsupporting

confidence: 85%

Unique Classes of Mutations in the Saccharomyces cerevisiae G-Protein Translation Elongation Factor 1A Suppress the Requirement for Guanine Nucleotide Exchange

et al. 2006

View full text Add to dashboard Cite

show abstract

“…A85K Mutant of Raf Induces the High Affinity Conformation of Ras Switch I-It has been shown earlier that Ras⅐GppNHp exists in two conformational states, with state 1 a low affinity state for effectors and state 2 a high affinity effector binding state (47)(48)(49). Ras mutations of Thr 35 lead to a shift of the switch I conformational equilibrium to state 1, resulting in lower affinity for Raf or other effectors, as was shown for different Ras⅐nucleotide complexes (48,49,55,56).…”

Section: Discussionmentioning

confidence: 75%

“…Ras mutations of Thr 35 lead to a shift of the switch I conformational equilibrium to state 1, resulting in lower affinity for Raf or other effectors, as was shown for different Ras⅐nucleotide complexes (48,49,55,56). It is assumed that only in state 2 does switch I occur in the correct conformation for effector binding.…”

Section: Discussionmentioning

confidence: 95%

“…This indicates that for the Raf mutants, the improvement in binding is more pronounced for Ras⅐GDP interaction than for Ras⅐GppNHp (Fig. 3B) hydrogen bonds with the ␥-phosphate of the nucleotide, and this conformation (state 2) is shown to be stabilized by effector binding (47)(48)(49). After hydrolysis and loss of the ␥-phosphate, Ras⅐GDP relaxes to other conformations of switch I, leading to much lower effector binding affinity.…”

Section: Computational Design Of Raf-rbd Mutants With Highermentioning

confidence: 96%

See 1 more Smart Citation

Improved Binding of Raf to Ras·GDP Is Correlated with Biological Activity

Kiel

Filchtinski

Spoerner

et al. 2009

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The GTP-binding protein Ras plays a central role in the regulation of various cellular processes, acting as a molecular switch that triggers signaling cascades. Only Ras bound to GTP is able to interact strongly with effector proteins like Raf kinase, phosphatidylinositol 3-kinase, and RalGDS, whereas in the GDP-bound state, the stability of the complex is strongly decreased, and signaling is interrupted. To determine whether this process is only controlled by the stability of the complex, we used computer-aided protein design to improve the interaction between Ras and effector. We challenged the Ras⅐Raf complex in this study because Raf among all effectors shows the highest Ras affinity and the fastest association kinetics. The proposed mutations were characterized as to their changes in dynamics and binding strength. We demonstrate that Ras-Raf interaction can only be improved at the cost of a loss in specificity of Ras⅐GTP versus Ras⅐GDP. As shown by NMR spectroscopy, the Raf mutation A85K leads to a shift of Ras switch I in the GTP-bound as well as in the GDP-bound state, thereby increasing the complex stability. In a luciferase-based reporter gene assay, Raf A85K is associated with higher signaling activity, which appears to be a mere matter of Ras-Raf affinity.Signal transduction across the cell is mediated by a network of interacting proteins leading to a controlled level of cellular response. Within the superfamily of small GTP-binding proteins, Ras appears to be a "master regulator" involved in cell proliferation, cell cycle progression, cell division, and apoptosis (1, 2). Attached to the inner leaflet of the cell membrane, Ras comes in two states; the inactive GDP-bound and the active GTP-bound state. The activation of Ras occurs by GDP/GTP nucleotide exchange, whereas hydrolysis of GTP leads to inactivation and interruption of signaling (3, 4). The activation of a distinct pathway occurs by the interaction of Ras with the responsible effector. Therefore, Ras has a large number of effector proteins, such as Raf kinase (5, 6), RalGDS (7), phosphatidylinositol 3-kinase (8 -10), and Nore1A (11, 12), representing different signal directions. These effectors have in common the so-called Ras binding domain (RBD), 2 enabling them to interact with Ras. Only the GTP-bound form of Ras binds strongly to effectors and leads to their activation.Zooming more into the molecular detail of the Ras/effector interactions reveals two regions of Ras to be responsive to the nucleotide state and thereby convey specific recognition by the effectors. The flexible region switch I (residues 30 -38) is mainly responsible for the interaction with the RBD of the effectors (13-15), whereas only a few effectors like Nore1A contact also the second flexible region of Ras, switch II (residues 60 -67) (16). The RBDs from most effectors comprise 80 -100 amino acids, and despite poor sequence homology, they all show the same topology (i.e. the ubiquitin fold) (17). In addition, the structures of various effector RBDs in complex with Ras s...

show abstract

“…2, D and E, respectively). It is interesting that in GMP-PCP-bound Cdc42, Thr-35 does not participate in Mg 2ϩ coordination, given that it has been suggested to be critical for the structural change imparted by GTP analogs within the Switch I loop of H-Ras (23,24). Specifically, Thr-35 appears to interact with the ␥-phosphate of GTP in Ras with this interaction being lost upon GTP hydrolysis and thereby possibly accounting for the observed change in the orientation of Switch I.…”

Section: The X-ray Crystal Structure For Gmp-pcp-bound Cdc42-mentioning

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

View full text Add to dashboard Cite

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...

show abstract

Conformational States of Ras Complexed with the GTP Analogue GppNHp or GppCH₂p: Implications for the Interaction with Effector Proteins

Cited by 87 publications

References 37 publications

Unique Classes of Mutations in the Saccharomyces cerevisiae G-Protein Translation Elongation Factor 1A Suppress the Requirement for Guanine Nucleotide Exchange

Unique Classes of Mutations in the Saccharomyces cerevisiae G-Protein Translation Elongation Factor 1A Suppress the Requirement for Guanine Nucleotide Exchange

Improved Binding of Raf to Ras·GDP Is Correlated with Biological Activity

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

Contact Info

Product

Resources

About

Conformational States of Ras Complexed with the GTP Analogue GppNHp or GppCH2p: Implications for the Interaction with Effector Proteins

Cited by 87 publications

References 37 publications

Unique Classes of Mutations in the Saccharomyces cerevisiae G-Protein Translation Elongation Factor 1A Suppress the Requirement for Guanine Nucleotide Exchange

Unique Classes of Mutations in the Saccharomyces cerevisiae G-Protein Translation Elongation Factor 1A Suppress the Requirement for Guanine Nucleotide Exchange

Improved Binding of Raf to Ras·GDP Is Correlated with Biological Activity

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

Contact Info

Product

Resources

About

Conformational States of Ras Complexed with the GTP Analogue GppNHp or GppCH₂p: Implications for the Interaction with Effector Proteins