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Publications and source records attributed to A Wittinghofer.
The recent independent structure elucidations of two heterotrimeric G proteins represent a milestone in our understanding of the regulation of this important class of signal switch molecules. The results show how the introduction of GTP into the heterotrimer produces two signalling molecules: the G alpha-GTP and G beta, gamma subunits.
The interactions of H-Ras, R-Ras, and Rap1A with the Ras-binding domains (RBD) of the c-Raf kinase and of the Ral guanine nucleotide exchange factor (RGF) was studied biochemically in solution. From deletion cloning the RGF-RBD was defined as a 97-amino acid-long fragment from the C-terminal end of the human RGF, which is an independent folding domain with high stability. Interestingly, whereas H-Ras binds with high affinity (KD = 20 nM) to Raf-RBD and with low affinity (KD = 1 microM) to RGF-RBD, Rap1A shows the opposite behavior. The binding of both RBDs to R-Ras is weak and shows no specificity. The interaction between Rap1A and RGF-RBD shows similar characteristics to the Ras-Raf interaction because it is blocked by mutations in the effector region (D38A) and it inhibits the dissociation of guanine nucleotide, which is the basis for the quantitative measurements in this work. Furthermore, the binding of RGF-RBD inhibits the interaction between Rap1A and Rap-GAP. As long as the cellular localizations of the different proteins and their biological functions are not clarified, these biochemical data seem to indicate that Ral-guanine nucleotide exchange factors is an effector molecule of Rap1A rather than of H-Ras.
The D119N mutation of p21ras was prepared by site-directed mutagenesis. Its nucleotide binding properties were investigated using fluorescently labelled guanosine and xanthosine nucleotides. Its affinity for guanosine nucleotides is severely reduced, with a concomitant increase in the affinity for xanthosine nucleotides, which leads to an almost complete reversal of base specificity. The protein is a GTPase as well as a XTPase and the hydrolysis reaction can be efficiently stimulated by GAP. Dissociation of XDP from the mutant is stimulated by the guanine nucleotide exchange factor Cdc25Mm in a similar manner to that of GDP from wildtype. The interaction of the mutant with the effector domain of c-Raf kinase or Ral-GEF is normal. In microinjection experiments in PC12 and NIH3T3 cells the protein behaves as an oncogenic mutant due to its high dissociation rate for GDP. However, when the protein is loaded with XDP before microinjection the onset of the oncogenic signal can be efficiently retarded. Thus, the protein behaves initially as wildtype and later as an oncogenic protein.
We have previously shown that the oncogene product p21 Ras is essential for the survival and neurite outgrowth-promoting activity of nerve growth factor on cultured chick embryonic sensory, but not sympathetic neurons. In order to extend our observations to the human system and to non-neuronal cells, we introduced the oncogenic form of p21 Ras into the cytoplasm of three different types of cultured human embryonic neural crest derivatives (8th-11th gestational week): dorsal root ganglion neurons, sympathetic neurons, and adrenal chromaffin cells. These cells are dependent on nerve growth factor for survival and/or fibre outgrowth in vitro. In dorsal root ganglion neurons, p21 Ras promoted survival and fibre outgrowth which was quantitatively and qualitatively comparable to the nerve growth factor effect (84% vs. 95%, control 18%). Sympathetic neurons showed a similar effect, albeit with a higher background survival (91% vs. 93%, control 58%). On chromaffin cells, which respond to nerve growth factor with pronounced fibre outgrowth in culture, the effect of p21 Ras was again comparable to that of nerve growth factor (35% vs. 30%, control 5%). The survival and fibre outgrowth-promoting effects of p21 Ras on human embryonic dorsal root ganglion neurons, sympathetic neurons and chromaffin cells suggest an involvement of p21 Ras in the intracellular signal transduction of nerve growth factor in human neural crest-derived cell populations.
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More and more effectors for the Ras-related protein superfamily are being discovered and it is emerging that these GTP-binding proteins interact with more than one effector to generate more than one cellular signal. Atomic details for the interaction of Rap/Ras with one of the effectors, the protein kinase c-Raf-1, have recently become available by X-ray structure analysis. The implications for the specificity of the signal transduction pathway, and how the GTP-dependent switch mechanism modulates the interaction with effectors will be discussed here, using Ras as a paradigm.
Binding of Ras to c-Raf-1 is a pivotal step of many mitogenic signalling pathways. Based on the recent crystal structure of the complex of Rap1A with the Ras-binding domain of Raf, mutations were introduced in c-Raf-1 and their effects on Ras/Raf binding affinity in vitro and Ras/Raf regulated gene expression in vivo were analysed. Our data reveal an empirical semilogarithmic correlation between dissociation constants and Raf-induced gene activity. The functional epitope that primarily determines binding affinity consists of residues Gln 66, Lys 84 and Arg 89 in Raf. This quantitative structure-activity investigation may provide a general approach to correlate structure-guided biochemical analysis with biological function of protein-protein interactions.
Members of the Ras subfamily of small GTP-binding proteins have been shown to be promiscuous towards a variety of putative effector molecules such as the protein kinase c-Raf and the Ral-specific guanine nucleotide exchange factor (Ral-GEF). To address the question of specificity of interactions we have introduced the mutations E30D and K31E into Rap and show biochemically, by X-ray structure analysis and by transfection in vivo that the identical core effector region of Ras and Rap (residues 32-40) is responsible for molecular recognition, but that residues outside this region are responsible for the specificity of the interaction. The major determinant for the switch in specificity is the opposite charge of residue 31--Lys in Rap, Glu in Ras--which creates a favourable complementary interface for the Ras-Raf interaction.
Charged amino acid residues of human RCC1 were converted to alanine and mutants which were unable to complement tsBN2 cells (a temperature-sensitive rcc1- mutant of the hamster BHK21 cell line) were selected. These RCC1 mutants were analyzed for the ability to inhibit premature chromatin condensation by microinjection into tsBN2 cells, and their steady-state kinetic parameters for guanine nucleotide exchange reaction were measured. Examined RCC1 mutants were unstable in tsBN2 cells at the restrictive temperature, yet they significantly inhibited premature chromatin condensation. Mutants located on the N-terminus of the RCC1 repeat showed an increased K(m), while their kcat values were comparable to that of wild-type RCC1. In contrast, mutants containing the conserved histidine residues in the C-terminus of the RCC1 repeat showed a value of K(m) similar to that of wild-type RCC1, while the kcat values of these mutants were reduced, depending upon the RCC1 repeats on which the mutation was located. These steady-state kinetic parameters of mutants indicate that the N-terminus and the C-terminus of RCC1 repeats play different roles in guanine nucleotide exchange on Ran. The comparison of kcat among the histidine mutants suggests that those histidine residues which are conserved in the RCC1 repeats and also through evolution comprise the catalytic site for the guanine nucleotide exchange reaction.
Could the recent elucidation of the structure of the Rap-Raf complex have been the first glimpse of a universal arrangement between GTPase switches and kinase cascades, as a number of recent reports show that Ras is not unique in its ability to start a signalling 'chain reaction'?
The X-ray structures of the 1:1 complexes formed between p21H-ras (residues 1 to 166) and the nucleotides P3-1-(2-nitrophenyl)ethyl guanosine triphosphate ("caged GTP"; pure R- and S-diastereomers) and 3'-O-(N-methylanthraniloyl)-2'-deoxyguanosine 5'-(beta, gamma-imido)-triphosphate ("mant dG-ppNHp"), have been refined to an R-factor of 21.4% (R-caged GTP, 1.85 A resolution), 18.9% (S-caged GTP, 2.5 A resolution) and 17.6% (mant dGppNHp, 2.7 A resolution), respectively. Details of the structure determination, refinement and the structures themselves are presented. The overall structures of the complexes are identical in terms of the general organization of their secondary structure elements and are also identical to that reported for the analogous complex of p21H-ras with GppNHp. The binding of the GTP part is not significantly affected by the additional aromatic group (cage and mant, respectively) in contrast to the original observation on p21:caged GTP using the racemic mixture of R- and S-caged GTP. The main differences in the structures are observed in the region of loop L2 (residues Glu31 to Thr35) where the additional aromatic group attached to the nucleotide comes very close to the side-chain of Tyr32, including backbone displacements of 2.6 A, 2.2 A and 0.3 A for the residues from Glu31 to Thr35 for R-caged, S-caged GTP and mant dGppNHp, respectively. The refined structures provide additional data for the design of new nucleotide analogs and the importance of their stereochemistry as well as for the design of new mutant forms of p21H-ras for further biochemical investigations. The binding mode of mant dGppNHp reveals significant features for the understanding of the fluorescence signals observed in solution.
The interaction of Ran, a Ras-related nuclear GTP-binding protein, with its guanine nucleotide exchange factor RCC1 has been studied by equilibrium and transient kinetic measurements using fluorescent nucleotides. The four-step mechanism of catalyzed nucleotide exchange involves the formation of ternary complexes consisting of Ran, RCC1, and GXP as well as a nucleotide-free dimeric Ran.RCC1 complex. This model is sufficient to describe all experimental data obtained, so that no additional reaction steps must be assumed. All the rate and equilibrium constants for the four-step mechanism have been determined either experimentally or from a simultaneous theoretical fit to all experimental data sets. The affinities of RCC1 to Ran.GDP and Ran.GTP are similar (1.3 x 10(5) and 1.8 x 10(5) M-1, respectively) and are high enough to allow formation of the ternary complex under appropriate concentration conditions. In the absence of excess nucleotide and at low Ran concentrations, GDP (or GTP) can be efficiently displaced by excess RCC1 and the ternary complex can be produced. The affinities of both nucleotides (GDP or GTP) to Ran in the corresponding ternary complexes are reduced by orders of magnitude in comparison with the respective binary complexes. The reduction of affinity of both nucleotides in the ternary complexes leads to a dramatic increase in the dissociation rate constants by similar orders of magnitude (from 1.5 x 10(-5) s(-1) to 21 s(-1) for GDP) and thus to facilitated nucleotide exchange. The quantitative results of the kinetic analysis suggest that the exchange reaction does not per se favor the formation of the Ran.GTP complex, but rather accelerates the formation of the equilibrium dictated by the relative affinities of Ran for GDP/GTP and the respective concentrations of the nucleotide in the cell. The extent of Ran.GTP formation in vivo can be calculated using the constants derived.
Ras plays the role of a molecular switch in many cellular signalling pathways. The Raf-kinase has been identified as the direct target molecule of Ras in mammalian cells. However, in recent reports other proteins have been characterised as putative Ras effectors which have neither a functional nor a structural relationship to each other. In addition it has been shown that also other members of the Ras family like Rap and R-Ras can interact with some of these proteins. To address the problem of specificity and of biological relevance of the interactions, they have to be carefully quantified and the cellular localisation of the proteins involved taken into account.
The X-ray crystal structure of the complex between the Ras-related protein Rap1A in the GTP-analogue (GppNHp) form and the Ras-binding domain (RBD) of the Ras effector molecule c-Raf1, a Ser/Thr-specific protein kinase, has been solved to a resolution of 2.2 A. It shows that RBD has the ubiquitin superfold and that the structure of Rap1A is very similar to that of Ras. The interaction between the two proteins is mediated by an apparent central antiparallel beta-sheet formed by strands B1-B2 from RBD and strands beta 2-beta 3 from Rap1A. Complex formation is mediated by main-chain and side-chain interactions of the so-called effector residues in the switch I region of Rap1A.
MxA is a GTPase encoded by an interferon-activated human gene which inhibits the multiplication of several RNA viruses. Recombinant histidine-tagged MxA protein (His-MxA) was expressed in Escherichia coli and purified to near homogeneity. Gel filtration showed that it formed high molecular weight oligomers. Purified His-MxA exhibited specific GTP hydrolysis rates of up to 350 nmol of GTP/min/mg of protein, corresponding to a turnover number of 27 min-1. The Km for this reaction was 260 microM. Guanine nucleotides did not copurify with His-MxA. Binding experiments in solution with fluorescent-labeled nucleotides confirmed that His-MxA binds guanine nucleotides rather weakly and further showed that the fluorescent GDP analog N-methylanthraniloyl (mant)-GDP had a much lower affinity for His-MxA (Kd 20 microM, koff 8.5 s-1) than the nonhydrolyzable GTP analog mant-5'-guanylyl-beta,gamma-imidotriphosphate (mant-GMP-PNP) (Kd 0.75 microM, koff 0.012 s-1). Competitive binding studies with nonlabeled nucleotides revealed a similar binding preference of His-MxA for GTP over GDP: the Kd for GTP was 20 microM, whereas the Kd for GDP was 100 microM. Thus, a high percentage of MxA molecules may be complexed with GTP in vivo.
Ran/TC4 is a ras-related GTP-binding protein predominantly located in the nucleus. Ran/TC4 is essential for nuclear transport and is involved in mitotic control. In Saccharomyces cerevisiae a gene highly homologous to Ran/TC4 has been identified and named GSP1. Like all ras-related GTP-binding proteins, Gsp1p undergoes cycles of GTP hydrolysis and GDP/GTP exchange. The switching between the two different nucleotide bound states regulates the function of these GTP-binding proteins. Here we identify the product of the yeast RNA1 gene as the GTPase-activating protein (GAP) of Gsp1p. RNA1 belongs to a group of genes which are conserved in a variety of different organisms. We have expressed and purified recombinant Gsp1p and Rna1p from Escherichia coli. The GTPase activity of Gsp1p is stimulated 10(7)-fold by Rna1p. In addition, we find that the previously identified human RanGAP1 and rna1p from Schizosaccharomyces pombe are also able to induce GTPase activity of Gsp1p. The GTP hydrolysis of Ran is induced by RanGAP1 and rna1p but not by Rna1p. Implications for the suggested functions of Ran/TC4/Gsp1p in nuclear transport and mitotic control are discussed.
UMP/CMP-kinase (UK) from the slime mold Dictyostelium discoideum has been purified to high homogeneity and co-crystallized with the bisubstrate inhibitor P1-(adenosine 5')-P5-(uridine 5')-pentaphosphate (UP5A). UP5A binds to UK with a dissociation constant (Kd) of 3 +/- 0.5 nM at 25 degrees C and pH 7.5. This is some 50-fold tighter than the binding of P1,P5-(diadenosine 5')-pentaphosphate (AP5A, Kd = 160 +/- 15 nM). AP5A is a bisubstrate inhibitor that is specific for adenylate kinase. The crystals have the symmetry of the tetragonal space group P4(1)2(1)2 or its enantiomorph P4(3)2(1)2. The unit cell dimensions are a = b = 78.5 A and c = 101.4 A. The crystals diffract to a Bragg spacing of 2.1 A.
The Ran proteins constitute a distinct branch of the superfamily of Ras-related GTP-binding proteins which function as molecular switches cycling between GTP-bound 'on' and GDP-bound 'off' states. Ran is located predominantly in the nucleus of eukaryotic cells and is involved in the nuclear import of proteins as well as in control of DNA synthesis and of cell-cycle progression. We report here the crystal structure at 2.3 A resolution of human Ran (Mr 24K) complexed with GDP and Mg2+. This structure reveals a similarity with the Ras core (G-domain) but with significant variations in regions involved in GDP and Mg2+ coordination (switch I and switch II regions in Ras), suggesting that there could be major conformational changes upon GTP binding. In addition to the G-domain, an extended chain and an alpha-helix were identified at the carboxy terminus. The amino-terminal (amino-acid residues MAAQGEP) stretch and the acidic tail (DEDDDL) appear to be flexible in the crystal structure.